Advertisement

C-terminal splice variants of P/Q-type Ca2+ channel CaV2.1 α1 subunits are differentially regulated by Rab3-interacting molecule proteins

  • Mitsuru Hirano
    Affiliations
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
    Search for articles by this author
  • Yoshinori Takada
    Affiliations
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
    Search for articles by this author
  • Chee Fah Wong
    Affiliations
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan

    Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjung Malim, Perak, Malaysia
    Search for articles by this author
  • Kazuma Yamaguchi
    Affiliations
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
    Search for articles by this author
  • Hiroshi Kotani
    Affiliations
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
    Search for articles by this author
  • Tatsuki Kurokawa
    Affiliations
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
    Search for articles by this author
  • Masayuki X. Mori
    Affiliations
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
    Search for articles by this author
  • Terrance P. Snutch
    Affiliations
    Michael Smith Laboratories and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
    Search for articles by this author
  • Michel Ronjat
    Affiliations
    LabEx Ion Channels, Science and Therapeutics, INSERM UMR1087/CNRS UMR6291, Institut du Thorax, Université de Nantes, Nantes F-44000, France
    Search for articles by this author
  • Michel De Waard
    Affiliations
    LabEx Ion Channels, Science and Therapeutics, INSERM UMR1087/CNRS UMR6291, Institut du Thorax, Université de Nantes, Nantes F-44000, France
    Search for articles by this author
  • Yasuo Mori
    Correspondence
    To whom correspondence should be addressed: Laboratory of Molecular Biology, Dept. of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan. Tel.: 81-75-383-2761; Fax: 81-75-383-2765
    Affiliations
    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan

    Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto 615-8510, Japan
    Search for articles by this author
Open AccessPublished:April 04, 2017DOI:https://doi.org/10.1074/jbc.M117.778829
      Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and β subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40–47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.

      Introduction

      Fine regulation of neurotransmitter release is integral to adaptive functions of the nervous system, including learning, memory, and cognition. Neurotransmitter release is triggered by depolarization-induced Ca2+ influx via voltage-dependent Ca2+ channels (VDCCs)
      The abbreviations used are: VDCC
      voltage-dependent Ca2+ channel
      VDI
      voltage-dependent inactivation
      polyQ
      polyglutamine
      AZ
      active zone
      SV
      synaptic vesicle
      AP
      action potential
      RIM
      Rab3 interacting molecule
      CTD
      C-terminal domain encoded by the exons 40–47 in the CaV2.1 gene
      co-IP
      co-immunoprecipitation
      IP
      immunoprecipitation
      WB
      Western blotting
      I-V
      current density-voltage
      RIM-BP
      RIM-binding protein
      PXXP
      proline-rich region
      EGFP
      enhanced GFP.
      in presynaptic active zones (AZs), where synaptic vesicles (SVs) dock in close vicinity to VDCCs at the presynaptic membrane (
      • Ackermann F.
      • Waites C.L.
      • Garner C.C.
      Presynaptic active zones in invertebrates and vertebrates.
      ,
      • Jahn R.
      • Fasshauer D.
      Molecular machines governing exocytosis of synaptic vesicles.
      ). Among different VDCC types, which are distinguished on the basis of their pharmacological and biophysical properties, L-, N-, R-, and P/Q-types have been reported to mediate Ca2+ influx responsible for neurotransmitter release (
      • Tsien R.W.
      • Lipscombe D.
      • Madison D.V.
      • Bley K.R.
      • Fox A.P.
      Multiple types of neuronal calcium channels and their selective modulation.
      ,
      • Birnbaumer L.
      • Campbell K.P.
      • Catterall W.A.
      • Harpold M.M.
      • Hofmann F.
      • Horne W.A.
      • Mori Y.
      • Schwartz A.
      • Snutch T.P.
      • Tanabe T.
      The naming of voltage-gated calcium channels.
      ,
      • Takahashi T.
      • Momiyama A.
      Different types of calcium channels mediate central synaptic transmission.
      ,
      • Wu L.G.
      • Borst J.G.
      • Sakmann B.
      R-type Ca2+ currents evoke transmitter release at a rat central synapse.
      ). Different VDCC types show distinct tissue expression patterns, subcellular localizations, activity-dependent properties, and amounts of Ca2+ influx, all of which contribute to the fine regulation of neurotransmitter release (
      • Fox A.P.
      • Nowycky M.C.
      • Tsien R.W.
      Single-channel recordings of three types of calcium channels in chick sensory neurones.
      ,
      • Weber A.M.
      • Wong F.K.
      • Tufford A.R.
      • Schlichter L.C.
      • Matveev V.
      • Stanley E.F.
      N-type Ca2+ channels carry the largest current: implications for nanodomains and transmitter release.
      ,
      • Davydova D.
      • Marini C.
      • King C.
      • Klueva J.
      • Bischof F.
      • Romorini S.
      • Montenegro-Venegas C.
      • Heine M.
      • Schneider R.
      • Schröder M.S.
      • Altrock W.D.
      • Henneberger C.
      • Rusakov D.A.
      • Gundelfinger E.D.
      • Fejtova A.
      Bassoon specifically controls presynaptic P/Q-type Ca2+ channels via RIM-binding protein.
      ,
      • Parajuli L.K.
      • Nakajima C.
      • Kulik A.
      • Matsui K.
      • Schneider T.
      • Shigemoto R.
      • Fukazawa Y.
      Quantitative regional and ultrastructural localization of the CaV2.3 subunit of R-type calcium channel in mouse brain.
      ,
      • Holderith N.
      • Lorincz A.
      • Katona G.
      • Rózsa B.
      • Kulik A.
      • Watanabe M.
      • Nusser Z.
      Release probability of hippocampal glutamatergic terminals scales with the size of the active zone.
      ,
      • Kulik A.
      • Nakadate K.
      • Hagiwara A.
      • Fukazawa Y.
      • Luján R.
      • Saito H.
      • Suzuki N.
      • Futatsugi A.
      • Mikoshiba K.
      • Frotscher M.
      • Shigemoto R.
      Immunocytochemical localization of the a1A subunit of the P/Q-type calcium channel in the rat cerebellum.
      ,
      • Forsythe I.D.
      • Tsujimoto T.
      • Barnes-Davies M.
      • Cuttle M.F.
      • Takahashi T.
      Inactivation of presynaptic calcium current contributes to synaptic depression at a fast central synapse.
      ,
      • Inchauspe C.G.
      • Martini F.J.
      • Forsythe I.D.
      • Uchitel O.D.
      Functional compensation of P/Q by N-type channels blocks short-term plasticity at the calyx of Held presynaptic terminal.
      ,
      • Ishikawa T.
      • Kaneko M.
      • Shin H.S.
      • Takahashi T.
      Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice.
      ,
      • Miki T.
      • Hirai H.
      • Takahashi T.
      Activity-dependent neurotrophin signaling underlies developmental switch of Ca2+ channel subtypes mediating neurotransmitter release.
      ,
      • Ertel E.A.
      • Campbell K.P.
      • Harpold M.M.
      • Hofmann F.
      • Mori Y.
      • Perez-Reyes E.
      • Schwartz A.
      • Snutch T.P.
      • Tanabe T.
      • Birnbaumer L.
      • Tsien R.W.
      • Catterall W.A.
      Nomenclature of voltage-gated calcium channels.
      ,
      • Catterall W.A.
      • Few A.P.
      Calcium channel regulation and presynaptic plasticity.
      ). In particular, the local Ca2+ concentration ([Ca2+]local) and spacing between VDCCs and SVs are tightly regulated by the molecular organization of presynaptic AZs and influence the dynamic properties of neurotransmitter release (
      • Jahn R.
      • Fasshauer D.
      Molecular machines governing exocytosis of synaptic vesicles.
      ,
      • Davydova D.
      • Marini C.
      • King C.
      • Klueva J.
      • Bischof F.
      • Romorini S.
      • Montenegro-Venegas C.
      • Heine M.
      • Schneider R.
      • Schröder M.S.
      • Altrock W.D.
      • Henneberger C.
      • Rusakov D.A.
      • Gundelfinger E.D.
      • Fejtova A.
      Bassoon specifically controls presynaptic P/Q-type Ca2+ channels via RIM-binding protein.
      ,
      • Catterall W.A.
      • Few A.P.
      Calcium channel regulation and presynaptic plasticity.
      ,
      • Hibino H.
      • Pironkova R.
      • Onwumere O.
      • Vologodskaia M.
      • Hudspeth A.J.
      • Lesage F.
      RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca2+ channels.
      ,
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Geppert M.
      • Goda Y.
      • Hammer R.E.
      • Li C.
      • Rosahl T.W.
      • Stevens C.F.
      • Südhof T.C.
      Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse.
      ,
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ,
      • Sheng Z.H.
      • Rettig J.
      • Takahashi M.
      • Catterall W.A.
      Identification of a syntaxin-binding site on N-type calcium channels.
      ,
      • Bezprozvanny I.
      • Scheller R.H.
      • Tsien R.W.
      Functional impact of syntaxin on gating of N-type and Q-type calcium channels.
      ,
      • Bachnoff N.
      • Cohen-Kutner M.
      • Trus M.
      • Atlas D.
      Intra-membrane signaling between the voltage-gated Ca2+-channel and cysteine residues of syntaxin 1A coordinates synchronous release.
      ,
      • Kaeser P.S.
      • Regehr W.G.
      Molecular mechanisms for synchronous, asynchronous, and spontaneous neurotransmitter release.
      ,
      • Wang L.Y.
      • Augustine G.J.
      Presynaptic nanodomains: a tale of two synapses.
      ). It is also understood that the number of open VDCCs, which determines the [Ca2+]local and release probability of SVs, depends on the efficiency of targeting and availability of VDCCs in the AZ (
      • Sheng J.
      • He L.
      • Zheng H.
      • Xue L.
      • Luo F.
      • Shin W.
      • Sun T.
      • Kuner T.
      • Yue D.T.
      • Wu L.G.
      Calcium-channel number critically influences synaptic strength and plasticity at the active zone.
      ). In response to membrane depolarization, VDCCs open to evoke [Ca2+]local rises and simultaneously close via inactivation. This negative feedback reduces the number of VDCCs available and restricts the amplitude of Ca2+ influx, which is important for the diversification of Ca2+ signaling (
      • Simms B.A.
      • Zamponi G.W.
      Neuronal voltage-gated calcium channels: structure, function, and dysfunction.
      ). Inactivation of VDCCs in the presynapse is largely dependent upon the inward Ca2+ current magnitude and displays only a weak voltage dependence (
      • Forsythe I.D.
      • Tsujimoto T.
      • Barnes-Davies M.
      • Cuttle M.F.
      • Takahashi T.
      Inactivation of presynaptic calcium current contributes to synaptic depression at a fast central synapse.
      ,
      • Stanley E.F.
      Syntaxin I modulation of presynaptic calcium channel inactivation revealed by botulinum toxin C1.
      ).
      In the P/Q-type, VDCCs are composed of the pore-forming α1 subunit (CaV2.1) and accessory α2δ, β, and γ subunits. CaV2.1 is the most abundantly expressed VDCC α1 subunit in the mammalian brain (
      • Mori Y.
      • Friedrich T.
      • Kim M.S.
      • Mikami A.
      • Nakai J.
      • Ruth P.
      • Bosse E.
      • Hofmann F.
      • Flockerzi V.
      • Furuichi T.
      • Mikoshiba K.
      • Imoto K.
      • Tanabe T.
      • Numa S.
      Primary structure and functional expression from complementary DNA of a brain calcium channel.
      ), and mutations in the CaV2.1 gene, cacna1a, cause several autosomal-dominant neurological disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6 (SCA6) (
      • Pietrobon D.
      CaV2.1 channelopathies.
      ,
      • Ophoff R.A.
      • Terwindt G.M.
      • Vergouwe M.N.
      • van Eijk R.
      • Oefner P.J.
      • Hoffman S.M.
      • Lamerdin J.E.
      • Mohrenweiser H.W.
      • Bulman D.E.
      • Ferrari M.
      • Haan J.
      • Lindhout D.
      • van Ommen G.J.
      • Hofker M.H.
      • Ferrari M.D.
      • Frants R.R.
      Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4.
      ,
      • Zhuchenko O.
      • Bailey J.
      • Bonnen P.
      • Ashizawa T.
      • Stockton D.W.
      • Amos C.
      • Dobyns W.B.
      • Subramony S.H.
      • Zoghbi H.Y.
      • Lee C.C.
      Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel.
      ). Multiple functional P/Q-type VDCC variants are generated by alternative splicing of subunit genes (
      • Mori Y.
      • Friedrich T.
      • Kim M.S.
      • Mikami A.
      • Nakai J.
      • Ruth P.
      • Bosse E.
      • Hofmann F.
      • Flockerzi V.
      • Furuichi T.
      • Mikoshiba K.
      • Imoto K.
      • Tanabe T.
      • Numa S.
      Primary structure and functional expression from complementary DNA of a brain calcium channel.
      ,
      • Bourinet E.
      • Soong T.W.
      • Sutton K.
      • Slaymaker S.
      • Mathews E.
      • Monteil A.
      • Zamponi G.W.
      • Nargeot J.
      • Snutch T.P.
      Splicing of α1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.
      ), different subunit compositions (
      • Stea A.
      • Tomlinson W.J.
      • Soong T.W.
      • Bourinet E.
      • Dubel S.J.
      • Vincent S.R.
      • Snutch T.P.
      Localization and functional properties of a rat brain α1A calcium channel reflect similarities to neuronal Q- and P-type channels.
      ), post-translational processing (
      • Yokoyama C.T.
      • Myers S.J.
      • Fu J.
      • Mockus S.M.
      • Scheuer T.
      • Catterall W.A.
      Mechanism of SNARE protein binding and regulation of Cav2 channels by phosphorylation of the synaptic protein interaction site.
      ), and association with interacting proteins (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Bezprozvanny I.
      • Scheller R.H.
      • Tsien R.W.
      Functional impact of syntaxin on gating of N-type and Q-type calcium channels.
      ,
      • Tsunemi T.
      • Saegusa H.
      • Ishikawa K.
      • Nagayama S.
      • Murakoshi T.
      • Mizusawa H.
      • Tanabe T.
      Novel CaV2.1 splice variants isolated from Purkinje cells do not generate P-type Ca2+ current.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ). Several types of P/Q-type VDCC complexes can be co-localized in a single neuron and are believed to contribute to the fine-tuning of neuronal processes, such as neurotransmitter release, because formation of each type of CaV2.1 channel complex is regulated in a different manner (
      • Bourinet E.
      • Soong T.W.
      • Sutton K.
      • Slaymaker S.
      • Mathews E.
      • Monteil A.
      • Zamponi G.W.
      • Nargeot J.
      • Snutch T.P.
      Splicing of α1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.
      ,
      • Randall A.
      • Tsien R.W.
      Pharmacological dissection of multiple types of Ca2+ channel currents in rat cerebellar granule neurons.
      ,
      • Wakamori M.
      • Yamazaki K.
      • Matsunodaira H.
      • Teramoto T.
      • Tanaka I.
      • Niidome T.
      • Sawada K.
      • Nishizawa Y.
      • Sekiguchi N.
      • Mori E.
      • Mori Y.
      • Imoto K.
      Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel.
      ,
      • Kanumilli S.
      • Tringham E.W.
      • Payne C.E.
      • Dupere J.R.
      • Venkateswarlu K.
      • Usowicz M.M.
      Alternative splicing generates a smaller assortment of CaV2.1 transcripts in cerebellar Purkinje cells than in the cerebellum.
      ).
      Rab3-interacting molecules (RIMs) are multidomain scaffolding proteins expressed in secretory cells (
      • Südhof T.C.
      The presynaptic active zone.
      ). Long isoform α-RIMs, including RIM1α and RIM2α, contain an N-terminal zinc finger domain, a central PDZ domain, and two C-terminal domains, C2A and C2B. Physiological experiments have shown that α-RIMs are essential for docking and priming of SVs and for recruiting and tethering VDCCs to the presynaptic AZ, thereby regulating VDCC function and short-term plasticity of neurotransmitter release (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ,
      • Schoch S.
      • Castillo P.E.
      • Jo T.
      • Mukherjee K.
      • Geppert M.
      • Wang Y.
      • Schmitz F.
      • Malenka R.C.
      • Südhof T.C.
      RIM1α forms a protein scaffold for regulating neurotransmitter release at the active zone.
      ,
      • Gracheva E.O.
      • Hadwiger G.
      • Nonet M.L.
      • Richmond J.E.
      Direct interactions between C. elegans RAB-3 and Rim provide a mechanism to target vesicles to the presynaptic density.
      ,
      • Koushika S.P.
      • Richmond J.E.
      • Hadwiger G.
      • Weimer R.M.
      • Jorgensen E.M.
      • Nonet M.L.
      A post-docking role for active zone protein Rim.
      ,
      • Deng L.
      • Kaeser P.S.
      • Xu W.
      • Südhof T.C.
      RIM proteins activate vesicle priming by reversing autoinhibitory homodimerization of Munc13.
      ,
      • Han Y.
      • Kaeser P.S.
      • Südhof T.C.
      • Schneggenburger R.
      RIM determines Ca2+ channel density and vesicle docking at the presynaptic active zone.
      ,
      • Castillo P.E.
      • Schoch S.
      • Schmitz F.
      • Südhof T.C.
      • Malenka R.C.
      RIM1α is required for presynaptic long-term potentiation.
      ). We have reported that α-RIMs increase neurotransmitter release by sustaining Ca2+ influx through strong inhibition of voltage-dependent inactivation (VDI) of VDCCs and by anchoring vesicles in the vicinity of VDCCs via interaction with VDCC β subunits (Fig. 1A) (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ). We have also revealed that the RIM C-terminal C2B domain is essential for RIM-β subunit interaction and inhibition of VDI of VDCCs (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ). Mutations in the gene encoding RIMs associated with autism and cone-rod dystrophy, CORD7, modify this interaction and/or the regulation of VDCC currents (
      • Takada Y.
      • Hirano M.
      • Kiyonaka S.
      • Ueda Y.
      • Yamaguchi K.
      • Nakahara K.
      • Mori M.X.
      • Mori Y.
      Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca2+ channels.
      ,
      • Miki T.
      • Kiyonaka S.
      • Uriu Y.
      • De Waard M.
      • Wakamori M.
      • Beedle A.M.
      • Campbell K.P.
      • Mori Y.
      Mutation associated with an autosomal dominant cone-rod dystrophy CORD7 modifies RIM1-mediated modulation of voltage-dependent Ca2+ channels.
      ). Functional coupling of RIM1α to β subunits of VDCCs is also essential for insulin secretion in non-neuronal cells (
      • Gandini M.A.
      • Sandoval A.
      • González-Ramírez R.
      • Mori Y.
      • de Waard M.
      • Felix R.
      Functional coupling of Rab3-interacting molecule 1 (RIM1) and L-type Ca2+ channels in insulin release.
      ). In addition to the β subunits, the PDZ domain of α-RIMs has been reported to interact with the PDZ-binding motif located at the end of the C terminus of α1 subunits to modulate localization of P/Q- and N-type VDCC complexes to presynaptic AZs (Fig. 1A) (
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ). Thus, α-RIMs may interact with VDCC complexes through multiple sites of the constituent subunits. However, the significance of multipoint interactions among VDCC α1 subunits, β subunits, and α-RIMs, as well as functional effects of interactions between α-RIMs and the CaV2.1 C terminus on VDCC currents, remains unclear.
      Figure thumbnail gr1
      Figure 1Characterization of splice variants of the CaV2.1 C terminus in the human cerebellum. A, schematic topology of VDCC CaV2.1 α1 subunit and β subunit and domain structures of CaV2.1 CTD splice variants. PolyQ is 11 glutamine (Q) repeats in the cDNA construct subjected to functional characterization. GenBankTM accession numbers are as follows: U79666 for CaV2.1 CTD (+44,47) and NM_001127221 for CaV2.1 CTD (+44,Δ47). The exon 44-coding region (GenBankTM accession number Z80152) is eliminated in CaV2.1 CTD (−44,47) and CaV2.1 CTD (−44,Δ47). The GGCAG sequence at the 5′-end of exon 47 is eliminated in CaV2.1 CTD (+44,Δ47) and CaV2.1 CTD (−44,Δ47). B, line represents the structure of the region of the human cacna1a gene containing exons 40–47 (top), exons 42–45 (bottom left), and exons 46 and 47 (bottom right). Constitutive exon is shown as black boxes, and alternative exon is shown as gray boxes. The alternatively spliced sequence GGCAG is indicated by dark gray boxes. Stop codons are indicated by asterisks. Alternative splicing patterns are indicated by lines connecting the exons. C, PCR products from human cerebellum cDNA library.
      To quantify the functional significance of multipoint interaction, it is interesting to focus on alternative splicing of exons 44 and 47, because this generates major CaV2.1 C-terminal splice variants expressed in the human cerebellum (Fig. 1B and Table 1). Introns 42–44 are flanked by GT/AG splice-site sequences, and alternative splicing leads to either the inclusion or exclusion of exons 43 and 44 (referred to as (+43 or43) and (+44 or44) in Fig. 1B) (
      • Soong T.W.
      • DeMaria C.D.
      • Alvania R.S.
      • Zweifel L.S.
      • Liang M.C.
      • Mittman S.
      • Agnew W.S.
      • Yue D.T.
      Systematic identification of splice variants in human P/Q-type channel α12.1 subunits: implications for current density and Ca2+-dependent inactivation.
      ). Insertion of a pentanucleotide GGCAG at the beginning of exon 47 allows in-frame translation of exon 47 to produce a long version of the C terminus (referred to as 47) in Fig. 1B). Otherwise, omission of the GGCAG in transcripts causes a frameshift, leading to stop codon termination near the beginning of exon 47 (referred to as (Δ47) in Fig. 1B) to generate the human homolog of the rabbit VDCC α1A subunit BI-I (Fig. 1B) (
      • Mori Y.
      • Friedrich T.
      • Kim M.S.
      • Mikami A.
      • Nakai J.
      • Ruth P.
      • Bosse E.
      • Hofmann F.
      • Flockerzi V.
      • Furuichi T.
      • Mikoshiba K.
      • Imoto K.
      • Tanabe T.
      • Numa S.
      Primary structure and functional expression from complementary DNA of a brain calcium channel.
      ,
      • Zhuchenko O.
      • Bailey J.
      • Bonnen P.
      • Ashizawa T.
      • Stockton D.W.
      • Amos C.
      • Dobyns W.B.
      • Subramony S.H.
      • Zoghbi H.Y.
      • Lee C.C.
      Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel.
      ,
      • Soong T.W.
      • DeMaria C.D.
      • Alvania R.S.
      • Zweifel L.S.
      • Liang M.C.
      • Mittman S.
      • Agnew W.S.
      • Yue D.T.
      Systematic identification of splice variants in human P/Q-type channel α12.1 subunits: implications for current density and Ca2+-dependent inactivation.
      ). The 12-amino acid region encoded by exon 44 starts with the arginine residue, which is located 292 amino acids downstream from the transmembrane segment S6 of repeat IV. The exon 44-encoded region is thought to have an AT-hook domain, which is a tripartite DNA-binding motif specific for AT-rich sequences that is typically found in nuclear proteins and DNA-binding proteins (
      • Giunti P.
      • Mantuano E.
      • Frontali M.
      • Veneziano L.
      Molecular mechanism of Spinocerebellar ataxia type 6: glutamine repeat disorder, channelopathy and transcriptional dysregulation. The multifaceted aspects of a single mutation.
      ,
      • Aravind L.
      • Landsman D.
      AT-hook motifs identified in a wide variety of DNA-binding proteins.
      ). The 244-amino acid region encoded by exon 47 with the GGCAG insertion starts with the glycine residue, which is located 451 amino acids downstream from S6 of repeat IV. The exon 47-encoded region has Src homology 3 and PDZ domain-binding motifs, which are targets of synaptic proteins such as CASK, Mint1, RIM-binding protein (RIM-BP), and α-RIMs (
      • Hibino H.
      • Pironkova R.
      • Onwumere O.
      • Vologodskaia M.
      • Hudspeth A.J.
      • Lesage F.
      RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca2+ channels.
      ,
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ,
      • Maximov A.
      • Südhof T.C.
      • Bezprozvanny I.
      Association of neuronal calcium channels with modular adaptor proteins.
      ). It is also known that expansion of the polyglutamine tract (polyQ), encoded by CAG trinucleotide repeats in exon 47 of human CaV2.1, causes the neurological disease, SCA6 (
      • Zhuchenko O.
      • Bailey J.
      • Bonnen P.
      • Ashizawa T.
      • Stockton D.W.
      • Amos C.
      • Dobyns W.B.
      • Subramony S.H.
      • Zoghbi H.Y.
      • Lee C.C.
      Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel.
      ).
      Table 1Characterization of CaV2.1 C-terminal splice variation in the human cerebellum
      Splice variant% of totaln
      −43087
      +43100
      −441487
      +4486
      Δ473485
      4766
      +43,+44,475680
      +43,−44,4711
      +43,+44,Δ4730
      +43,−44,Δ473
      Here, we studied the interactions between α-RIMs and the CaV2.1 C-terminal regions encoded by exons 44 and 47. We revealed the functional impacts of α-RIM interaction with different CaV2.1 C-terminal regions on its VDI. The 17 amino acid residues on the C-terminal side of the polyQ stretch play an essential role in the pronounced VDI suppression characteristic of RIM2α. In the CaV2.1 splice variant lacking exons 44 and 47, VDI suppression remained intact for RIM1α, but for RIM2α, it was reduced to a level comparable with that of RIM1α. These results suggest that the CTD region plays an important role in the α-RIM isoform-dependent potentiation of VDI suppression. Also, our data reveal that the interaction of α-RIMs with the CTD regions encoded by exons 44 and 47 is not essential for the suppressive effects of α-RIMs on VDI, further raising the possibility that interactions of the VDCC β subunits with α-RIMs underlie their strong suppressive effects on the VDI of VDCCs.

