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Familial Hemiplegic Migraine Type 1 Mutations K1336E, W1684R, and V1696I Alter Cav2.1 Ca2+ Channel Gating

EVIDENCE FOR β-SUBUNIT ISOFORM-SPECIFIC EFFECTS*
  • Carmen Müllner
    Affiliations
    Abteilung Pharmakologie und Toxikologie, Institut für Pharmazie, Universität Innsbruck, Peter-Mayrstrasse 1/I, A-6020 Innsbruck, Austria and the
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  • Ludo A.M. Broos
    Affiliations
    Leiden University Medical Center, Departments of
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  • Arn M.J.M. van den Maagdenberg
    Affiliations
    Departments of Human Genetics and

    Departments of Neurology, Leiden 2300, The Netherlands
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  • Jörg Striessnig
    Correspondence
    To whom correspondence should be addressed. Tel.: 43-512-507-5600; Fax: 43-512-507-2931;
    Affiliations
    Abteilung Pharmakologie und Toxikologie, Institut für Pharmazie, Universität Innsbruck, Peter-Mayrstrasse 1/I, A-6020 Innsbruck, Austria and the
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  • Author Footnotes
    * This work was supported by the FWF (P-17109 to J. S.), the Österreichische Nationalbank and the European Community (HPRN-CT-2000-00082). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:September 23, 2004DOI:https://doi.org/10.1074/jbc.M408756200
      Mutations in the Cav2.1 α1-subunit of P/Q-type Ca2+channels cause human diseases, including familial hemiplegic migraine type-1 (FHM1). FHM1 mutations alter channel gating and enhanced channel activity at negative potentials appears to be a common pathogenetic mechanism. Different β-subunit isoforms (primarily β4 and β3) participate in the formation of Cav2.1 channel complexes in mammalian brain. Here we investigated not only whether FHM1 mutations K1336E (KE), W1684R (WR), and V1696I (VI) can affect Cav2.1 channel function but focused on the important question whether mutation-induced changes on channel gating depend on the β-subunit isoform. Mutants were co-expressed in Xenopus oocytes together with β1, β3, or β4 and α2δ1 subunits, and channel function was analyzed using the two-electrode voltage-clamp technique. WR shifted the voltage dependence for steady-state inactivation of Ba2+ inward currents (IBa) to more negative voltages with all β-subunits tested. In contrast, a similar shift was observed for KE only when expressed with β3. All mutations promoted IBa decay during pulse trains only when expressed with β1 or β3 but not with β4. Enhanced decay could be explained by delayed recovery from inactivation. KE accelerated IBa inactivation only when co-expressed with β3, and VI slowed inactivation only with β1 or β3. KE and WR shifted channel activation of IBa to more negative voltages. As the β-subunit composition of Cav2.1 channels varies in different brain regions, our data predict that the functional FHM1 phenotype also varies between different neurons or even within different neuronal compartments.
      Voltage-gated Cav2.1 (P/Q-type) channels are the most abundant isoforms in mammalian brain (
      • Jun K.
      • Piedras-Renteria E.S.
      • Smith S.M.
      • Wheeler D.B.
      • Lee S.B.
      • Lee T.G.
      • Chin H.
      • Adams M.E.
      • Scheller R.H.
      • Tsien R.W.
      • Shin H.S.
      ). They cluster in nerve terminals where they control fast neurotransmitter release and physically interact with SNARE
      The abbreviations used are: SNARE, soluble NSF attachment protein receptor; EA2, episodic ataxia type 2; FHM1, familial hemiplegic migraine type 1; IBa, inward Ba2+ current; I-V, current-voltage; V0.5,act, V0.5,inact, half-maximal voltage for activation and inactivation, respectively; kact, kinact, steepness of the curve at V0.5,act, V0.5,inact; PCS, permanent cerebellar signs; WT, wild-type; nt, nucleotide(s); ANOVA, analysis of variance; KE, K1336E; WR, W1684R; VI, V1696I.