      Results

      Characterization of CaV2.1 C-terminal splice variation in the human cerebellum

      We have previously demonstrated that the RIM C-terminal region containing the C2B domain interacts with VDCC β subunits (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ,
      • Takada Y.
      • Hirano M.
      • Kiyonaka S.
      • Ueda Y.
      • Yamaguchi K.
      • Nakahara K.
      • Mori M.X.
      • Mori Y.
      Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca2+ channels.
      ). It has also been reported that PDZ domains of α-RIMs interact with the PDZ-binding motif located at the C-terminal end of CaV2.1 and CaV2.2 (Fig. 1A) (
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ,
      • Wong F.K.
      • Nath A.R.
      • Chen R.H.
      • Gardezi S.R.
      • Li Q.
      • Stanley E.F.
      Synaptic vesicle tethering and the CaV2.2 distal C-terminal.
      ). The C-terminal region is highly divergent in VDCC α1 subunits because of multiple alternative splice sites (
      • Lipscombe D.
      • Pan J.Q.
      • Gray A.C.
      Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of CaVa1.
      ). In particular, splicing out exon 47 generates CaV2.1 splice variants that lack the most C-terminal region, including the PDZ-binding motif. It is important to quantitatively assess the relative significance of α-RIM interactions with the C-terminal region of CaV2.1 and the VDCC β subunit by comparing α-RIM actions on P/Q-type VDCCs containing different CaV2.1 splice variants carrying the C terminus with and without the α-RIM-interacting region. Previous studies have revealed that exons 43, 44, and 47 contribute to C-terminal splice variations in human CaV2.1 (
      • Soong T.W.
      • DeMaria C.D.
      • Alvania R.S.
      • Zweifel L.S.
      • Liang M.C.
      • Mittman S.
      • Agnew W.S.
      • Yue D.T.
      Systematic identification of splice variants in human P/Q-type channel α12.1 subunits: implications for current density and Ca2+-dependent inactivation.
      ), but relative levels of splice variants with different combinations of these exons have not been quantified. We performed sequence analysis of PCR products from a cDNA library of the human cerebellum, in which abundant expressions of α-RIMs and CaV2.1 mRNAs were reported (
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ,
      • Schoch S.
      • Mittelstaedt T.
      • Kaeser P.S.
      • Padgett D.
      • Feldmann N.
      • Chevaleyre V.
      • Castillo P.E.
      • Hammer R.E.
      • Han W.
      • Schmitz F.
      • Lin W.
      • Südhof T.C.
      Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release.
      ,
      • Ludwig A.
      • Flockerzi V.
      • Hofmann F.
      Regional expression and cellular localization of the α1 and β subunit of high voltage-activated calcium channels in rat brain.
      ). A set of PCR oligonucleotide primers were located in exon 42 (forward) and exon 47 (reverse). Agarose gel electrophoresis (1%) revealed a broad band of PCR products of ∼1,000 bp consistent with the predicted sizes ranging from 834 to 989 bp (Fig. 1C). This DNA band was subcloned into a vector, and the relative levels of splice variants were determined by counting the number of clones containing each exon. The relative proportions of individual splice variants of exon +43/−43, +44/−44, and 47/Δ47 were 100/0, 86/14, and 66/34%, as reported previously (Table 1) (
      • Soong T.W.
      • DeMaria C.D.
      • Alvania R.S.
      • Zweifel L.S.
      • Liang M.C.
      • Mittman S.
      • Agnew W.S.
      • Yue D.T.
      Systematic identification of splice variants in human P/Q-type channel α12.1 subunits: implications for current density and Ca2+-dependent inactivation.
      ). CaV2.1-containing regions encoded by exons 44 and 47 (+44, 47) were detected at the highest relative proportion (56%) (Fig. 1A and Table 1). Relative proportions of CaV2.1 (+44,Δ47), CaV2.1 (−44,47), and CaV2.1 (−44,Δ47) were 30, 11, and 3%, respectively (Fig. 1A and Table 1).

      Interaction between α-RIMs and CaV2.1 C-terminal splice variants

      We next performed yeast two-hybrid screening of a human brain cDNA library using the C-terminal domain encoded by exons 40–47 of human CaV2.1, CaV2.1 CTD (+44,47), as bait, and we identified an interaction between CaV2.1 CTD and the amino acid residues 487–1349 of human RIM2α (GenBankTM accession number NM_001100117) (Fig. 2A). We also performed co-immunoprecipitation (co-IP) experiments to confirm RIM-CaV2.1 CTD interactions (Fig. 2B). As a control, we chose the VDCC β4 subunit, because β4 is abundantly expressed in the brain and the spontaneous β4 mutant lethargic mouse (cacnb4lh) has clear neurological defects, supporting the physiological significance of β4 in the brain (
      • Burgess D.L.
      • Biddlecome G.H.
      • McDonough S.I.
      • Diaz M.E.
      • Zilinski C.A.
      • Bean B.P.
      • Campbell K.P.
      • Noebels J.L.
      β subunit reshuffling modifies N- and P/Q-type Ca2+ channel subunit compositions in lethargic mouse brain.
      ,
      • Burgess D.L.
      • Jones J.M.
      • Meisler M.H.
      • Noebels J.L.
      Mutation of the Ca2+ channel β subunit gene Cchb4 is associated with ataxia and seizures in the lethargic (lh) mouse.
      ). YFP-tagged β4 was co-immunoprecipitated with FLAG-tagged α-RIMs, RIM1α and RIM2α, in HEK293T cells (Fig. 2C), as reported previously (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ). Next, we performed co-IP between YFP-tagged α-RIMs and FLAG-tagged CTDs of CaV2.1 variants derived from alternative splicing of exons 44 and 47 in HEK293T cells. RIM1α and RIM2α were co-immunoprecipitated with the CaV2.1 CTD splice variants except for the variant lacking exons 44 and 47 (Fig. 2D). These results suggest that the two regions encoded by exons 44 and 47 contribute significantly to the interaction between the CaV2.1 CTD and α-RIMs.
      Figure thumbnail gr2
      Figure 2α-RIMs interact with the β4 subunit and the CaV2.1 C-terminal splice variants. A, yeast two-hybrid results indicating the interaction level of CaV2.1 CTD (+44,47) with amino acid residues 487–1349 of human RIM2α (GenBankTM accession number NM_001100117.1) as a function of time. The interactions are scored by β-galactosidase activity judged on a scale of one to five, with one meaning “low activity” and five meaning “high activity.” B, domain structures of mouse α-RIMs. The arrows indicate molecules interacting with RIM proteins at the following domains: Zn2+ finger-like domain (Zn2+), PDZ domain (PDZ), first and second C2 domains (C2A and C2B), and proline-rich region (PXXP). Primary β subunit-binding site (RIM1α(1079–1257)) and β subunit modulatory region (RIM1α(1258–1463)) are indicated (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ). C, interactions of the YFP-tagged β4 subunit with FLAG-tagged α-RIMs in HEK293T cells. D, interactions of the FLAG-tagged CaV2.1 CTD splice variants with YFP-tagged α-RIMs in HEK293T cells. The interactions are evaluated by co-IP with monoclonal anti-FLAG antibody, followed by WB with polyclonal anti-YFP antibody. Input is 10% of the amount of cell lysate used for co-IP and is analyzed by WB using polyclonal anti-YFP antibody. Immunoprecipitation (IP) of FLAG-tagged CaV2.1 CTD splice variants or α-RIMs with monoclonal anti-FLAG antibody is analyzed by WB using polyclonal anti-FLAG antibody.

      Functional impacts of RIM-CaV2.1 C-terminal interaction on CaV2.1 channel properties

      We have previously reported that RIMs strongly suppress VDI of neuronal VDCCs by interacting with the β subunits (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ,
      • Takada Y.
      • Hirano M.
      • Kiyonaka S.
      • Ueda Y.
      • Yamaguchi K.
      • Nakahara K.
      • Mori M.X.
      • Mori Y.
      Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca2+ channels.
      ). It has also been reported that the RIM-CaV2.1 C-terminal interaction modulates localization of VDCCs to presynaptic AZs (
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ). However, the effects of RIM-CaV2.1 C-terminal interaction on VDCC properties have not been examined. We characterized whole-cell Ba2+ currents through recombinant P/Q-type VDCCs containing the CaV2.1 splice variants, β4 and α2δ-1 subunits, in HEK293 cells. We chose Ba2+ as a charge carrier, because CaV2.1 splice variants show different Ca2+-dependent properties in HEK293 cells (
      • Chaudhuri D.
      • Chang S.Y.
      • DeMaria C.D.
      • Alvania R.S.
      • Soong T.W.
      • Yue D.T.
      Alternative splicing as a molecular switch for Ca2+/calmodulin-dependent facilitation of P/Q-type Ca2+ channels.
      ). Voltage dependence of inactivation at different voltages (inactivation curve) (Fig. 3A) was first examined in CaV2.1 (+44,47)-expressing and CaV2.1 (−44,Δ47)-expressing cells. No significant difference was detected between inactivation curves of CaV2.1 (+44,47) and CaV2.1 (−44,Δ47) channels (Fig. 3B and Table 2), which is consistent with previous reports (
      • Chaudhuri D.
      • Chang S.Y.
      • DeMaria C.D.
      • Alvania R.S.
      • Soong T.W.
      • Yue D.T.
      Alternative splicing as a molecular switch for Ca2+/calmodulin-dependent facilitation of P/Q-type Ca2+ channels.
      ,
      • Adams P.J.
      • Garcia E.
      • David L.S.
      • Mulatz K.J.
      • Spacey S.D.
      • Snutch T.P.
      CaV2.1 P/Q-type calcium channel alternative splicing affects the functional impact of familial hemiplegic migraine mutations: implications for calcium channelopathies.
      ). In cells co-expressing α-RIMs and CaV2.1 (−44,Δ47), we detected remarkable inactivation curve shifts toward depolarizing potentials, as described previously (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ). Co-expression of RIM2α induced a more significant inactivation curve shift toward depolarizing potentials compared with that for RIM1α in CaV2.1 (+44,47)-expressing cells, whereas this difference between α-RIMs was not observed in CaV2.1 (-44,Δ47)-expressing cells (the half-inactivation potentials (V0.5) for CaV2.1 (+44,47) and CaV2.1 (−44,Δ47) with RIM2α were −6.7 ± 2.3 and −17.2 ± 2.1 mV, respectively) (Fig. 3B and Table 2). Considering that the previously reported PDZ domain-binding region is not present in the C-terminal end of CaV2.1 (−44,Δ47), RIM-β subunit interaction may play major roles in suppressing VDI of VDCCs, whereas RIM-CaV2.1 C-terminal interaction potentiates this regulatory effect exerted by α-RIMs on VDCCs.
      Figure thumbnail gr3
      Figure 3Effects of α-RIMs on VDI of P/Q-type CaV2.1 channels. A, voltage protocol to determine the inactivation curve and representative traces for Ba2+ currents. To determine the voltage dependence of inactivation, currents are evoked by a 40-ms test pulse to 10 mV after the 10-ms repolarization to −90 mV following 1-s Vpre displacements (conditioning pulses) from −80 to 20 mV with 10-mV increments. B, effects of α-RIMs on inactivation curves of Ba2+ currents mediated by P/Q-type CaV2.1 splice variants in HEK293 cells expressing β4 and α2δ-1 subunits. The inactivation curve in cells co-expressing RIM2α and CaV2.1 (−44,Δ47) (dashed gray line) is taken from left panel and is shown for comparison. *, p < 0.05, and **, p < 0.01, statistical significance of differences between RIM1α- and RIM2α-expressing cells. #, p < 0.05, and ##, p < 0.01, statistical significance of differences versus cells co-expressing RIM2α and CaV2.1 (−44,Δ47). See for statistical significance of the differences. Error bars, S.E.
      Table 2Effect of α-RIMs on inactivation properties of P/Q-type VDCCs in HEK293 cells expressing CaV2.1s, α2/δ-1, and β4
      Inactivation parameters
      AV0.5k
      mVmV
      CaV2.1 (+44,47)Vector0.91 ± 0.02 (5)−30.0 ± 1.9 (5)−7.6 ± 0.4 (5)
      RIM1α0.63 ± 0.04 (11)***−16.9 ± 2.4 (11)**−6.6 ± 0.7 (11)
      RIM2α0.53 ± 0.04 (13)***−6.7 ± 2.3 (13)***, ##, ††−9.9 ± 1.0 (13)#
      CaV2.1 (−44,Δ47)Vector0.94 ± 0.01 (5)−27.1 ± 0.6 (5)−6.2 ± 0.2 (5)
      RIM1α0.67 ± 0.06 (5)**−16.2 ± 4.2 (5)*−8.4 ± 0.9 (5)
      RIM2α0.61 ± 0.02 (15)***−17.2 ± 2.1 (15)*−7.7 ± 0.6 (15)
      CaV2.1 (−44,47)Vector0.92 ± 0.03 (5)−24.8 ± 1.5 (5)−6.2 ± 1.0 (5)
      RIM2α0.51 ± 0.07 (7)**−8.6 ± 2.0 (7)***, †−8.4 ± 1.1 (7)
      CaV2.1 (+44,Δ47)Vector0.92 ± 0.03 (4)−23.0 ± 0.7 (4)−6.0 ± 0.2 (4)
      RIM2α0.69 ± 0.03 (5)**−15.4 ± 2.4 (5)*−6.9 ± 1.1 (5)
      CaV2.1 (+44,47) Gln-40Vector0.96 ± 0.02 (4)−30.6 ± 2.0 (4)−7.1 ± 0.6 (4)
      RIM2α0.42 ± 0.08 (8)***, ††−11.0 ± 2.2 (8)***−8.5 ± 0.8 (8)
      To further assess the importance of interactions via the regions encoded by exons 44 and 47 in this potentiation characteristic of RIM2α in suppression of VDI, we examined the effect of RIM2α on CaV2.1 (−44,47) and CaV2.1 (+44,Δ47) inactivation curves. A significantly enhanced shift in the inactivation curve toward depolarizing potentials was detected in cells co-expressing RIM2α and CaV2.1 (−44,47), but not in cells co-expressing RIM2α and CaV2.1 (+44,Δ47), compared with cells co-expressing RIM2α and CaV2.1 (−44,Δ47) (V0.5 of CaV2.1 (−44,47) and CaV2.1 (+44,Δ47) with RIM2α was −8.6 ± 2.0 and −15.4 ± 2.4 mV, respectively) (Fig. 3B and Table 2). We could not observe a significant difference between CaV2.1 (+44,47) and CaV2.1 (−44,Δ47) in current density-voltage (I-V) relationships, with or without RIM2α co-expression (Fig. 4 and Table 3). These results suggest that the RIM-CaV2.1 C-terminal interaction via the region encoded by exon 47 is important for the potentiated suppressive effect of RIM2α on VDI.
      Figure thumbnail gr4
      Figure 4Effects of α-RIMs on the I-V relationships of P/Q-type CaV2.1 channels. I-V relationships of CaV2.1 splice variants in HEK293 cells expressing β4 and α2δ-1 subunits. Left, representative traces for Ba2+ currents on application of test pluses from −40 to 60 mV with 10-mV increments from a holding potential (Vh) of −90 mV. Right, I-V relationships of CaV2.1 splice variants. See for statistical significance of the differences. Error bars, S.E.
      Table 3Effect of α-RIMs on the I-V relationships of P/Q-type VDCCs in HEK293 cells expressing CaV2.1s, α2/δ-1, and β4
      Current density
      Ba2+ currents evoked by depolarizing pulse to 10 mV from a Vh of −90 mV are divided by capacitance.
      V0.5k
      pA/picofaradmVmV
      CaV2.1 (+44,47)Vector−71.7 ± 10.8 (5)−3.0 ± 1.3 (5)4.5 ± 0.2 (5)
      RIM1α−97.5 ± 21.3 (5)−5.1 ± 1.9 (5)4.5 ± 0.4 (5)
      RIM2α−83.1 ± 7.5 (7)−3.7 ± 1.1 (7)3.7 ± 0.6 (7)
      CaV2.1 (−44,Δ47)Vector−111.5 ± 13.8 (4)−4.0 ± 1.6 (4)3.0 ± 0.3 (4)
      RIM1α−123.8 ± 30.6 (4)−3.2 ± 1.7 (4)3.6 ± 0.6 (4)
      RIM2α−102.5 ± 7.4 (7)−6.5 ± 1.0 (7)3.6 ± 0.3 (7)
      a Ba2+ currents evoked by depolarizing pulse to 10 mV from a Vh of −90 mV are divided by capacitance.
      Inactivation kinetics of P/Q-type VDCCs was characterized by analyzing the decay phase of Ba2+ currents evoked by 1-s test pulses in HEK293 cells (Fig. 5A). The decay phase was well fitted by two exponential functions with a non-inactivating component (Fig. 5B) as reported previously (
      • Wakamori M.
      • Yamazaki K.
      • Matsunodaira H.
      • Teramoto T.
      • Tanaka I.
      • Niidome T.
      • Sawada K.
      • Nishizawa Y.
      • Sekiguchi N.
      • Mori E.
      • Mori Y.
      • Imoto K.
      Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel.
      ). The two exponential time constants (τfast and τslow) and the ratio of fast, slow, and non-inactivating components were similar in CaV2.1 (+44,47)- and CaV2.1 (−44,Δ47)-expressing cells at test potentials of 0, 10, and 20 mV (Fig. 5, C and D, and Table 4). Co-expression of α-RIMs exerted common effects on Ba2+ currents in CaV2.1 (+44,47)- and CaV2.1 (−44,Δ47)-expressing cells. The fast-inactivating component was significantly decreased at 0 and 10 mV, whereas the non-inactivating component as well as τslow were significantly increased by α-RIMs at 0, 10, and 20 mV (Fig. 5, C and D, and Table 4). Thus, it is unlikely that these effects are mediated by interaction of α-RIMs with the CaV2.1 C-terminal region encoded by exons 44 and 47. In cells co-expressing RIM2α and CaV2.1 (+44,47), increases in the non-inactivating component at 0, 10, and 20 mV and τfast at 0 and 10 mV were more pronounced compared with cells co-expressing RIM2α and CaV2.1 (−44,Δ47) (Fig. 5, C and D, and Table 4). This effect can be mediated by the RIM2α-CaV2.1 C-terminal interaction. Furthermore, currents evoked by trains (100 Hz) of action potential (AP)-like waveforms for 3 s, a more physiological voltage-clamp protocol used to determine closed-state inactivation (
      • Patil P.G.
      • Brody D.L.
      • Yue D.T.
      Preferential closed-state inactivation of neuronal calcium channels.
      ), showed a more rapid decrease in amplitude in cells co-expressing RIM2α and CaV2.1 (−44,Δ47) compared with cells co-expressing RIM2α and CaV2.1 (+44,47) (Fig. 5E). These results suggest that RIM2α-CaV2.1 C-terminal interaction is not essential for but enhances the VDI suppression induced by α-RIMs.
      Figure thumbnail gr5
      Figure 5Effects of α-RIMs on VDI kinetics of P/Q-type CaV2.1 channels. A, effects of α-RIMs on inactivation of Ba2+ currents mediated by P/Q-type CaV2.1 splice variants in HEK293 cells expressing β4 and α2δ-1 subunits. The peak amplitudes are normalized for Ba2+ currents elicited by 1-s pulses to 10 mV from a Vh of −90 mV. B, Ba2+ current evoked by 1-s test pulse to 10 mV from a Vh of −90 mV in HEK293 cells expressing β4, α2δ-1 subunits, and RIM1α. Current decay is fitted by a sum of two exponential functions with time constants of 55 and 995 ms, whose fraction of components is 0.19 and 0.61, respectively. The fraction of its non-inactivating component is 0.24. C, voltage dependence of the two inactivation time constants, τfast and τslow. The mean inactivation time constants are plotted as a function of test potential from 0 to 20 mV. D, voltage dependence of the fraction of the three components, fast-, slow-, and non-inactivating components. The fractions of the components are plotted against test potentials. †, p < 0.05; ††, p < 0.01; statistical significance of differences between cells co-expressing CaV2.1 (−44,Δ47) and RIM2α and cells co-expressing CaV2.1 (+44,47) and RIM2α. See for statistical significance of the differences. Error bars, S.E. E, left, effects of RIM2α on Ba2+ currents mediated by P/Q-type CaV2.1 splice variants in response to 100 Hz AP-like voltage command for 3 s in HEK293 cells expressing β4 and α2δ-1 subunits (AP-like waveform, −80 to 33 mV; rise time, 0.4 ms; decay time, 1.1 ms). Right, percentage of currents in response to the last stimulus compared with currents in response to the first stimulus (CaV2.1 (+44,47) RIM2α, 88 ± 4%, n = 4; CaV2.1 (−44,Δ47) RIM2α, 67 ± 15%, n = 8). Data are presented in scatter plots. The vertical error bars represent standard deviations, and the mean values are indicated with horizontal bars. *, p < 0.05; statistical significance of difference between cells co-expressing CaV2.1 (−44,Δ47) and RIM2α and cells co-expressing CaV2.1 (+44,47) and RIM2α.
      Table 4Effect of α-RIMs on inactivation kinetics of P/Q-type VDCCs in HEK293 cells expressing CaV2.1s, α2/δ-1, and β4
      Fraction of componentsTime constants
      mVFastSlowSustainedτfastτslow
      CaV2.1 (+44,47)Vector (8)00.84 ± 0.050.25 ± 0.050.10 ± 0.020.08 ± 0.010.41 ± 0.09
      100.79 ± 0.100.33 ± 0.060.07 ± 0.010.07 ± 0.010.26 ± 0.05
      200.44 ± 0.050.61 ± 0.050.03 ± 0.010.04 ± 0.010.16 ± 0.02
      RIM1α (11)00.41 ± 0.04***0.38 ± 0.030.32 ± 0.02***0.10 ± 0.040.88 ± 0.13*
      100.39 ± 0.06**0.50 ± 0.060.19 ± 0.02**0.07 ± 0.020.78 ± 0.09***
      200.42 ± 0.070.48 ± 0.040.17 ± 0.03**0.09 ± 0.030.74 ± 0.09***
      RIM2α (11)00.20 ± 0.03***, ##0.28 ± 0.050.53 ± 0.06***, ##, †0.71 ± 0.19*, ##, ††1.12 ± 0.11***
      100.30 ± 0.04***0.36 ± 0.040.37 ± 0.04***, ##, †0.40 ± 0.14#, †0.94 ± 0.08***
      200.30 ± 0.040.40 ± 0.02***0.33 ± 0.03***, ##, ††0.19 ± 0.060.95 ± 0.12***
      CaV2.1 (−44,Δ47)Vector (5)00.95 ± 0.050.22 ± 0.040.06 ± 0.010.09 ± 0.010.39 ± 0.08
      100.84 ± 0.100.27 ± 0.080.05 ± 0.010.09 ± 0.010.29 ± 0.04
      200.66 ± 0.120.51 ± 0.080.04 ± 0.010.08 ± 0.010.24 ± 0.05
      RIM1α (5)00.45 ± 0.08***0.38 ± 0.050.22 ± 0.03***0.16 ± 0.080.88 ± 0.15*
      100.45 ± 0.07*0.39 ± 0.050.22 ± 0.04**0.18 ± 0.080.74 ± 0.13*
      200.43 ± 0.100.49 ± 0.110.14 ± 0.01***0.06 ± 0.010.64 ± 0.18*
      RIM2α (13)00.26 ± 0.10***0.39 ± 0.050.32 ± 0.04**0.12 ± 0.031.08 ± 0.07***
      100.26 ± 0.10**0.45 ± 0.04*0.25 ± 0.02***0.08 ± 0.01#0.83 ± 0.05***
      200.29 ± 0.07*0.46 ± 0.040.22 ± 0.02***, #0.08 ± 0.010.72 ± 0.09**
      It is important to note that, without the interaction of α-RIMs, these splice variants were indistinguishable in channel properties such as inactivation curve (Fig. 3 and Table 2), inactivation kinetics (Fig. 5 and Table 4), and I-V relationship (Fig. 4 and Table 3). This underscores the significance of protein-protein interaction in differentiating functional properties of alternatively spliced CaV2.1 variants.