      1The abbreviations used are: SNARE, soluble NSF attachment protein receptor; EA2, episodic ataxia type 2; FHM1, familial hemiplegic migraine type 1; IBa, inward Ba2+ current; I-V, current-voltage; V0.5,act, V0.5,inact, half-maximal voltage for activation and inactivation, respectively; kact, kinact, steepness of the curve at V0.5,act, V0.5,inact; PCS, permanent cerebellar signs; WT, wild-type; nt, nucleotide(s); ANOVA, analysis of variance; KE, K1336E; WR, W1684R; VI, V1696I.
      proteins (
      • Spafford J.D.
      • Zamponi G.W.
      ,
      • Mochida S.
      • Westenbroek R.E.
      • Yokoyama C.T.
      • Zhong H.
      • Myers S.J.
      • Scheuer T.
      • Itoh K.
      • Catterall W.A.
      ). They are also found at somatodendritic locations allowing them to modulate other neuronal Ca2+-dependent processes (
      • Westenbroek R.E.
      • Sakurai T.
      • Elliott E.M.
      • Hell J.W.
      • Starr T.V.
      • Snutch T.P.
      • Catterall W.A.
      ), including neuronal firing (
      • Bayliss D.A.
      • Li Y.-W.
      • Talley E.M.
      ).
      Disease-relevant structural defects in the gene encoding Cav2.1 α1-subunits have been described in mice and humans (
      • Pietrobon D.
      ). In humans Cav2.1 α1 mutations cause e.g. familial hemiplegic migraine type-1 (FHM1), episodic ataxia type-2 (EA2), and inherited forms of epilepsy (
      • Ophoff R.A.
      • Terwindt G.M.
      • Vergouwe M.N.
      • Eijk R.v.
      • Oefner P.J.
      • Hoffmann S.M.G.
      • Lamerdin J.E.
      • Mohrenweiser H.W.
      • Bulman D.E.
      • Ferrari M.
      • Haan J.
      • Lindhout D.
      • van Ommen G.J.B.
      • Hofker M.H.
      • Ferrari M.D.
      • Frants R.R.
      ,
      • Pietrobon D.
      • Striessnig J.
      ,
      • Jouvenceau A.
      • Eunson L.H.
      • Spauschus A.
      • Ramesh V.
      • Zuberi S.M.
      • Kullmann D.M.
      • Hanna M.G.
      ). FHM1 is a rare form of migraine with aura characterized by often unilateral obligatory motor aura symptoms (motor weakness, paralysis) (
      • Pietrobon D.
      • Striessnig J.
      ). In some patients EA2, FHM1 (
      • Jen J.
      • Yue Q.
      • Nelson S.F.
      • Yu H.
      • Litt M.
      • Nutt J.
      • Baloh R.W.
      ), or epileptic symptoms (
      • Jouvenceau A.
      • Eunson L.H.
      • Spauschus A.
      • Ramesh V.
      • Zuberi S.M.
      • Kullmann D.M.
      • Hanna M.G.
      ) co-exist, suggesting that these allelic diseases form part of a broad disease spectrum. These mutations provide us with the opportunity to relate well defined structural changes of a single Ca2+ channel subunit to (paroxysmal) neurological dysfunction and thereby also gain insight into the neurobiology of more common forms of migraine.
      Heterologous expression of EA2 mutants demonstrated that EA2 is caused by complete or severe loss of mutated channel function (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      ,
      • Jen J.
      • Wan J.
      • Graves M.
      • Yu H.
      • Mock A.F.
      • Coulin C.J.
      • Kim G.
      • Yue Q.
      • Papazian D.M.
      • Baloh R.W.
      ,
      • Guida S.
      • Trettel F.
      • Pagnutti S.
      • Mantuano E.
      • Tottene A.
      • Veneziano L.
      • Fellin T.
      • Spadaro M.
      • Stauderman K.
      • Williams M.
      • Volsen S.
      • Ophoff R.
      • Frants R.
      • Jodice C.
      • Frontali M.
      • Pietrobon D.
      ). In contrast, a common functional feature of FHM1 mutations is a shift of the Cav2.1 activation curve to more hyperpolarized voltages (
      • Pietrobon D.
      • Striessnig J.
      ,
      • Tottene A.
      • Fellin T.
      • Pagnutti S.
      • Luvisetto S.
      • Striessnig J.
      • Fletcher C.
      • Pietrobon D.
      ). This implies the existence of a gain-of-function phenotype allowing Ca2+ influx through mutant channels in response to small depolarizations that are insufficient to open wild-type (WT) channels and an increase of Ca2+ influx through single mutant channels over a large voltage range (
      • Pietrobon D.
      • Striessnig J.
      ). This has recently been confirmed in cerebellar neurons isolated from mice containing a FHM1 mutation (
      • van den Maagdenberg A.M.
      • Pietrobon D.
      • Pizzorusso T.
      • Kaja S.
      • Broos L.A.
      • Cesetti T.
      • van de Ven R.C.
      • Tottene A.
      • van der Kaa J.
      • Plomp J.J.
      • Frants R.R.
      • Ferrari M.D.
      ). Increased action potential-evoked Ca2+ influx and neurotransmitter release can explain the facilitation of cortical spreading depression (
      • van den Maagdenberg A.M.
      • Pietrobon D.
      • Pizzorusso T.
      • Kaja S.
      • Broos L.A.
      • Cesetti T.
      • van de Ven R.C.
      • Tottene A.
      • van der Kaa J.
      • Plomp J.J.
      • Frants R.R.
      • Ferrari M.D.
      ) and phenomena of enhanced cortical network hyperexcitability associated with common forms of migraine (
      • Pietrobon D.
      • Striessnig J.
      ).
      The validity of this gain-of-function hypothesis is challenged by two important questions: (i) As Cav2.1 channels can associate with different β-subunits in mammalian brain (mainly β3, β4, and β1 (
      • Liu H.
      • De Waard M.
      • Scott V.E.S.
      • Gurnett C.A.
      • Lennon V.A.
      • Campbell K.P.
      ,
      • Pichler M.
      • Cassidy T.N.
      • Reimer D.
      • Haase H.
      • Kraus R.
      • Ostler D.
      • Striessnig J.
      ,
      • Ludwig A.
      • Flockerzi V.
      • Hofmann F.
      )) the functional effects of FHM1 mutations may vary depending on the associated β-subunit; (ii) so far only seven of at least sixteen known FHM1 mutations have been analyzed functionally. It remains unclear whether all FHM1 mutants can also induce the observed negative shift in inactivation gating.
      Here we directly addressed these questions by studying the functional consequences of three FHM1 mutants, K1336E (KE), W1684R (WR), and V1696I (VI) (
      • Ducros A.
      • Denier C.
      • Joutel A.
      • Cecillon M.
      • Lescoat C.
      • Vahedi K.
      • Darcel F.
      • Vicaut E.
      • Bousser M.G.
      • Tournier-Lasserve E.
      ), expressed in Xenopus laevis oocytes together with α2δ1 and different β-subunit isoforms (β1, β3, or β4). We not only demonstrate that KE, WR, and VI cause significant changes in Cav2.1 gating, but our systematic analysis of β-subunits also revealed isoform-selective effects on all three FHM1 mutants. This suggests that the functional FHM1 phenotype varies between different neurons or even within different neuronal compartments.

      MATERIALS AND METHODS

      Generation of Cav2.1 Mutants—Mutations were introduced into human full-length cDNA, encoding Cav2.1 α1-subunit, cloned in expression plasmid pGFP. This Cav2.1 α1-subunit cDNA corresponds to the Q-type channel splice isoform (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      ) (GenBank™ accession number AF004883 lacking Val726-Ala728). Briefly, mutant PCR products harboring either mutation K1336E, W1684R, or V1696I were generated by “gene SOEing” technique (
      • Grabner M.
      • Wang Z.
      • Hering S.
      • Striessnig J.
      • Glossmann H.
      ) using Pfu Turbo DNA polymerase (Stratagene). PCR product containing the K1336E mutation was digested with SunI (nt 3284)-EcoRV (nt 5178) and cloned in SunI-EcoRV-digested vector pGFP, resulting in mutant KE. Similarly, W1684R or V1696I containing PCR products were digested with EcoRV (nt 5178)-BglII (nt 6116) and cloned in EcoRV-BglII-digested vector, resulting in Cav2.1 α1 mutants WR and VI, respectively. All PCR-derived sequences were verified by DNA sequencing.