      Biochemical characterization of the RIM2α-CaV2.1 C-terminal interaction

      The CaV2.1 mutant, CaV2.1 (+44,47) ΔDDWC, which lacks four amino acids (DDWC) in the C-terminal end was constructed, because these four amino acids bind to the PDZ domain of α-RIMs (
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ,
      • Wong F.K.
      • Nath A.R.
      • Chen R.H.
      • Gardezi S.R.
      • Li Q.
      • Stanley E.F.
      Synaptic vesicle tethering and the CaV2.2 distal C-terminal.
      ). Inactivation curves of whole-cell Ba2+ currents in HEK293 cells co-expressing RIM2α and CaV2.1 (+44,47) ΔDDWC were indistinguishable from those in cells co-expressing RIM2α and CaV2.1 (+44,47), showing shifts toward depolarizing potentials compared with the inactivation curves in cells co-expressing RIM2α and CaV2.1 (−44,Δ47) (V0.5 of CaV2.1 (+44,47) ΔDDWC, CaV2.1 (+44,47), and CaV2.1 (−44,Δ47) were −6.7 ± 3.2 mV, −6.7 ± 2.3 mV, and −17.2 ± 2.1 mV, respectively) (Fig. 6A and Table 5). In co-IP experiments, YFP-tagged RIM2α showed association with FLAG-tagged CaV2.1 CTD (+44,47) ΔDDWC in HEK293T cells (Fig. 6B). The intensity of the co-IP band for RIM2α normalized to the IP band for the CTD was significantly decreased in cells co-expressing RIM2α and CaV2.1 CTD (+44,47) ΔDDWC, compared with cells co-expressing RIM2α and CaV2.1 CTD (+44,47) (Fig. 6C). These data suggest that DDWC in the region encoded by exon 47 contributes to the RIM2α-CaV2.1 C-terminal interaction, as reported previously (
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ,
      • Wong F.K.
      • Nath A.R.
      • Chen R.H.
      • Gardezi S.R.
      • Li Q.
      • Stanley E.F.
      Synaptic vesicle tethering and the CaV2.2 distal C-terminal.
      ), but not to potentiation of VDI suppression by RIM2α.
      Figure thumbnail gr6
      Figure 6Effects of RIM2α on VDI of the P/Q-type CaV2.1 deletion mutant lacking DDWC. A, effects of RIM2α on inactivation curves of Ba2+ currents mediated by P/Q-type CaV2.1 (+44,47) ΔDDWC, which lacks DDWC at the C-terminal end of CaV2.1 (+44,47), in HEK293 cells expressing β4 and α2δ-1 subunits. The inactivation curve in cells co-expressing RIM2α and CaV2.1 (+44,47) (dashed dark gray line) is taken from and is shown for comparison. See for statistical significance of the differences. Error bars, S.E. B, interaction of FLAG-tagged CaV2.1 CTD (+44,47) or CaV2.1 CTD (+44,47) ΔDDWC with YFP-tagged RIM2α in HEK293T cells. The interactions are evaluated by co-IP with monoclonal anti-FLAG antibody, followed by WB with polyclonal anti-YFP antibody. Input is 10% of the amount of cell lysate used for co-IP and is analyzed by WB using polyclonal anti-YFP antibody. IP of FLAG-tagged CaV2.1 CTDs with monoclonal anti-FLAG antibody is analyzed by WB using polyclonal anti-FLAG antibody. C, quantification of the data shown in B. The intensity of the co-IP band for RIM2α normalized to the IP band for the CTD (CaV2.1 CTD (+44,47), 100%; CaV2.1 CTD (+44,47) ΔDDWC, 55 ± 20%) (
      • Lin D.T.
      • Makino Y.
      • Sharma K.
      • Hayashi T.
      • Neve R.
      • Takamiya K.
      • Huganir R.L.
      Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation.
      ). Data for nine experiments are presented in scatter plots. The vertical error bars represent standard deviations, and the mean values are indicated with horizontal bars. ***, p < 0.001; statistical significance of difference between CTD (+44,47) and CTD (+44,47) ΔDDWC.
      Table 5Effects of chimeric RIM and RIM2α deletion mutant on inactivation properties of P/Q-type VDCCs in HEK293 cells expressing CaV2.1, α2/δ-1, and β4
      Inactivation parameters
      aV0.5k
      mVmV
      CaV2.1 (+44,47)RIM2α0.53 ± 0.04 (13)−6.7 ± 2.3 (13)**−9.9 ± 1.0 (13)
      CaV2.1 (−44,Δ47)RIM2α0.61 ± 0.02 (15)−17.2 ± 2.1 (15)−7.7 ± 0.6 (15)
      CaV2.1 (+44,47) ΔDDWCRIM2α0.55 ± 0.05 (7)−6.7 ± 3.2 (7)*−7.2 ± 1.1 (7)
      CaV2.1 (+44,47) 2366XRIM2α0.53 ± 0.07 (6)−6.3 ± 2.3 (6)**−5.7 ± 0.7 (6)
      CaV2.1 (+44,47) 2344XRIM2α0.54 ± 0.04 (6)−8.3 ± 3.0 (6)*−8.2 ± 0.9 (6)
      CaV2.1 (+44,47) 2327XRIM2α0.70 ± 0.03 (6)−14.1 ± 2.4 (6)−8.4 ± 0.9 (6)
      CaV2.1 (+44,47) 2315XRIM2α0.67 ± 0.04 (8)−13.5 ± 1.2 (8)−6.8 ± 0.5 (8)
      CaV2.1 (+44,47)RIM1–2 chimera0.49 ± 0.07 (7)−4.7 ± 2.6 (7)**, #−8.3 ± 0.7 (7)
      RIM2α (1183–1572)0.43 ± 0.06 (7)**, ††−1.9 ± 3.2 (7)***, †−5.3 ± 1.2 (7)
      CaV2.1 (−44,Δ47)RIM1–2 chimera0.59 ± 0.03 (3)−15.9 ± 3.3 (3)−7.6 ± 1.5 (3)
      RIM2α (1183–1572)0.69 ± 0.04 (6)−14.2 ± 4.0 (6)−7.0 ± 1.4 (6)
      To clarify the RIM2α-interacting regions responsible for the potentiation of VDI suppression by RIM2α, additional deletion mutants of CaV2.1 were constructed (Fig. 7A). In cells co-expressing RIM2α with CaV2.1 (+44,2364X) or CaV2.1 (+44,2344X), in which C-terminal residues 2365–2505 or 2345–2505 were deleted, respectively, the inactivation curves were similar to those in cells co-expressing RIM2α with CaV2.1 (+44,47). In contrast, in cells co-expressing RIM2α with CaV2.1 (+44,2327X) or CaV2.1 (+44,2313X), in which C-terminal residues 2328–2505 or 2314–2505 were deleted, respectively, the inactivation curves were similar to those in cells co-expressing RIM2α and CaV2.1 (−44,Δ47) (Fig. 7B and Table 5). These data suggest that 17 amino acid residues, 2328–2344 (RPGRAATSGPRRYPGPT), on the C-terminal side of the polyQ stretch of CaV2.1 (+44,47) is a RIM2α-interacting region that potentiates the suppressive effect on VDI.
      Figure thumbnail gr7
      Figure 7Identification of RIM2α-binding regions in the CaV2.1 C terminus. A, domain structures of CaV2.1 CTD deletion mutants. B, effects of RIM2α on inactivation curves of Ba2+ currents mediated by the deletion mutants of CaV2.1 (+44,47) in HEK293 cells expressing β4 and α2δ-1 subunits. The inactivation curve in cells co-expressing RIM2α and CaV2.1 (+44,47) (dashed dark gray line) is taken from and is shown for comparison. *, p < 0.05; **, p < 0.01; statistical significance of differences versus cells co-expressing RIM2α and CaV2.1 (+44,47). See for statistical significance of the differences. Error bars, S.E. C, interactions of FLAG-tagged CaV2.1 CTD (+44) deletion mutants with YFP-tagged RIM2α in HEK293T cells. D, interactions of FLAG-tagged CaV2.1 CTD (−44) deletion mutants with YFP-tagged RIM2α in HEK293T cells. E, interactions of FLAG-tagged CaV2.1 CTD (−44,Δ2264–2280) deletion mutants with YFP-tagged RIM2α in HEK293T cells. The interactions are evaluated by co-IP with monoclonal anti-FLAG antibody, followed by WB with polyclonal anti-YFP antibody. Input is 10% of the amount of cell lysate used for co-IP and is analyzed by WB using polyclonal anti-YFP antibody. IP of FLAG-tagged CaV2.1 CTDs with monoclonal anti-FLAG antibody is analyzed by WB using polyclonal anti-FLAG antibody. Dashed lines divide mutants in terms of co-IP level (blue) and inactivation curve (red).
      In co-IP experiments, however, YFP-tagged RIM2α showed a comparable level of co-IP with FLAG-tagged CTD of CaV2.1 (+44,2344X) and FLAG-tagged CTD of CaV2.1 (+44,2327X) but a decreased level of co-IP with CTD of CaV2.1 (+44,2275X) (Fig. 7C). To eliminate possible contributions of exon 44-encoded amino acid residues to the interaction of RIM2α with CTD mutants, we next constructed deletion mutants of the CTD of CaV2.1 (−44,47). The co-IP was nearly abolished for the CTD of CaV2.1 (−44,2263X), in which 2263X corresponds to 2275X in CaV2.1 (+44,47) (Fig. 7D). This result suggests that the amino acid residues 2264–2280 (GTSTPRRGRRQLPQTPS) of CaV2.1 (−44,47), which correspond to 2276–2292 of CaV2.1 (+44,47), are an important region for the RIM2α-CaV2.1 CTD interaction. However, the co-IP experiments failed to unveil interaction between the 17 amino acid residues, 2328–2344, of CaV2.1 (+44,47) with RIM2α, although this region is supposed to be essential for potentiated VDI suppression by RIM2α.
      To demonstrate interaction of the 2328–2344 amino acid residues, contributions of the exon 44 region and residues 2276–2292 of CaV2.1 (+44,47) were eliminated in deletion mutants based upon CaV2.1 (−44,Δ2264–2280). The level of co-IP was markedly diminished for the CTD of CaV2.1 (−44,Δ2264–2280,2315X) and CaV2.1 (−44,Δ2264–2280,2301X) compared with the CTD of CaV2.1 (−44,Δ2264–2280,2332X) and CaV2.1 (−44,Δ2264–2280,2352X) (Fig. 7E). This result suggests that the 17 amino acid residues, 2316–2332, of CaV2.1 (−44,47), which correspond to 2328–2344 of CaV2.1 (+44,47), indeed constitute an important binding region for RIM2α to exert a potentiated suppressive effect on VDI.
      It is interesting to note that deletion of the region encoded by exon 44 failed to elicit suppression of interaction of the CaV2.1 CTD containing the region encoded by exon 47 (compare the bands of CaV2.1 CTD (−44,47) and CaV2.1 CTD (+44,47) in Fig. 2D). In contrast, deletion of the region encoded by exon 47 suppressed the interaction of the CaV2.1 CTD containing the region encoded by exon 44 (compare the bands of CaV2.1 CTD (+44,Δ47) and CaV2.1 (+44,47) in Fig. 7C). These results suggest that the region encoded by exon 47 binds more strongly to α-RIMs compared with the region encoded by exon 44. Thus, formation of RIM2α-CaV2.1 complexes is mediated by multipoint interaction.

      Characterization of the RIM2α region responsible for interaction with the CaV2.1 C terminus

      To identify the region in RIM2α that interacts with the CaV2.1 C terminus, we constructed the chimeric RIM, RIM1–2 chimera, with amino acid residues from the N terminus to the C2A domain of RIM1α and from the C2A domain of RIM2α (Fig. 8A). We examined effects of the RIM1–2 chimera on whole-cell Ba2+ currents mediated by CaV2.1 (+44,47)- and CaV2.1 (−44,Δ47)-containing VDCCs (Fig. 8B). Significant enhancement of inactivation curve shift by the RIM1–2 chimera toward depolarizing potentials was observed for CaV2.1 (+44,47) compared with CaV2.1 (−44,Δ47) (V0.5 for CaV2.1 (+44,47) and CaV2.1 (−44,Δ47) with RIM1–2 chimera was −4.7 ± 2.6 and −15.9 ± 3.3 mV, respectively) (Fig. 8B and Table 5). This tendency of the RIM1–2 chimera suggests that the region on the C-terminal side of the C2A domain is important for potentiating the suppressive effect of RIM2α on VDI. We also tested a mutant of RIM2α(1183–1572) composed of the amino acid residues 1183–1572 of RIM2α corresponding to the β subunit-binding region in RIM1α (residues 1079–1463 of RIM1α) (Figs. 2B and 8A) (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ). In cells co-expressing RIM2α(1183–1572) and CaV2.1 (+44,47), we also detected potentiation of the suppressive effect on VDI as observed in cells co-expressing RIM2α(1183–1572) and CaV2.1 (−44,Δ47) (V0.5 and the rates of the inactivating component were −1.9 ± 3.2 mV and 0.43 ± 0.06, respectively, for CaV2.1 (+44,47) with RIM2α(1183–1572) and 14.2 ± 4.0 mV and 0.69 ± 0.04, respectively, for CaV2.1 (−44,Δ47) with RIM2α(1183–1572)) (Fig. 8C and Table 5). Associations between YFP-tagged RIM2α(1183–1572) and FLAG-tagged mutants based upon CaV2.1 CTD (−44,Δ2264–2280), which has a deletion of the region corresponding to 2328–2344 of CaV2.1 (+44,47), were tested by co-IP experiments. RIM2α(1183–1572) showed co-IP with the CTD of CaV2.1 (−44,Δ2264–2280, 2332X) but not with the CTD of CaV2.1 (−44,Δ2264–2280, 2315X) or CaV2.1 (−44,Δ2264–2280, 2301X) (Fig. 8D). These data suggest that the C-terminal region containing the C2B domain is critical for RIM2α to potentiate suppression of VDI and binds with 2328–2344 of CaV2.1 (+44,47) in addition to the β subunits (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ).
      Figure thumbnail gr8
      Figure 8Identification of CaV2.1 C-terminal-binding region in RIM2α. A, domain structures of α-RIMs, RIM1–2 chimera, and RIM2α(1183–1572). B and C, effects of RIM1–2 chimera and RIM2α(1183–1572) on inactivation curves of Ba2+ currents mediated by CaV2.1 splice variants in HEK293 cells expressing β4 and α2δ-1 subunits. *, p < 0.05; **, p < 0.01; statistical significance of differences between CaV2.1 (−44,Δ47)-expressing and CaV2.1 (+44,47)-expressing cells. See for statistical significance of the differences. Error bars, S.E. D, interactions of the deletion mutants of FLAG-tagged CaV2.1 (−44,Δ2264–2280) with YFP-tagged RIM2α(1183–1572) in HEK293T cells. The interactions are evaluated by co-IP with monoclonal anti-FLAG antibody, followed by WB with polyclonal anti-YFP antibody. Input is 10% of the amount of cell lysate used for co-IP and is analyzed by WB using polyclonal anti-YFP antibody. IP of FLAG-tagged CaV2.1 CTDs with monoclonal anti-FLAG antibody is analyzed by WB using polyclonal anti-FLAG antibody.

      Effects of polyQ elongation in the CaV2.1 C-terminal region on regulation of VDI by RIM2α

      The polyQ stretch is an interesting characteristic of the CaV2.1 C-terminal primary structure. SCA6 is caused by expansion of the polyQ tract in the human CaV2.1 gene from a normal repeat size range of 4–17 to a size range of 20–33 (
      • Zhuchenko O.
      • Bailey J.
      • Bonnen P.
      • Ashizawa T.
      • Stockton D.W.
      • Amos C.
      • Dobyns W.B.
      • Subramony S.H.
      • Zoghbi H.Y.
      • Lee C.C.
      Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel.
      ,
      • Giunti P.
      • Mantuano E.
      • Frontali M.
      • Veneziano L.
      Molecular mechanism of Spinocerebellar ataxia type 6: glutamine repeat disorder, channelopathy and transcriptional dysregulation. The multifaceted aspects of a single mutation.
      ,
      • Matsuyama Z.
      • Kawakami H.
      • Maruyama H.
      • Izumi Y.
      • Komure O.
      • Udaka F.
      • Kameyama M.
      • Nishio T.
      • Kuroda Y.
      • Nishimura M.
      • Nakamura S.
      Molecular features of the CAG repeats of spinocerebellar ataxia 6 (SCA6).
      ). To confirm the effect of polyQ expansion on the interaction between the CaV2.1 C terminus and RIM2α, association between YFP-tagged RIM2α and FLAG-tagged CTD of CaV2.1 (+44,47) Gln-40 with an elongated polyQ stretch of 40 residues was tested by co-IP in HEK293T cells (Fig. 9A). The intensity of the co-IP band for RIM2α normalized to the IP band for the CTD was moderately but significantly decreased for CaV2.1 CTD (+44,47) Gln-40 compared with that for CaV2.1 CTD (+44,47) with a polyQ stretch of 11 residues (Fig. 9B). To examine the effect of polyQ elongation on suppression of VDI by RIM2α, a recombinant P/Q-type VDCC was expressed as a complex of CaV2.1 (+44,47) with polyQ expansion (CaV2.1 (+44,47) Gln-40), β4, and α2δ-1 subunits in HEK293 cells (Fig. 9C). Without co-expression of α-RIMs, inactivation curves of whole-cell Ba2+ currents elicited by CaV2.1 (+44,47) and CaV2.1 (+44,47) Gln-40 were indistinguishable (Fig. 9C and Table 2). This contradicts with our previous report that polyQ expansion itself causes a hyperpolarizing shift of the inactivation curve for rabbit CaV2.1 channels carrying the β1 subunit (
      • Matsuyama Z.
      • Wakamori M.
      • Mori Y.
      • Kawakami H.
      • Nakamura S.
      • Imoto K.
      Direct alteration of the P/Q-type Ca2+ channel property by polyglutamine expansion in spinocerebellar ataxia 6.
      ). It is possible that the effect of polyQ expansion on VDI depends on species and β subtypes (
      • Chen H.
      • Piedras-Rentería E.S.
      Altered frequency-dependent inactivation and steady-state inactivation of polyglutamine-expanded α1A in SCA6.
      ). When RIM2α was co-expressed, inactivation curves of CaV2.1 (+44,47) and CaV2.1 (+44,47) Gln-40 were indistinguishable (V0.5 of CaV2.1 (+44,47) Gln-40 and CaV2.1 (+44,47) were −11.0 ± 2.2 mV and −6.7 ± 2.3 mV, respectively) (Fig. 9C and Table 2). These data indicate that polyQ expansion reduced the binding affinity of CaV2.1 C terminus for RIM2α but not the suppressive effect of RIM2α on the VDI of CaV2.1 (+44,47).
      Figure thumbnail gr9
      Figure 9Effects of RIM2α on VDI of polyQ-elongated P/Q-type CaV2.1 channels. A, interaction of FLAG-tagged CaV2.1 CTD (+44,47) or (+44,47) Gln-40 (Q40) with YFP-tagged RIM2α in HEK293T cells. The interactions are evaluated by co-IP with monoclonal anti-FLAG antibody, followed by WB with polyclonal anti-YFP antibody. Input is 10% of the amount of cell lysate used for co-IP and is analyzed by WB using polyclonal anti-YFP antibody. IP of FLAG-tagged CaV2.1 CTDs with monoclonal anti-FLAG antibody is analyzed by WB using polyclonal anti-FLAG antibody. B, quantification of the data shown in A. The intensity of the co-IP band for RIM2α normalized to the IP band for the CTD (CaV2.1 CTD (+44,47), 100%; CaV2.1 CTD (+44,47) Gln-40, 73 ± 18%) (
      • Lin D.T.
      • Makino Y.
      • Sharma K.
      • Hayashi T.
      • Neve R.
      • Takamiya K.
      • Huganir R.L.
      Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation.
      ). Data for nine experiments are presented in scatter plots. The vertical error bars represent standard deviations, and the mean values are indicated with horizontal bars. ***, p < 0.001; statistical significance of difference between CTD (+44,47) and CTD (+44,47) Gln-40. C, effects of RIM2α on inactivation curves of P/Q-type CaV2.1 (+44,47) Gln-40 currents in HEK293 cells expressing β4 and α2δ-1 subunits. The inactivation curves in cells co-expressing vector and CaV2.1 (+44,47) (dashed gray line) and in cells co-expressing RIM2α and CaV2.1 (+44,47) (dashed dark gray line) are taken from and are shown for comparison. See for statistical significance of the differences. Error bars, S.E.

      Functional impacts of α-RIMs on VDI of N-type CaV2.2 and R-type CaV2.3 channels

      CaV2 VDCCs are a major source of presynaptic Ca2+ influx (
      • Catterall W.A.
      Voltage-gated calcium channels.
      ,
      • Wu L.G.
      • Saggau P.
      Presynaptic inhibition of elicited neurotransmitter release.
      ,
      • Ricoy U.M.
      • Frerking M.E.
      Distinct roles for CaV2.1–2.3 in activity-dependent synaptic dynamics.
      ), and α-RIMs also interact with the C terminus of CaV2.2 (
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ,
      • Wong F.K.
      • Nath A.R.
      • Chen R.H.
      • Gardezi S.R.
      • Li Q.
      • Stanley E.F.
      Synaptic vesicle tethering and the CaV2.2 distal C-terminal.
      ). To explore the generality of our findings using CaV2.1, associations of the C-terminal region of CaV2.2 encoded by exons 41–47 of the CaV2.2 gene (CaV2.2 CTD) and the C-terminal region of CaV2.3 encoded by exons 41–48 of the CaV2.3 gene (CaV2.3 CTD) with α-RIMs were tested by co-IP experiments. Both the YFP-tagged CTD of CaV2.2 and the YFP-tagged CTD of CaV2.3 were co-immunoprecipitated with FLAG-tagged α-RIMs in HEK293T cells (Fig. 10A). Thus, the C-terminal interaction with α-RIMs is shared by CaV2.2 and CaV2.3.
      Figure thumbnail gr10
      Figure 10Effects of α-RIMs on VDI of N-type CaV2.2 and R-type CaV2.3 channels. A, interactions of YFP-tagged CaV2.2 CTD or CaV2.3 CTD with FLAG-tagged α-RIMs in HEK293T cells. The interactions are evaluated by co-IP with monoclonal anti-FLAG antibody, followed by WB with polyclonal anti-YFP antibody. Input is 10% of the amount of cell lysate used for co-IP and is analyzed by WB using polyclonal anti-YFP antibody. IP of FLAG-tagged α-RIMs with monoclonal anti-FLAG antibody is analyzed by WB using polyclonal anti-FLAG antibody. B, effects of α-RIMs on inactivation curves of N-type CaV2.2 and R-type CaV2.3 currents in HEK293 cells expressing β4 and α2δ-1 subunits. See for statistical significance of the differences. *, p < 0.05; **, p < 0.01; statistical significance of differences between RIM1α- and RIM2α-expressing cells. Error bars, S.E.
      We also examined the effect of α-RIMs on the VDI of N-type VDCCs containing the CaV2.2, β4, and α2δ-1 subunits and of R-type VDCCs containing the CaV2.3, β4, and α2δ-1 subunits. In CaV2.2-expressing cells, α-RIMs significantly shifted V0.5 values of the component susceptible to inactivation at high voltages (V0.5high) in inactivation curves of Ba2+ currents toward depolarizing potentials (V0.5high of CaV2.2 co-transfected with vector, RIM1α, and RIM2α were −46.2 ± 3.0, −8.2 ± 14.8, and −2.8 ± 5.7 mV, respectively) (Fig. 10B and Table 6), as reported previously (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ). RIM2α induced significant reduction of low voltage-inactivated phases (the ratios of low voltage-inactivating phases of CaV2.2 co-transfected with vector, RIM1α, and RIM2α were 0.64 ± 0.11, 0.63 ± 0.12, and 0.29 ± 0.05, respectively) (Fig. 10B and Table 6). Suppressive effects of α-RIMs on VDI were also observed for CaV2.3 (the ratios of inactivating components of CaV2.3 with co-transfection of vector, RIM1α, and RIM2α were 0.90 ± 0.03, 0.63 ± 0.09, and 0.67 ± 0.08, respectively). We failed to detect significant differences between the effect of RIM1α and RIM2α on VDI of CaV2.3 channels (Fig. 10B and Table 6). Thus, N-type CaV2.2 channels but not R-type CaV2.3 channels are susceptible to RIM2α-mediated potentiation of VDI suppression.
      Table 6Effect of α-RIMs on inactivation properties of N-type and R-type VDCCs in HEK293 cells expressing CaV2.2 or CaV2.3, α2/δ-1, and β4
      Inactivation parameters
      aV0.5lowKlowbV0.5highKhigh
      mVmVmVmV
      CaV2.2Vector0.64 ± 0.11 (4)−50.2 ± 1.6 (4)−7.4 ± 0.8 (4)0.32 ± 0.11 (4)−46.2 ± 3.0 (4)−3.9 ± 1.7 (4)
      RIM1α0.63 ± 0.12 (4)−52.7 ± 5.7 (4)−8.4 ± 2.1 (4)0.06 ± 0.14 (4)−8.2 ± 14.8 (4)*−10.6 ± 6.3 (4)
      RIM2α0.29 ± 0.05 (5)*, #−46.5 ± 7.6 (5)−11.1 ± 4.4 (5)0.21 ± 0.07 (5)−2.8 ± 5.7 (5)***−2.0 ± 1.6 (5)
      CaV2.3Vector0.90 ± 0.03 (4)−40.5 ± 2.6 (4)−8.5 ± 0.6 (4)
      RIM1α0.63 ± 0.09 (4)*−36.0 ± 2.5 (4)−9.1 ± 1.2 (4)
      RIM2α0.67 ± 0.08 (4)*−37.3 ± 3.8 (4)−9.3 ± 1.7 (4)