      For expression in oocytes WT and mutant Cav2.1 α1-subunits were constructed in the polyadenylating transcription plasmid pNKS2 (a gift of O. Pongs). K1336E/pNKS2 was constructed using an AatII-Acc65I cassette (nt 2870–4445). W1684R/pNKS2 and V1696I/pNKS2 were constructed using a BglII-Acc65I cassette (nt 4445–5870). All mutations were verified by sequence analysis (MWGBiotech).
      Expression of Cav2.1 Mutants in X. laevis Oocytes—This was performed as described previously (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      ). Capped run-off poly(A+) cRNA transcripts from XbaI-linearized cDNA templates were synthesized according to Krieg and Melton (
      • Krieg P.A.
      • Melton D.A.
      ). α1 cRNA (20 ng) was co-injected with α2δ1 (6 ng) and β-subunit (6 ng each) cRNAs into stage V–VI oocytes from X. laevis. To exclude effects of endogenous Ca2+-activated Cl currents kinetics experiments were carried out in oocytes previously injected with 10–20 nl of a 0.1 m 1,2-bis-(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid solution.
      Electrophysiological Recordings in X. laevis Oocytes—1–2 days after cRNA injection IBa was measured at 19–23 °C using the two-microelectrode voltage clamp technique with the two-electrode voltage-clamp Turbo TEC 01C amplifier (NPI Electronics, Germany) (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      ). Data analysis and acquisition was performed by using the pClamp software package version 9.0 (Axon Instruments) after adjusting current traces by a conversion factor calculated from the difference between the leak at –80 and –90 mV, respectively. Microelectrodes were filled with 2.8 m CsCl, 0.2 m CsOH, 10 mm HEPES, and 10 mm EGTA (pH 7.4 with HCl), were pulled as described above and had pipette resistances between 0.3 and 1.1 MΩ. The extracellular solution contained 10 mm Ba(OH)2, 50 mm NaOH, 2 mm CsOH, and 5 mm HEPES (pH 7.4 with methanesulfonic acid).
      To quantify endogenous Ca2+ channel currents, oocytes injected only with β1, β3, or β4 subunits together with α2δ1 were analyzed. All α1-injected peak IBa currents were at least 25-fold higher than endogenous currents.
      Voltage-dependent inactivation during depolarization was estimated during 3-s pulses from a holding potential of –80 mV to a test potential 10 mV positive to the peak potential (Vmax) of the I-V relations of the respective cell. Decrease of maximal IBa during pulse trains was determined by applying 1-Hz trains of 15 100-ms pulses to the Vmax from a holding potential of –60 mV as described (
      • Kraus R.L.
      • Sinnegger M.J.
      • Glossmann H.
      • Hering S.
      • Striessnig J.
      ,
      • Kraus R.L.
      • Sinnegger M.J.
      • Koschak A.
      • Glossmann H.
      • Stenirri S.
      • Carrera P.
      • Striessnig J.
      ).
      The voltage dependence of activation was determined from current-voltage (I-V) curves at a holding potential of –80 mV which were fitted according to Equation 1,
      I=Gmax(VVrev)/{1+exp[(V0.5,actV)/kact]}
      (Eq. 1)


      where Vrev is the extrapolated reversal potential of IBa, V is the membrane potential, I is the peak current, Gmax is the maximum conductance of the cell, V0.5,act is the voltage for half-maximal activation, and kact is the slope factor of the Boltzmann term.
      Recovery of IBa from inactivation was studied using a double pulse protocol. After a 3-s depolarizing prepulse from holding potentials of –80 mV to Vmax, the time course of IBa recovery was determined at –60 mV by applying 300-ms test pulses to Vmax at various time intervals (between 0.05 and 20 s) after the prepulse. Peak IBa was normalized to the peak current amplitude measured during the prepulse. The double-pulse protocol was repeated for each recovery time interval in the same oocyte. Between protocols oocytes were held at –100 mV for 1 min. The time course of recovery was fit to a biexponential decay yielding time constants for the fast (τfast) and slow (τslow) component and the contribution of the fast component (%τfast). The voltage dependence of inactivation (steady-state inactivation, holding potential of –80 mV) was estimated from normalized inward currents elicited during steps to Vmax after 10-s steps to various holding potentials (conditioning pulses) between –90 and +30 mV. The half-maximal voltage (V0.5,inact) and the slope factor for steady-state inactivation (kinact) were obtained by fitting the data to the following Boltzmann equation (Equation 2),
      I=ISS+(1ISS)×(1+exp[(VV0.5,inact)/kinact])
      (Eq. 2)


      where ISS is the non-inactivating current component.