      Discussion

      In presynaptic AZs, where SVs dock in close vicinity to VDCCs at the presynaptic membrane, depolarization-induced Ca2+ influx via VDCCs triggers neurotransmitter release (
      • Ackermann F.
      • Waites C.L.
      • Garner C.C.
      Presynaptic active zones in invertebrates and vertebrates.
      ,
      • Jahn R.
      • Fasshauer D.
      Molecular machines governing exocytosis of synaptic vesicles.
      ). Previous proteomic analysis has shown that P/Q-, N-, and R-type VDCCs are embedded into protein networks assembled from a pool of 200 proteins (
      • Müller C.S.
      • Haupt A.
      • Bildl W.
      • Schindler J.
      • Knaus H.G.
      • Meissner M.
      • Rammner B.
      • Striessnig J.
      • Flockerzi V.
      • Fakler B.
      • Schulte U.
      Quantitative proteomics of the Cav2 channel nano-environments in the mammalian brain.
      ). Unveiling the manner of protein-protein interactions in protein networks and their functional consequences is important for understanding the process of synapse formation and the modulation of synaptic transmission (
      • Kim J.H.
      • Huganir R.L.
      Organization and regulation of proteins at synapses.
      ). Previously, we reported that α-RIMs increase neurotransmitter release by sustaining Ca2+ influx through strong inhibition of VDI of VDCCs and by anchoring vesicles in the vicinity of VDCCs via the RIM-β subunit interaction (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ). It has also been reported that α-RIMs interact with the CaV2.1 C-terminal region and modulate VDCCs targeting to presynaptic AZs (
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ). These previous studies have shown that RIM-VDCC interactions are key to the protein assembly responsible for stimulus-secretion coupling in presynaptic AZs.
      Multipoint interaction plays important roles in the regulation of properties such as stabilization of protein complexes. In RIM-VDCC complexes, the significance of multipoint interaction has not yet been resolved. To approach this question, the contributions of each interaction should be quantitatively assessed. Our data strongly indicate that RIM-β subunit interaction is sufficient and necessary for α-RIMs to exert prominent suppressive effects of VDI of VDCCs, because in the CaV2.1 splice variant with the deletion of exons 44 and 47 (CaV2.1 (−44,Δ47)), strong VDI suppression remains intact for RIM1α, but for RIM2α it is attenuated to a level comparable with that of RIM1α. In the presynapse, VDCCs display a weak VDI (
      • Forsythe I.D.
      • Tsujimoto T.
      • Barnes-Davies M.
      • Cuttle M.F.
      • Takahashi T.
      Inactivation of presynaptic calcium current contributes to synaptic depression at a fast central synapse.
      ,
      • Stanley E.F.
      Syntaxin I modulation of presynaptic calcium channel inactivation revealed by botulinum toxin C1.
      ). Moreover, in the calyx of Held nerve terminals of RIM1 and RIM2 conditional double knock-out mice, depolarization pre-pulses induced stronger inactivation of VDCCs compared with wild-type mice (
      • Han Y.
      • Kaeser P.S.
      • Südhof T.C.
      • Schneggenburger R.
      RIM determines Ca2+ channel density and vesicle docking at the presynaptic active zone.
      ).
      CaV2.1 is known to be inactivated through at least two voltage-dependent mechanisms (fast and slow inactivation) (
      • Hering S.
      • Berjukow S.
      • Sokolov S.
      • Marksteiner R.
      • Weiss R.G.
      • Kraus R.
      • Timin E.N.
      Molecular determinants of inactivation in voltage-gated Ca2+ channels.
      ). The mechanism underlying inactivation is not completely understood but may involve “hinged lid” or pore block-type mechanisms (
      • Oz S.
      • Tsemakhovich V.
      • Christel C.J.
      • Lee A.
      • Dascal N.
      CaBP1 regulates voltage-dependent inactivation and activation of CaV1.2 (L-type) calcium channels.
      ,
      • Stotz S.C.
      • Jarvis S.E.
      • Zamponi G.W.
      Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels.
      ,
      • Babich O.
      • Matveev V.
      • Harris A.L.
      • Shirokov R.
      Ca2+-dependent inactivation of CaV1.2 channels prevents Gd3+ block: does Ca2+ block the pore of inactivated channels?.
      ). It has also been reported that fast and slow inactivation represents structurally independent conformational changes (
      • Sokolov S.
      • Weiss R.G.
      • Timin E.N.
      • Hering S.
      Modulation of slow inactivation in class A Ca2+ channels by β-subunits.
      ). Our kinetic analyses of current decay showed that both RIM1α and RIM2α decreased the fast inactivation component and increased τslow regardless of the presence of the exon 44 and 47 regions in CaV2.1 (Fig. 5, C and D), although only RIM2α (but not RIM1α) increased τfast in the presence of the exon 44 and 47 regions in CaV2.1 (Fig. 5C). These findings may suggest that the interaction between CaV2.1 CTD and RIM2α induces conformational changes of the VDCC CaV2.1 α1 subunit in addition to those induced by the interaction between β subunit and α-RIMs.
      By focusing on the splice variants of the CaV2.1 C-terminal region, we have deepened our understanding of the interaction between VDCCs and α-RIMs. α-RIMs interact with the CaV2.1 C-terminal region via the regions encoded by exons 44 and 47, the alternative splicing of which generates major C-terminal variants in the human cerebellum. Our experiments revealed at least four regions in the CaV2.1 CTD involved in interaction with RIM2α: a site encoded by exon 44 and three sites in the region encoded by exon 47. Analysis of relative mRNA levels of these CaV2.1 C-terminal splice variants indicates that 56, 86, or 67% of the total CaV2.1 mRNA carries either both exons 44 and 47, exon 44 alone, or exon 47 alone, respectively (Table 1). These spliced mRNAs are capable of encoding the CaV2.1 variants that interact with α-RIMs. The effect of RIM2α-CaV2.1 C-terminal interaction on VDI can be generated from 67% of the total CaV2.1 mRNA. Interestingly, there is a wide range of fundamental properties for individual synapses, including release probability, unitary response, and effects of previous stimulation on subsequent response (
      • Craig A.M.
      • Boudin H.
      Molecular heterogeneity of central synapses: afferent and target regulation.
      ). This suggests that CaV2.1 C-terminal splice variants may contribute to the heterogeneous molecular composition and function of VDCCs in presynaptic AZs. Importantly, a recent study has suggested that tissue-regulated alternatively spliced exons are significantly enriched in flexible regions of proteins that form conserved interaction surfaces to establish tissue-dependent protein-protein interaction networks (
      • Ellis J.D.
      • Barrios-Rodiles M.
      • Colak R.
      • Irimia M.
      • Kim T.
      • Calarco J.A.
      • Wang X.
      • Pan Q.
      • O'Hanlon D.
      • Kim P.M.
      • Wrana J.L.
      • Blencowe B.J.
      Tissue-specific alternative splicing remodels protein-protein interaction networks.
      ). It is therefore possible that arrangements of presynaptic proteins that include VDCC subunits may be precisely coordinated via protein-protein interactions through the regions encoded by alternatively spliced exons 44 and 47. In terms of the effect of α-RIMs on VDCC channel properties, the interaction between RIM2α and the 17 amino acid residues, 2328–2344, of CaV2.1 (+44,47) in the region encoded by exon 47 of CaV2.1 is responsible for potentiation of VDI suppression (Fig. 7, B and E). In contrast, the interaction between α-RIMs and the region encoded by exon 44 of CaV2.1 failed to exert any significant effect on VDCC channel properties (Fig. 3B). Alternative splicing of exons 44 and 47 may contribute to the fine adjustment of RIM-VDCC complexes that can affect neurotransmitter release from AZs of different types of presynapses.
      The interaction between the CaV2.1 C terminus and RIM-BPs, which bind to α-RIMs, is important for the coupling of SVs, VDCCs, and the SV fusion machinery (
      • Hibino H.
      • Pironkova R.
      • Onwumere O.
      • Vologodskaia M.
      • Hudspeth A.J.
      • Lesage F.
      RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca2+ channels.
      ,
      • Liu K.S.
      • Siebert M.
      • Mertel S.
      • Knoche E.
      • Wegener S.
      • Wichmann C.
      • Matkovic T.
      • Muhammad K.
      • Depner H.
      • Mettke C.
      • Bückers J.
      • Hell S.W.
      • Müller M.
      • Davis G.W.
      • Schmitz D.
      • Sigrist S.J.
      RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release.
      ). The AZ protein Bassoon interacts with both β subunits and RIM-BPs to regulate CaV2.1 targeting to the AZ (
      • Davydova D.
      • Marini C.
      • King C.
      • Klueva J.
      • Bischof F.
      • Romorini S.
      • Montenegro-Venegas C.
      • Heine M.
      • Schneider R.
      • Schröder M.S.
      • Altrock W.D.
      • Henneberger C.
      • Rusakov D.A.
      • Gundelfinger E.D.
      • Fejtova A.
      Bassoon specifically controls presynaptic P/Q-type Ca2+ channels via RIM-binding protein.
      ,
      • Chen J.
      • Billings S.E.
      • Nishimune H.
      Calcium channels link the muscle-derived synapse organizer laminin β2 to Bassoon and CAST/Erc2 to organize presynaptic active zones.
      ,
      • Nishimune H.
      • Numata T.
      • Chen J.
      • Aoki Y.
      • Wang Y.
      • Starr M.P.
      • Mori Y.
      • Stanford J.A.
      Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise.
      ,
      • tom Dieck S.
      • Sanmartí-Vila L.
      • Langnaese K.
      • Richter K.
      • Kindler S.
      • Soyke A.
      • Wex H.
      • Smalla K.H.
      • Kämpf U.
      • Fränzer J.T.
      • Stumm M.
      • Garner C.C.
      • Gundelfinger E.D.
      Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals.
      ). The β subunits are also known to directly interact with the CaV2.1 C terminus (
      • Walker D.
      • Bichet D.
      • Campbell K.P.
      • De Waard M.
      A β4 isoform-specific interaction site in the carboxyl-terminal region of the voltage-dependent Ca2+ channel α1A subunit.
      ). These findings may inform the physiological significance of interactions among the CaV2.1 C-terminal region, β subunits, and α-RIMs. Interestingly, the RIM-BP-binding region in the CaV2.1 C-terminal region is the proline-rich region (PXXP) in amino acid residues 2276–2292 of CaV2.1 (+44,47), which is a RIM2α-binding region in the region encoded by exon 47 (
      • Hibino H.
      • Pironkova R.
      • Onwumere O.
      • Vologodskaia M.
      • Hudspeth A.J.
      • Lesage F.
      RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca2+ channels.
      ). Previous studies have revealed that the amino acid residues 2502–2505 of CaV2.1 (+44,47) bind to α-RIMs (
      • Kaeser P.S.
      • Deng L.
      • Wang Y.
      • Dulubova I.
      • Liu X.
      • Rizo J.
      • Südhof T.C.
      RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
      ) and are essential for interaction with Mint1 (
      • Maximov A.
      • Südhof T.C.
      • Bezprozvanny I.
      Association of neuronal calcium channels with modular adaptor proteins.
      ). These results raise the possibility that the CaV2.1 C-terminal region is the target of AZ scaffolding proteins and that their competitive binding underlies dynamic properties of AZ protein networks in the molecular processes of neurotransmitter release.
      Notably, both RIM1α and RIM2α bind to the C-terminal regions of CaV2.1, CaV2.2, and CaV2.3 (Figs. 2D and 10A), but potentiation of the suppressive effect on VDI was only observed for CaV2.1 or CaV2.2 combined with RIM2α (Figs. 3B and 10B). As already mentioned above, among four regions in the CaV2.1 CTD involved in interaction with RIM2α, the two regions of CaV2.1 CTD (the amino acid residues 2276–2292 and 2502–2505 of CaV2.1 (+44,47)) are highly conserved among the three CaV2 isoforms (supplemental Fig. 1). It has been shown that Bassoon localizes CaV2.1 but not CaV2.2 to AZs via molecular interaction with RIM-BPs (
      • Davydova D.
      • Marini C.
      • King C.
      • Klueva J.
      • Bischof F.
      • Romorini S.
      • Montenegro-Venegas C.
      • Heine M.
      • Schneider R.
      • Schröder M.S.
      • Altrock W.D.
      • Henneberger C.
      • Rusakov D.A.
      • Gundelfinger E.D.
      • Fejtova A.
      Bassoon specifically controls presynaptic P/Q-type Ca2+ channels via RIM-binding protein.
      ), despite the fact that RIM-BPs can also interact with the PXXP motif of CaV2.2 (
      • Hibino H.
      • Pironkova R.
      • Onwumere O.
      • Vologodskaia M.
      • Hudspeth A.J.
      • Lesage F.
      RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca2+ channels.
      ). Furthermore, mutation of the PDZ-binding motif of EGFP-tagged CaV2.1 failed to affect the localization pattern of CaV2.1 in cultured mouse hippocampal neurons (
      • Hu Q.
      • Saegusa H.
      • Hayashi Y.
      • Tanabe T.
      The carboxy-terminal tail region of human Cav2.1 (P/Q-type) channel is not an essential determinant for its subcellular localization in cultured neurones.
      ). These results indicate that the functions of some protein-protein interactions are dependent on other components and that use of appropriate assay systems and neuronal types is necessary to reveal their functionality.
      From a pathological point of view, it is interesting that mutations associated with genetic diseases in the genes encoding RIMs modify their function in regulating VDCC currents (
      • Takada Y.
      • Hirano M.
      • Kiyonaka S.
      • Ueda Y.
      • Yamaguchi K.
      • Nakahara K.
      • Mori M.X.
      • Mori Y.
      Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca2+ channels.
      ,
      • Miki T.
      • Kiyonaka S.
      • Uriu Y.
      • De Waard M.
      • Wakamori M.
      • Beedle A.M.
      • Campbell K.P.
      • Mori Y.
      Mutation associated with an autosomal dominant cone-rod dystrophy CORD7 modifies RIM1-mediated modulation of voltage-dependent Ca2+ channels.
      ). In SCA6 patients, the relative mRNA level of the CaV2.1 splice variant, which possesses exon 47, is increased in cerebellar Purkinje cells but not in granule cells (
      • Tsunemi T.
      • Ishikawa K.
      • Jin H.
      • Mizusawa H.
      Cell-type-specific alternative splicing in spinocerebellar ataxia type 6.
      ). In several episodic ataxia type 2 patients, mutations in CaV2.1 result in the loss of the regions encoded by exons 44 and 47 (
      • Pietrobon D.
      CaV2.1 channelopathies.
      ). These two diseases have similar symptoms, such as ataxic gait and loss of limb coordination. Although our results showed that the polyQ elongation itself does not affect functional regulation of VDCCs by RIM2α, increase in the relative proportion of the CaV2.1 splice variants that possess the region encoded by exon 47 may cause excessive RIM2α functional regulation. Dysregulation of the molecular organization of presynaptic AZs containing VDCC complexes may lead to abnormalities in different functional hierarchies of nervous system.

      Experimental procedures

      cDNA cloning and construction of expression vectors

      Mouse RIM1α, mouse RIM2α, mouse CaV2.2, rabbit α2δ, human CaV2.1 (−44,Δ47), and rat CaV2.3 have been described previously (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ,
      • Takada Y.
      • Hirano M.
      • Kiyonaka S.
      • Ueda Y.
      • Yamaguchi K.
      • Nakahara K.
      • Mori M.X.
      • Mori Y.
      Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca2+ channels.
      ,
      • Sutton K.G.
      • McRory J.E.
      • Guthrie H.
      • Murphy T.H.
      • Snutch T.P.
      P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A.
      ,
      • Soong T.W.
      • Stea A.
      • Hodson C.D.
      • Dubel S.J.
      • Vincent S.R.
      • Snutch T.P.
      Structure and functional expression of a member of the low voltage-activated calcium channel family.
      ). Human VDCC β4 subunit (GenBankTM accession number NM_001005747) was cloned using PCR from human whole-brain Marathon-Ready cDNA (Clontech) and was subcloned into pcDNATM3.1 (−) vector (Thermo Fisher Scientific). For production of YFP fusion proteins for RIM1α, RIM2α, CTD (amino acid residues 1855–2327) of CaV2.2 (GenBankTM accession number NM_001042528), and CTD (amino acid residues 1860–2295) of CaV2.3 (GenBankTM accession number NM_019294), cDNAs for these constructs and the YFP were subcloned together into the pCI-neo vector (Promega). For production of YFP fusion protein for β4, cDNAs for β4 construct and the YFP were subcloned together into the pcDNATM3.1(−) vector. CTD (amino acid residues 1966–2505) of human CaV2.1 (+44,47) (GenBankTM accession number U79666) was cloned using PCR from human whole-brain Marathon-Ready cDNA (Clontech) and was subcloned into the FLAG-tagged vector pCMV-tag2 (Stratagene) and human CaV2.1 (−44,Δ47) clone. To construct CTD of CaV2.1 (+44,47) Gln-40, the ApaI(7152)–MscI(7196) fragment containing 11 CAG repeats was replaced with synthetic oligonucleotides containing 40 CAG repeats. CTD of human CaV2.1 splice variants with different combinations of exons 44 and 47 were constructed by PCR and were subcloned into pCMV-tag2 and human CaV2.1 (+44,47) clone. Mutants or chimeras of CTD of CaV2.1 and α-RIMs were constructed by PCR.

      Cell culture and cDNA expression in HEK293 or HEK293T cells

      HEK293 and HEK293T cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 30 units/ml penicillin, and 30 μg/ml streptomycin at 37 °C under 5% CO2. Transfection of cDNA plasmids was carried out using SuperFect Transfection Reagent (Qiagen). For electrophysiological measurements, recombinant plasmids were co-transfected with pIRES2-EGFP (Clontech), and HEK293 cells with green fluorescence were analyzed. Transfected cells were grown for 36–48 h before electrophysiological measurements and co-IP assay.

      Characterization of splice variation of CaV2.1 C terminus in the human cerebellum by sequence analysis

      We designed PCR oligonucleotide primers as follows: forward in the exon 42, 5′-GCTGGTCACACCTCACAAG-3′, and reverse in the exon 47, 5′-GCTGGGCTTCCACTTACG-3′. Temperature cycles were as follows. Temperature initially 98 °C for 2 min was followed by 25 cycles at 98 °C for 10 s, 64 °C for 30 s, and 68 °C for 1 min. The PCR products from human cerebellum cDNA (Takara, 9523) were electrophoresed on an 1% agarose gel, cut out, purified using the QiaexII gel extraction kit (Qiagen), and ligated into the EcoRV-digested pBluescript II SK(−) (Stratagene). The ligation products were transformed into competent Escherichia coli DH5α cells and screened on Luria-Bertani plates containing ampicillin, X-Gal, and isopropyl β-d-1-thiogalactopyranoside for 15–20 h at 37 °C. White colonies were picked randomly and subjected to sequencing analyses using T7 and M13-reverse universal sequencing primers. The sequencing results were analyzed by using BioEdit software version 7.2.5.

      Yeast two-hybrid screening and β-galactosidase assay

      We subcloned CTD of human CaV2.1 (+44,47) into pGBK-T7 and used it as a bait to screen a human brain pACT2 library (Clontech) in the yeast strain AH109 (Clontech). We plated transformants (2.5 × 106) on synthetic medium lacking adenine, histidine, leucine, and tryptophan and assayed His+ colonies for β-galactosidase activity with a filter assay. Of the transformants, 56 were His+, and 6 of these were also LacZ+. We isolated prey clone encoding amino acid residues 487–1349 of RIM2α (GenBankTM accession number NM_001100117).

      co-IP assay in HEK293T cells

      36–48 h after transfection, HEK293T cells were solubilized in Nonidet P-40 buffer (150 mm NaCl, 50 mm Tris, 1% Nonidet P-40, and 1 mm PMSF) and then centrifuged at 17,400 × g for 20 min. The cell lysate was incubated with anti-FLAG M2 monoclonal antibody (Sigma, F3165), and then the immunocomplexes were incubated with protein A-agarose beads (Santa Cruz Biotechnology, SC-2001), and the beads were washed with Nonidet P-40 buffer. The co-immunoprecipitated and immunoprecipitated proteins were characterized by Western blotting (WB) with anti-YFP antibody (Clontech, 632592) and anti-FLAG antibody (Sigma, F7425), respectively. Input was 10% of the amount of cell lysate used for co-IP and was characterized by WB with anti-YFP antibody. The chemiluminescence intensities of the bands were measured by Multigauge version 3.0 (Fuji film).

      Current recordings

      Whole-cell mode of the patch-clamp technique was carried out at 22–25 °C with an EPC-10 (HEKA Elektronik) patch-clamp amplifier as described previously (
      • Uriu Y.
      • Kiyonaka S.
      • Miki T.
      • Yagi M.
      • Akiyama S.
      • Mori E.
      • Nakao A.
      • Beedle A.M.
      • Campbell K.P.
      • Wakamori M.
      • Mori Y.
      Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
      ,
      • Wakamori M.
      • Yamazaki K.
      • Matsunodaira H.
      • Teramoto T.
      • Tanaka I.
      • Niidome T.
      • Sawada K.
      • Nishizawa Y.
      • Sekiguchi N.
      • Mori E.
      • Mori Y.
      • Imoto K.
      Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel.
      ,
      • Nakao A.
      • Miki T.
      • Shimono K.
      • Oka H.
      • Numata T.
      • Kiyonaka S.
      • Matsushita K.
      • Ogura H.
      • Niidome T.
      • Noebels J.L.
      • Wakamori M.
      • Imoto K.
      • Mori Y.
      Compromised maturation of GABAergic inhibition underlies abnormal network activity in the hippocampus of epileptic Ca2+ channel mutant mice, tottering.
      ). Patch pipettes were made from borosilicate glass capillaries (1.5-mm outer diameter, 0.87-mm inner diameter; Hilgenberg) using a model P-97 Flaming-Brown micropipette puller (Sutter Instrument Co.). The patch electrodes were fire-polished. Pipette resistance ranged from 2 to 4 megohms when filled with the pipette solutions described below. The series resistance was electronically compensated to >60%, and both the leakage and the remaining capacitance were subtracted by the −P/4 method. Currents were sampled at 10 kHz after low-pass filtering at 3.0 kHz (3 db) in the experiments of inactivation kinetics and AP-like trains, otherwise sampled at 20 kHz after low-pass filtering at 3.0 kHz (3 db). Data were collected and analyzed using Patchmaster (HEKA Elektronik) software. An external solution contained 5 mm BaCl2, 148 mm tetraethylammonium chloride, 10 mm HEPES, and 10 mm glucose (pH 7.4-adjusted with tetraethylammonium-OH). The pipette solution contained 95 mm CsOH, 95 mm aspartate, 40 mm CsCl, 4 mm MgCl2, 5 mm EGTA, 2 mm disodium ATP, 5 mm HEPES, and 8 mm creatine phosphate (pH 7.2-adjusted with CsOH).

      Voltage dependence of inactivation

      To determine the inactivation curve of VDCCs, Ba2+ currents were evoked by 40-ms test pulse to 10 mV after the 10-ms repolarization to −90 mV following 1-s (300 ms for CaV2.3) prepulse voltage (Vpre) displacement (conditioning pulse) from −80 to 20 mV with 10-mV increments. Amplitudes of currents elicited by the test pulses were normalized to those elicited by the test pulse after a 1-s Vpre displacement to −80 mV. The mean values were plotted against potentials of the 1-s Vpre displacement. When the inactivation curve was monophasic, the mean values were fitted to the single Boltzmann equation, h(Vpre) = (1 − a) + a/(1 + exp((V0.5Vpre)/k)), where a is the rate of inactivating component; V0.5 is the potential to give a half-value of inactivation; and k is the slope factor. Otherwise, the mean values were fitted to the sum of two Boltzmann equations: h(Vpre) = (1 − ab) + a/(1 + exp((V0.5lowVpre)/klow)) +b/(1 + exp((V0.5highVpre)/khigh)), where a, b, and (1 − ab) are the ratios of a low voltage-induced phase, a high voltage-induced phase, and a non-inactivating phase; V0.5low and V0.5high are the potentials that give a half-value of components susceptible to inactivation at low voltages in inactivation curves and at high voltages; and klow and khigh are the slope factors. The decay phase of Ba2+ currents evoked by 1-s test pulses was fitted by two (fast and slow) exponential functions with a non-inactivating component: −I(t) = a + bexp(−λ1t) + cexp(−λ2t), where I(t) is the inactivating current as a function of time; a is the current amplitude at t = ∞; b and c are the amplitudes of the time-dependent components; and λ1 and λ2 are the reciprocals of the fast (τfast) and the slow (τslow) time constants of inactivation, respectively (
      • Wakamori M.
      • Yamazaki K.
      • Matsunodaira H.
      • Teramoto T.
      • Tanaka I.
      • Niidome T.
      • Sawada K.
      • Nishizawa Y.
      • Sekiguchi N.
      • Mori E.
      • Mori Y.
      • Imoto K.
      Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel.
      ,
      • Gutnick M.J.
      • Lux H.D.
      • Swandulla D.
      • Zucker H.
      Voltage-dependent and calcium-dependent inactivation of calcium channel current in identified snail neurones.
      ). APs began at −80 mV and peaked at 33 mV. Rising and falling slopes were 283 and −103 V/s, respectively (
      • Kiyonaka S.
      • Wakamori M.
      • Miki T.
      • Uriu Y.
      • Nonaka M.
      • Bito H.
      • Beedle A.M.
      • Mori E.
      • Hara Y.
      • De Waard M.
      • Kanagawa M.
      • Itakura M.
      • Takahashi M.
      • Campbell K.P.
      • Mori Y.
      RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
      ,
      • Patil P.G.
      • Brody D.L.
      • Yue D.T.
      Preferential closed-state inactivation of neuronal calcium channels.
      ). Leaks and capacitive transients were subtracted by a −P/4 protocol.

      I-V relationships

      The individual activation data were fitted to standard Boltzmann equation in the form IBa = Gmax(VmVrev)/(1 + exp(−(VmV0.5)/k)), where Gmax is the maximal conductance; Vm is the membrane voltage; Vrev is the IBa reversal potential; V0.5 is the half-activation potential, and k is the slope factor.

      Statistical analysis

      All data are expressed as the means ± S.E. The statistical analyses were performed using Student's t test. A value of p < 0.05 was considered significant.

      Author contributions

      M. H. designed the study, conducted most of the experiments, analyzed the results, and wrote most of the paper. Y. T. collected preliminary data and constructed vectors. K. Y. and C. F. W. conducted and analyzed the experiments on co-IP. H. K. conducted the sequence analysis of PCR products from the human cerebellum cDNA library. T. K., M. X. M., T. P. S., M. R., M. D. W., and Y. M. directed the research, wrote the manuscript, analyzed, interpreted the data, and critically reviewed the manuscript.

      Acknowledgments

      We thank S. Kiyonaka, T. Numata, R. Sakaguchi, and N. Ogawa for experimental advice and helpful discussions.