      Transient Expression of Cav2.1 Mutants in Mammalian tsA-201 Cells—Expression and transfection of cells was performed as described previously (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      ). Human WT and mutant Cav2.1 subunits were cotransfected together with rabbit α2δ1 subunit and rabbit β1A (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      ), rat β3 (
      • Castellano A.
      • Wei X.
      • Birnbaumer L.
      • Perez-Reyes E.
      ), and β4 (
      • Castellano A.
      • Wei X.
      • Birnbaumer L.
      • Perez-Reyes E.
      ) subunit cDNAs. After transfection, the cells were incubated at 37 °C and 5% CO2 for 2–3 days prior to membrane preparation.
      Immunoblotting and Preparation of Affinity-purified Sequence-directed Antibodies—Experiments were carried out as described using a sequence directed antibody (anti-Cav2.1α11141–1156) against a synthetic peptide corresponding to residues 1141–1156 of the rabbit Cav2.1 α1 sequence (GenBank™ accession number X57477 (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      )). Antibody binding was visualized using the ECL-system (Pierce) employing a horseradish peroxidase labeled anti-rabbit antibody.
      Statistics—All data are presented as mean ± S.E. for the indicated number of experiments. Statistical significance was determined by one-way ANOVA followed by the Bonferroni post test using Sigma Plot 2001 (SPSS Inc.) or GraphPad Prism 4 (GraphPad software Inc.).

      RESULTS

      Fig. 1 illustrates the positions of the mutations within the α1 subunit. KE converts a positive to a negative charge in the S3b–S4 linker of repeat III, which forms the putative voltage-sensor paddle of the channel (
      • Jiang Y.
      • Lee A.
      • Chen J.
      • Ruta V.
      • Cadene M.
      • Chait B.T.
      • MacKinnon R.
      ). WR introduces an additional positive charge in the S4–S5 linker of repeat IV, which is believed to confer movements of the paddle to the pore structure (
      • Jiang Y.
      • Lee A.
      • Chen J.
      • Ruta V.
      • Cadene M.
      • Chait B.T.
      • MacKinnon R.
      ). VI in helix IVS5 is the first FHM1 mutation in a S5 helix subjected to electrophysiological analysis.
      Figure thumbnail gr1
      Fig. 1Location of mutations KE, WR, and VI in the Cav2.1 α1-subunit. Numbering is according to human Cav2.1 α1 GenBank™ accession number X99897. The proposed folding structure is drawn according to Ref.
      • Jiang Y.
      • Lee A.
      • Chen J.
      • Ruta V.
      • Cadene M.
      • Chait B.T.
      • MacKinnon R.
      . The approximate position of K1336E (KE), W1684R (WR), and V1696I (VI) are indicated by squares and bold letters. Mutations functionally characterized in previous studies are indicated by triangles. Black-filled symbols indicate mutations associated with permanent cerebellar signs.
      We introduced these mutations into the human Cav2.1 α1-subunit and expressed them together with α2δ1 and β1, β3, or β4 β-subunits in X. laevis oocytes. This expression system has proven to be very reliable to rapidly screen for FHM1-induced changes of whole cell Cav2.1 Ca2+ channel currents using the two-electrode voltage clamp technique (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      ,
      • Kraus R.L.
      • Sinnegger M.J.
      • Glossmann H.
      • Hering S.
      • Striessnig J.
      ,
      • Kraus R.L.
      • Sinnegger M.J.
      • Koschak A.
      • Glossmann H.
      • Stenirri S.
      • Carrera P.
      • Striessnig J.
      ). β3 and β4 subunits were selected for co-expression, because Cav2.1 channels predominantly associate with these isoforms in mammalian brain (
      • Liu H.
      • De Waard M.
      • Scott V.E.S.
      • Gurnett C.A.
      • Lennon V.A.
      • Campbell K.P.
      ,
      • Pichler M.
      • Cassidy T.N.
      • Reimer D.
      • Haase H.
      • Kraus R.
      • Ostler D.
      • Striessnig J.
      ). β1-subunit containing channels are a minor component of the Cav2.1 channel population (
      • Liu H.
      • De Waard M.
      • Scott V.E.S.
      • Gurnett C.A.
      • Lennon V.A.
      • Campbell K.P.
      ,
      • Pichler M.
      • Cassidy T.N.
      • Reimer D.
      • Haase H.
      • Kraus R.
      • Ostler D.
      • Striessnig J.
      ,
      • Ludwig A.
      • Flockerzi V.
      • Hofmann F.
      ) but were also tested to allow comparison with other mutations analyzed in earlier studies with β1 (
      • Kraus R.L.
      • Sinnegger M.J.
      • Glossmann H.
      • Hering S.
      • Striessnig J.
      ,
      • Kraus R.L.
      • Sinnegger M.J.
      • Koschak A.
      • Glossmann H.
      • Stenirri S.
      • Carrera P.
      • Striessnig J.
      ).
      All three mutant α1-subunits yielded robust IBa 1–2 days after cRNA injection. In Western blots of tsA-201 cell-expressed wild-type (WT) and mutant α1-subunits (together with β3 and α2δ1), α1 immunoreactivities migrated with the expected molecular mass and showed indistinguishable protein expression densities (see also legend to Fig. 2).
      Figure thumbnail gr2
      Fig. 2Expression of WT and FHM1 mutants in tsA-201 cells. Cells were transfected with equal amounts of Cav2.1 WT and KE, WR or VI mutants together with β3- and α2δ1-subunit cDNA and expressed α1-subunit protein was quantified by immunoblotting as described under “Materials and Methods.” 15 μg of membrane protein was loaded per lane and separated on a 7% SDS-polyacrylamide gel. Immunoreactivity was visualized using anti-Cav2.1α11141–1156 antibody (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      ). No immunoreactivity was present in mock-transfected cells on the same gel (data not shown). Antibody staining intensity was quantified by digital image analysis of antibody stained Cav2.1 bands. When staining intensity was corrected for protein load (Coomassie Blue staining of immunoblots) and normalized to WT signal intensity, the following relative expression densities were obtained: KE, 1.07 ± 0.21; WR, 1.21 ± 0.13; VI, 0.95 ± 0.10 (means ± S.E., n = 4; no statistically significant difference between mutants and WT).
      Previous studies revealed that FHM1 mutations increase channel activity at negative membrane potentials, an effect that was also evident as a shift of the half-maximal activation voltage (V0.5,act) in six of seven mutations studied by us previously in Xenopus oocytes (
      • Kraus R.L.
      • Sinnegger M.J.
      • Glossmann H.
      • Hering S.
      • Striessnig J.
      ). A statistically significant negative shift of the half-maximal activation voltage (V0.5,act) was indeed found for KE and WR independent of the co-expressed β-subunit (Table I). For VI a small (–4 mV) negative shift was only seen upon β1 co-expression, but this trend did not reach statistical significance (Fig. 3A; but see also legend to Table I). The differences in V0.5,act could not be attributed to voltage errors due to different current amplitudes, because the statistically significant difference remained when subgroups of oocytes in each group with current amplitudes within a 0.3-μA range (0.4–0.7 μA) were compared (not shown). The apparent reversal potentials for WT and all mutants ranged between 51.6 and 55.8 mV indicating that the permeability for Ba2+ was not affected by the mutations.
      Table IEffects of FHM1 mutations on IBa activation parameters
      β1β3β4
      V0.5,actkactnV0.5,actkactnV0.5,actkactn
      WT0.61 ± 1.39-3.12 ± 0.10100.52 ± 0.82-2.92 ± 0.12111.60 ± 0.90-3.40 ± 0.2017
      KE-6.93 ± 0.80
      p < 0.001.
      -3.21 ± 0.0913-5.66 ± 1.15
      p < 0.05.