      Supplementary Material

      References

        • Ackermann F.
        • Waites C.L.
        • Garner C.C.
        Presynaptic active zones in invertebrates and vertebrates.
        EMBO Rep. 2015; 16: 923-938
        • Jahn R.
        • Fasshauer D.
        Molecular machines governing exocytosis of synaptic vesicles.
        Nature. 2012; 490: 201-207
        • Tsien R.W.
        • Lipscombe D.
        • Madison D.V.
        • Bley K.R.
        • Fox A.P.
        Multiple types of neuronal calcium channels and their selective modulation.
        Trends Neurosci. 1988; 11: 431-438
        • Birnbaumer L.
        • Campbell K.P.
        • Catterall W.A.
        • Harpold M.M.
        • Hofmann F.
        • Horne W.A.
        • Mori Y.
        • Schwartz A.
        • Snutch T.P.
        • Tanabe T.
        The naming of voltage-gated calcium channels.
        Neuron. 1994; 13: 505-506
        • Takahashi T.
        • Momiyama A.
        Different types of calcium channels mediate central synaptic transmission.
        Nature. 1993; 366: 156-158
        • Wu L.G.
        • Borst J.G.
        • Sakmann B.
        R-type Ca2+ currents evoke transmitter release at a rat central synapse.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 4720-4725
        • Fox A.P.
        • Nowycky M.C.
        • Tsien R.W.
        Single-channel recordings of three types of calcium channels in chick sensory neurones.
        J. Physiol. 1987; 394: 173-200
        • Weber A.M.
        • Wong F.K.
        • Tufford A.R.
        • Schlichter L.C.
        • Matveev V.
        • Stanley E.F.
        N-type Ca2+ channels carry the largest current: implications for nanodomains and transmitter release.
        Nat. Neurosci. 2010; 13: 1348-1350
        • Davydova D.
        • Marini C.
        • King C.
        • Klueva J.
        • Bischof F.
        • Romorini S.
        • Montenegro-Venegas C.
        • Heine M.
        • Schneider R.
        • Schröder M.S.
        • Altrock W.D.
        • Henneberger C.
        • Rusakov D.A.
        • Gundelfinger E.D.
        • Fejtova A.
        Bassoon specifically controls presynaptic P/Q-type Ca2+ channels via RIM-binding protein.
        Neuron. 2014; 82: 181-194
        • Parajuli L.K.
        • Nakajima C.
        • Kulik A.
        • Matsui K.
        • Schneider T.
        • Shigemoto R.
        • Fukazawa Y.
        Quantitative regional and ultrastructural localization of the CaV2.3 subunit of R-type calcium channel in mouse brain.
        J. Neurosci. 2012; 32: 13555-13567
        • Holderith N.
        • Lorincz A.
        • Katona G.
        • Rózsa B.
        • Kulik A.
        • Watanabe M.
        • Nusser Z.
        Release probability of hippocampal glutamatergic terminals scales with the size of the active zone.
        Nat. Neurosci. 2012; 15: 988-997
        • Kulik A.
        • Nakadate K.
        • Hagiwara A.
        • Fukazawa Y.
        • Luján R.
        • Saito H.
        • Suzuki N.
        • Futatsugi A.
        • Mikoshiba K.
        • Frotscher M.
        • Shigemoto R.
        Immunocytochemical localization of the a1A subunit of the P/Q-type calcium channel in the rat cerebellum.
        Eur. J. Neurosci. 2004; 19: 2169-2178
        • Forsythe I.D.
        • Tsujimoto T.
        • Barnes-Davies M.
        • Cuttle M.F.
        • Takahashi T.
        Inactivation of presynaptic calcium current contributes to synaptic depression at a fast central synapse.
        Neuron. 1998; 20: 797-807
        • Inchauspe C.G.
        • Martini F.J.
        • Forsythe I.D.
        • Uchitel O.D.
        Functional compensation of P/Q by N-type channels blocks short-term plasticity at the calyx of Held presynaptic terminal.
        J. Neurosci. 2004; 24: 10379-10383
        • Ishikawa T.
        • Kaneko M.
        • Shin H.S.
        • Takahashi T.
        Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice.
        J. Physiol. 2005; 568: 199-209
        • Miki T.
        • Hirai H.
        • Takahashi T.
        Activity-dependent neurotrophin signaling underlies developmental switch of Ca2+ channel subtypes mediating neurotransmitter release.
        J. Neurosci. 2013; 33: 18755-18763
        • Ertel E.A.
        • Campbell K.P.
        • Harpold M.M.
        • Hofmann F.
        • Mori Y.
        • Perez-Reyes E.
        • Schwartz A.
        • Snutch T.P.
        • Tanabe T.
        • Birnbaumer L.
        • Tsien R.W.
        • Catterall W.A.
        Nomenclature of voltage-gated calcium channels.
        Neuron. 2000; 25: 533-535
        • Catterall W.A.
        • Few A.P.
        Calcium channel regulation and presynaptic plasticity.
        Neuron. 2008; 59: 882-901
        • Hibino H.
        • Pironkova R.
        • Onwumere O.
        • Vologodskaia M.
        • Hudspeth A.J.
        • Lesage F.
        RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca2+ channels.
        Neuron. 2002; 34: 411-423
        • Kiyonaka S.
        • Wakamori M.
        • Miki T.
        • Uriu Y.
        • Nonaka M.
        • Bito H.
        • Beedle A.M.
        • Mori E.
        • Hara Y.
        • De Waard M.
        • Kanagawa M.
        • Itakura M.
        • Takahashi M.
        • Campbell K.P.
        • Mori Y.
        RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.
        Nat. Neurosci. 2007; 10: 691-701
        • Geppert M.
        • Goda Y.
        • Hammer R.E.
        • Li C.
        • Rosahl T.W.
        • Stevens C.F.
        • Südhof T.C.
        Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse.
        Cell. 1994; 79: 717-727
        • Kaeser P.S.
        • Deng L.
        • Wang Y.
        • Dulubova I.
        • Liu X.
        • Rizo J.
        • Südhof T.C.
        RIM proteins tether Ca2+ channels to presynaptic active zones via a direct PDZ-domain interaction.
        Cell. 2011; 144: 282-295
        • Sheng Z.H.
        • Rettig J.
        • Takahashi M.
        • Catterall W.A.
        Identification of a syntaxin-binding site on N-type calcium channels.
        Neuron. 1994; 13: 1303-1313
        • Bezprozvanny I.
        • Scheller R.H.
        • Tsien R.W.
        Functional impact of syntaxin on gating of N-type and Q-type calcium channels.
        Nature. 1995; 378: 623-626
        • Bachnoff N.
        • Cohen-Kutner M.
        • Trus M.
        • Atlas D.
        Intra-membrane signaling between the voltage-gated Ca2+-channel and cysteine residues of syntaxin 1A coordinates synchronous release.
        Sci. Rep. 2013; 31620
        • Kaeser P.S.
        • Regehr W.G.
        Molecular mechanisms for synchronous, asynchronous, and spontaneous neurotransmitter release.
        Annu. Rev. Physiol. 2014; 76: 333-363
        • Wang L.Y.
        • Augustine G.J.
        Presynaptic nanodomains: a tale of two synapses.
        Front. Cell. Neurosci. 2014; 8: 455
        • Sheng J.
        • He L.
        • Zheng H.
        • Xue L.
        • Luo F.
        • Shin W.
        • Sun T.
        • Kuner T.
        • Yue D.T.
        • Wu L.G.
        Calcium-channel number critically influences synaptic strength and plasticity at the active zone.
        Nat. Neurosci. 2012; 15: 998-1006
        • Simms B.A.
        • Zamponi G.W.
        Neuronal voltage-gated calcium channels: structure, function, and dysfunction.
        Neuron. 2014; 82: 24-45
        • Stanley E.F.
        Syntaxin I modulation of presynaptic calcium channel inactivation revealed by botulinum toxin C1.
        Eur. J. Neurosci. 2003; 17: 1303-1305
        • Mori Y.
        • Friedrich T.
        • Kim M.S.
        • Mikami A.
        • Nakai J.
        • Ruth P.
        • Bosse E.
        • Hofmann F.
        • Flockerzi V.
        • Furuichi T.
        • Mikoshiba K.
        • Imoto K.
        • Tanabe T.
        • Numa S.
        Primary structure and functional expression from complementary DNA of a brain calcium channel.
        Nature. 1991; 350: 398-402
        • Pietrobon D.
        CaV2.1 channelopathies.
        Pflugers Arch. 2010; 460: 375-393
        • Ophoff R.A.
        • Terwindt G.M.
        • Vergouwe M.N.
        • van Eijk R.
        • Oefner P.J.
        • Hoffman S.M.
        • Lamerdin J.E.
        • Mohrenweiser H.W.
        • Bulman D.E.
        • Ferrari M.
        • Haan J.
        • Lindhout D.
        • van Ommen G.J.
        • Hofker M.H.
        • Ferrari M.D.
        • Frants R.R.
        Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4.
        Cell. 1996; 87: 543-552
        • Zhuchenko O.
        • Bailey J.
        • Bonnen P.
        • Ashizawa T.
        • Stockton D.W.
        • Amos C.
        • Dobyns W.B.
        • Subramony S.H.
        • Zoghbi H.Y.
        • Lee C.C.
        Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel.
        Nat. Genet. 1997; 15: 62-69
        • Bourinet E.
        • Soong T.W.
        • Sutton K.
        • Slaymaker S.
        • Mathews E.
        • Monteil A.
        • Zamponi G.W.
        • Nargeot J.
        • Snutch T.P.
        Splicing of α1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.
        Nat. Neurosci. 1999; 2: 407-415
        • Stea A.
        • Tomlinson W.J.
        • Soong T.W.
        • Bourinet E.
        • Dubel S.J.
        • Vincent S.R.
        • Snutch T.P.
        Localization and functional properties of a rat brain α1A calcium channel reflect similarities to neuronal Q- and P-type channels.
        Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 10576-10580
        • Yokoyama C.T.
        • Myers S.J.
        • Fu J.
        • Mockus S.M.
        • Scheuer T.
        • Catterall W.A.
        Mechanism of SNARE protein binding and regulation of Cav2 channels by phosphorylation of the synaptic protein interaction site.
        Mol. Cell. Neurosci. 2005; 28: 1-17
        • Tsunemi T.
        • Saegusa H.
        • Ishikawa K.
        • Nagayama S.
        • Murakoshi T.
        • Mizusawa H.
        • Tanabe T.
        Novel CaV2.1 splice variants isolated from Purkinje cells do not generate P-type Ca2+ current.
        J. Biol. Chem. 2002; 277: 7214-7221
        • Uriu Y.
        • Kiyonaka S.
        • Miki T.
        • Yagi M.
        • Akiyama S.
        • Mori E.
        • Nakao A.
        • Beedle A.M.
        • Campbell K.P.
        • Wakamori M.
        • Mori Y.
        Rab3-interacting molecule γ isoforms lacking the Rab3-binding domain induce long lasting currents but block neurotransmitter vesicle anchoring in voltage-dependent P/Q-type Ca2+ channels.
        J. Biol. Chem. 2010; 285: 21750-21767
        • Randall A.
        • Tsien R.W.
        Pharmacological dissection of multiple types of Ca2+ channel currents in rat cerebellar granule neurons.
        J. Neurosci. 1995; 15: 2995-3012
        • Wakamori M.
        • Yamazaki K.
        • Matsunodaira H.
        • Teramoto T.
        • Tanaka I.
        • Niidome T.
        • Sawada K.
        • Nishizawa Y.
        • Sekiguchi N.
        • Mori E.
        • Mori Y.
        • Imoto K.
        Single tottering mutations responsible for the neuropathic phenotype of the P-type calcium channel.
        J. Biol. Chem. 1998; 273: 34857-34867
        • Kanumilli S.
        • Tringham E.W.
        • Payne C.E.
        • Dupere J.R.
        • Venkateswarlu K.
        • Usowicz M.M.
        Alternative splicing generates a smaller assortment of CaV2.1 transcripts in cerebellar Purkinje cells than in the cerebellum.
        Physiol. Genomics. 2006; 24: 86-96
        • Südhof T.C.
        The presynaptic active zone.
        Neuron. 2012; 75: 11-25
        • Schoch S.
        • Castillo P.E.
        • Jo T.
        • Mukherjee K.
        • Geppert M.
        • Wang Y.
        • Schmitz F.
        • Malenka R.C.
        • Südhof T.C.
        RIM1α forms a protein scaffold for regulating neurotransmitter release at the active zone.
        Nature. 2002; 415: 321-326
        • Gracheva E.O.
        • Hadwiger G.
        • Nonet M.L.
        • Richmond J.E.
        Direct interactions between C. elegans RAB-3 and Rim provide a mechanism to target vesicles to the presynaptic density.
        Neurosci. Lett. 2008; 444: 137-142
        • Koushika S.P.
        • Richmond J.E.
        • Hadwiger G.
        • Weimer R.M.
        • Jorgensen E.M.
        • Nonet M.L.
        A post-docking role for active zone protein Rim.
        Nat. Neurosci. 2001; 4: 997-1005
        • Deng L.
        • Kaeser P.S.
        • Xu W.
        • Südhof T.C.
        RIM proteins activate vesicle priming by reversing autoinhibitory homodimerization of Munc13.
        Neuron. 2011; 69: 317-331
        • Han Y.
        • Kaeser P.S.
        • Südhof T.C.
        • Schneggenburger R.
        RIM determines Ca2+ channel density and vesicle docking at the presynaptic active zone.
        Neuron. 2011; 69: 304-316
        • Castillo P.E.
        • Schoch S.
        • Schmitz F.
        • Südhof T.C.
        • Malenka R.C.
        RIM1α is required for presynaptic long-term potentiation.
        Nature. 2002; 415: 327-330
        • Takada Y.
        • Hirano M.
        • Kiyonaka S.
        • Ueda Y.
        • Yamaguchi K.
        • Nakahara K.
        • Mori M.X.
        • Mori Y.
        Rab3 interacting molecule 3 mutations associated with autism alter regulation of voltage-dependent Ca2+ channels.
        Cell Calcium. 2015; 58: 296-306
        • Miki T.
        • Kiyonaka S.
        • Uriu Y.
        • De Waard M.
        • Wakamori M.
        • Beedle A.M.
        • Campbell K.P.
        • Mori Y.
        Mutation associated with an autosomal dominant cone-rod dystrophy CORD7 modifies RIM1-mediated modulation of voltage-dependent Ca2+ channels.
        Channels. 2007; 1: 144-147
        • Gandini M.A.
        • Sandoval A.
        • González-Ramírez R.
        • Mori Y.
        • de Waard M.
        • Felix R.
        Functional coupling of Rab3-interacting molecule 1 (RIM1) and L-type Ca2+ channels in insulin release.
        J. Biol. Chem. 2011; 286: 15757-15765
        • Soong T.W.
        • DeMaria C.D.
        • Alvania R.S.
        • Zweifel L.S.
        • Liang M.C.
        • Mittman S.
        • Agnew W.S.
        • Yue D.T.
        Systematic identification of splice variants in human P/Q-type channel α12.1 subunits: implications for current density and Ca2+-dependent inactivation.
        J. Neurosci. 2002; 22: 10142-10152
        • Giunti P.
        • Mantuano E.
        • Frontali M.
        • Veneziano L.
        Molecular mechanism of Spinocerebellar ataxia type 6: glutamine repeat disorder, channelopathy and transcriptional dysregulation. The multifaceted aspects of a single mutation.
        Front. Cell. Neurosci. 2015; 9: 36
        • Aravind L.
        • Landsman D.
        AT-hook motifs identified in a wide variety of DNA-binding proteins.
        Nucleic Acids Res. 1998; 26: 4413-4421
        • Maximov A.
        • Südhof T.C.
        • Bezprozvanny I.
        Association of neuronal calcium channels with modular adaptor proteins.
        J. Biol. Chem. 1999; 274: 24453-24456
        • Wong F.K.
        • Nath A.R.
        • Chen R.H.
        • Gardezi S.R.
        • Li Q.
        • Stanley E.F.
        Synaptic vesicle tethering and the CaV2.2 distal C-terminal.
        Front. Cell. Neurosci. 2014; 8: 71
        • Lipscombe D.
        • Pan J.Q.
        • Gray A.C.
        Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of CaVa1.
        Mol. Neurobiol. 2002; 26: 21-44
        • Schoch S.
        • Mittelstaedt T.
        • Kaeser P.S.
        • Padgett D.
        • Feldmann N.
        • Chevaleyre V.
        • Castillo P.E.
        • Hammer R.E.
        • Han W.
        • Schmitz F.
        • Lin W.
        • Südhof T.C.
        Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release.
        EMBO J. 2006; 25: 5852-5863
        • Ludwig A.
        • Flockerzi V.
        • Hofmann F.
        Regional expression and cellular localization of the α1 and β subunit of high voltage-activated calcium channels in rat brain.
        J. Neurosci. 1997; 17: 1339-1349
        • Burgess D.L.
        • Biddlecome G.H.
        • McDonough S.I.
        • Diaz M.E.
        • Zilinski C.A.
        • Bean B.P.
        • Campbell K.P.
        • Noebels J.L.
        β subunit reshuffling modifies N- and P/Q-type Ca2+ channel subunit compositions in lethargic mouse brain.
        Mol. Cell. Neurosci. 1999; 13: 293-311
        • Burgess D.L.
        • Jones J.M.
        • Meisler M.H.
        • Noebels J.L.
        Mutation of the Ca2+ channel β subunit gene Cchb4 is associated with ataxia and seizures in the lethargic (lh) mouse.
        Cell. 1997; 88: 385-392
        • Chaudhuri D.
        • Chang S.Y.
        • DeMaria C.D.
        • Alvania R.S.
        • Soong T.W.
        • Yue D.T.
        Alternative splicing as a molecular switch for Ca2+/calmodulin-dependent facilitation of P/Q-type Ca2+ channels.
        J. Neurosci. 2004; 24: 6334-6342
        • Adams P.J.
        • Garcia E.
        • David L.S.
        • Mulatz K.J.
        • Spacey S.D.
        • Snutch T.P.
        CaV2.1 P/Q-type calcium channel alternative splicing affects the functional impact of familial hemiplegic migraine mutations: implications for calcium channelopathies.
        Channels. 2009; 3: 110-121
        • Patil P.G.
        • Brody D.L.
        • Yue D.T.
        Preferential closed-state inactivation of neuronal calcium channels.
        Neuron. 1998; 20: 1027-1038
        • Matsuyama Z.
        • Kawakami H.
        • Maruyama H.
        • Izumi Y.
        • Komure O.
        • Udaka F.
        • Kameyama M.
        • Nishio T.
        • Kuroda Y.
        • Nishimura M.
        • Nakamura S.
        Molecular features of the CAG repeats of spinocerebellar ataxia 6 (SCA6).
        Hum. Mol. Genet. 1997; 6: 1283-1287
        • Matsuyama Z.
        • Wakamori M.
        • Mori Y.
        • Kawakami H.
        • Nakamura S.
        • Imoto K.
        Direct alteration of the P/Q-type Ca2+ channel property by polyglutamine expansion in spinocerebellar ataxia 6.
        J. Neurosci. 1999; 19: RC14
        • Chen H.
        • Piedras-Rentería E.S.
        Altered frequency-dependent inactivation and steady-state inactivation of polyglutamine-expanded α1A in SCA6.
        Am. J. Physiol. Cell Physiol. 2007; 292: C1078-C1086
        • Catterall W.A.
        Voltage-gated calcium channels.
        Cold Spring Harb. Perspect. Biol. 2011; 3a003947
        • Wu L.G.
        • Saggau P.
        Presynaptic inhibition of elicited neurotransmitter release.
        Trends Neurosci. 1997; 20: 204-212
        • Ricoy U.M.
        • Frerking M.E.
        Distinct roles for CaV2.1–2.3 in activity-dependent synaptic dynamics.
        J. Neurophysiol. 2014; 111: 2404-2413
        • Müller C.S.
        • Haupt A.
        • Bildl W.
        • Schindler J.
        • Knaus H.G.
        • Meissner M.
        • Rammner B.
        • Striessnig J.
        • Flockerzi V.
        • Fakler B.
        • Schulte U.
        Quantitative proteomics of the Cav2 channel nano-environments in the mammalian brain.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 14950-14957
        • Kim J.H.
        • Huganir R.L.
        Organization and regulation of proteins at synapses.
        Curr. Opin. Cell Biol. 1999; 11: 248-254
        • Hering S.
        • Berjukow S.
        • Sokolov S.
        • Marksteiner R.
        • Weiss R.G.
        • Kraus R.
        • Timin E.N.
        Molecular determinants of inactivation in voltage-gated Ca2+ channels.
        J. Physiol. 2000; 528: 237-249
        • Oz S.
        • Tsemakhovich V.
        • Christel C.J.
        • Lee A.
        • Dascal N.
        CaBP1 regulates voltage-dependent inactivation and activation of CaV1.2 (L-type) calcium channels.
        J. Biol. Chem. 2011; 286: 13945-13953
        • Stotz S.C.
        • Jarvis S.E.
        • Zamponi G.W.
        Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels.
        J. Physiol. 2004; 554: 263-273
        • Babich O.
        • Matveev V.
        • Harris A.L.
        • Shirokov R.
        Ca2+-dependent inactivation of CaV1.2 channels prevents Gd3+ block: does Ca2+ block the pore of inactivated channels?.
        J. Gen. Physiol. 2007; 129: 477-483
        • Sokolov S.
        • Weiss R.G.
        • Timin E.N.
        • Hering S.
        Modulation of slow inactivation in class A Ca2+ channels by β-subunits.
        J. Physiol. 2000; 527: 445-454
        • Craig A.M.
        • Boudin H.
        Molecular heterogeneity of central synapses: afferent and target regulation.
        Nat. Neurosci. 2001; 4: 569-578
        • Ellis J.D.
        • Barrios-Rodiles M.
        • Colak R.
        • Irimia M.
        • Kim T.
        • Calarco J.A.
        • Wang X.
        • Pan Q.
        • O'Hanlon D.
        • Kim P.M.
        • Wrana J.L.
        • Blencowe B.J.
        Tissue-specific alternative splicing remodels protein-protein interaction networks.
        Mol. Cell. 2012; 46: 884-892
        • Liu K.S.
        • Siebert M.
        • Mertel S.
        • Knoche E.
        • Wegener S.
        • Wichmann C.
        • Matkovic T.
        • Muhammad K.
        • Depner H.
        • Mettke C.
        • Bückers J.
        • Hell S.W.
        • Müller M.
        • Davis G.W.
        • Schmitz D.
        • Sigrist S.J.
        RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release.
        Science. 2011; 334: 1565-1569
        • Chen J.
        • Billings S.E.
        • Nishimune H.
        Calcium channels link the muscle-derived synapse organizer laminin β2 to Bassoon and CAST/Erc2 to organize presynaptic active zones.
        J. Neurosci. 2011; 31: 512-525
        • Nishimune H.
        • Numata T.
        • Chen J.
        • Aoki Y.
        • Wang Y.
        • Starr M.P.
        • Mori Y.
        • Stanford J.A.
        Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise.
        PLoS One. 2012; 7e38029
        • tom Dieck S.
        • Sanmartí-Vila L.
        • Langnaese K.
        • Richter K.
        • Kindler S.
        • Soyke A.
        • Wex H.
        • Smalla K.H.
        • Kämpf U.
        • Fränzer J.T.
        • Stumm M.
        • Garner C.C.
        • Gundelfinger E.D.
        Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals.
        J. Cell Biol. 1998; 142: 499-509
        • Walker D.
        • Bichet D.
        • Campbell K.P.
        • De Waard M.
        A β4 isoform-specific interaction site in the carboxyl-terminal region of the voltage-dependent Ca2+ channel α1A subunit.
        J. Biol. Chem. 1998; 273: 2361-2367
        • Hu Q.
        • Saegusa H.
        • Hayashi Y.
        • Tanabe T.
        The carboxy-terminal tail region of human Cav2.1 (P/Q-type) channel is not an essential determinant for its subcellular localization in cultured neurones.
        Genes Cells. 2005; 10: 87-96
        • Tsunemi T.
        • Ishikawa K.
        • Jin H.
        • Mizusawa H.
        Cell-type-specific alternative splicing in spinocerebellar ataxia type 6.
        Neurosci. Lett. 2008; 447: 78-81
        • Sutton K.G.
        • McRory J.E.
        • Guthrie H.
        • Murphy T.H.
        • Snutch T.P.
        P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A.
        Nature. 1999; 401: 800-804
        • Soong T.W.
        • Stea A.
        • Hodson C.D.
        • Dubel S.J.
        • Vincent S.R.
        • Snutch T.P.
        Structure and functional expression of a member of the low voltage-activated calcium channel family.
        Science. 1993; 260: 1133-1136
        • Nakao A.
        • Miki T.
        • Shimono K.
        • Oka H.
        • Numata T.
        • Kiyonaka S.
        • Matsushita K.
        • Ogura H.
        • Niidome T.
        • Noebels J.L.
        • Wakamori M.
        • Imoto K.
        • Mori Y.
        Compromised maturation of GABAergic inhibition underlies abnormal network activity in the hippocampus of epileptic Ca2+ channel mutant mice, tottering.
        Pflugers Arch. 2015; 467: 737-752
        • Gutnick M.J.
        • Lux H.D.
        • Swandulla D.
        • Zucker H.
        Voltage-dependent and calcium-dependent inactivation of calcium channel current in identified snail neurones.
        J. Physiol. 1989; 412: 197-220
        • Lin D.T.
        • Makino Y.
        • Sharma K.
        • Hayashi T.
        • Neve R.
        • Takamiya K.
        • Huganir R.L.
        Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation.
        Nat. Neurosci. 2009; 12: 879-887