      -3.21 ± 0.1310-4.04 ± 1.12
      p < 0.01.
      -3.70 ± 0.1112
      WR-8.02 ± 1.73
      p < 0.001.
      -2.99 ± 0.1111-9.22 ± 0.62
      p < 0.001.
      -3.15 ± 0.1015-10.9 ± 0.94
      p < 0.001.
      -3.28 ± 0.259
      VI-3.40 ± 1.16
      For VI+β1 the difference to WT did not reach statistical significance by appropriate analysis with one-way ANOVA, but it did (p < 0.05) so by unpaired Student's t-test. Hence, a statistical significance would only have been obtained if mutation VI had been studied alone.
      -2.92 ± 0.0813-0.18 ± 0.81-3.07 ± 0.18141.28 ± 0.99-3.98 ± 0.1312
      a p < 0.001.
      b p < 0.05.
      c p < 0.01.
      d For VI+β1 the difference to WT did not reach statistical significance by appropriate analysis with one-way ANOVA, but it did (p < 0.05) so by unpaired Student's t-test. Hence, a statistical significance would only have been obtained if mutation VI had been studied alone.
      Figure thumbnail gr3
      Fig. 3Effect of FHM1 mutations on voltage-dependent activation and inactivation properties. Representative experiments are illustrated. For complete statistics for all mutants and β-subunits see Tables and . A, I-V relationship of WT and mutant Cav2.1 α1-subunits co-expressed with α2δ1 and β4 (10 mm Ba2+ as charge carrier). Currents were elicited by depolarizing pulses from a holding potential of –90 mV to test potentials shown between –40 mV and +50 mV. For complete statistics of all mutants and β-subunits see . The following V0.5,act and kact values were obtained by fitting the data to the Boltzmann equation (in mV): WT, 1.22, –3.20; KE, –1.68, –4.43; WR, –10.29, 3.36; VI, 1.29, 4.03. The inset shows activation curves calculated from these parameters (sigmoidal Boltzmann function). B, steady-state inactivation of WT and WR expressed with β4-subunits and α2δ1. Steady-state inactivation parameters were determined after 10-s conditioning pre-pulses as described under “Materials and Methods.” The following V0.5,inact values were obtained by fitting the data to the Boltzmann equation (in mV): WT+b4, –18.1; WR+β4, –24.7.
      Next we investigated the inactivation kinetics of the mutant channels in comparison to WT for all three β-subunits. The half-maximal voltage for steady-state inactivation (V0.5,inact) induced by 3-s conditioning prepulses to Vmax (holding potential of –80 mV) was significantly altered by the WR and KE mutations (Table II and Fig. 3B). Mutation WR induced a significant shift of V0.5,inact to more negative potentials with all three β-subunits, which was most pronounced with β3 (–8.1 mV). Interestingly, for mutation KE this occurred in a β-subunit-dependent manner as a significant shift (–4.5 mV) was only observed with co-expressed β3-subunits, but not with β1 and β4. Mutation VI did not change V0.5,inact.
      Table IIEffects of FHM1 mutations on IBa inactivation properties
      β1β3β4
      V0.5,inactkinactnV0.5,inactkinactnV0.5,inactkinactn
      WT-27.6 ± 0.856.48 ± 0.1014-25.4 ± 0.674.99 ± 0.1213-20.3 ± 1.086.43 ± 0.3612
      KE-31.4 ± 1.406.21 ± 0.1614-29.9 ± 1.29
      p < 0.01.
      5.36 ± 0.2410-21.2 ± 1.097.44 ± 0.90
      p < 0.05.
      12
      WR-35.0 ± 1.12
      p < 0.001.
      6.60 ± 0.1012-33.5 ± 0.52
      p < 0.001.
      5.26 ± 0.0615-25.3 ± 0.75
      p < 0.01.
      7.26 ± 0.1510
      VI-29.1 ± 1.226.49 ± 0.1212-24.8 ± 0.935.55 ± 0.15
      p < 0.05.
      15-20.8 ± 0.858.18 ± 0.15
      p < 0.01.
      6
      a p < 0.01.
      b p < 0.05.
      c p < 0.001.
      To examine the possibility that other biophysical properties are also affected in a β-subunit isoform-specific manner, we determined the effects of the mutants on the kinetics of voltage-dependent inactivation. We measured the decay of IBa during 3-s depolarizations to voltages 10 mV positive to Vmax (holding potential –80 mV). Mutation WR did not affect inactivation kinetics with any of the co-expressed β-subunits (for statistics see Fig. 4). KE significantly accelerated inactivation when coexpressed with β3 (Fig. 4, A and D) but not with β4 or β1 (Fig. 4, C and E). Although VI did not change activation and inactivation voltage, it significantly slowed the inactivation time course with β1 and β3 (Fig. 4, B–D) but not with β4 (Fig. 4E). All three mutations also affected the recovery of Cav2.1 channels from inactivation. This was investigated using a double-pulse protocol (Fig. 5A) in which time-dependent recovery of IBa was measured at increasing time intervals after a conditioning 3-s prepulse to Vmax (holding potential –80 mV). All three mutations significantly slowed the bi-exponential recovery from inactivation, but this effect was mainly observed upon co-expression with β1 and β3 (Fig. 5, C and D, and Table III). With β4 (Fig. 5E) a small but significant inhibition of the late phase of IBa recovery (after 10, 15, and 20 s) was evident only for mutation WR but not for KE and VI. Non-linear fits of the bi-exponential recovery data (Table III) revealed that this slowed recovery could be explained by a corresponding increase of the time constants and/or a smaller contribution of the fast recovering component and/or an increase of the non-recovering component.
      Figure thumbnail gr4
      Fig. 4Effects of FHM1 mutations on voltage-dependent inactivation during test pulses. A and B, representative current traces illustrating voltage-dependent inactivation of KE and VI mutants compared with WT co-expressed with β3and α2δ1-subunits. IBa was elicited by 3-s pulses from a holding potential of –80 mV to a depolarizing test potential 10 mV positive to Vmax. Traces were normalized to the peak current amplitude. C–E, for statistical analysis IBa inactivation of normalized current traces of mutants expressed with α2δ1 and β1 (C), β3 (D), or β4 (E) was calculated for the indicated time points and expressed as means ± S.E. 1, p < 0.05; 2, p < 0.01; 3, p < 0.001, statistically significant differences to WT determined by one-way ANOVA followed by the Bonferroni post test for n = 9–15.
      Figure thumbnail gr5
      Fig. 5Effects of FHM1 mutations on IBa recovery from inactivation. Recovery was determined using a double pulse protocol as described under “Materials and Methods.” A and B, the pulse protocol (for determining recovery after 20 s) is illustrated on top of panel A. Representative current traces for WT (A) and WR (B) channels expressed with α2δ1- and β3-subunits are shown. C–E, fractional IBa recovery from inactivation after various time periods in seconds of WT and all three mutants co-expressed with α2δ and β1 (C), and β3 (D), β4 (E) are shown. Means ± S.E. for n = 7–15 are given. 1, p < 0.05; 2, p < 0.01; 3, p < 0.001, statistically significant differences to WT determined by one-way ANOVA followed by the Bonferroni post test. Statistical significant differences against WT (0 for no significance) are indicated for individual data points in the order KE, WR, then VI.
      Table IIIEffects of FHM1 mutations on IBa recovery from inactivation kinetics
      β1β3β4
      τfastτslowτfastNCCnτfastτslowτfastNCCnτfastτslowτfastNCCn
      %%%
      WT0.55 ± 0.043.27 ± 0.2859.7 ± 2.1012.7 ± 0.03120.45 ± 0.054.10 ± 0.4364.8 ± 2.227.80 ± 0.01130.54 ± 0.104.43 ± 0.3257.1 ± 3.7212.1 ± 0.028
      KE0.81 ± 0.114.58 ± 0.6849.6 ± 3.3226.2 ± 0.01
      p < 0.05.
      81.04 ± 0.13
      p < 0.001.
      6.92 ± 0.70
      p < 0.01.
      49.4 ± 5.33
      p < 0.01.
      21.2 ± 0.04
      p < 0.01.
      60.43 ± 0.044.54 ± 0.2748.7 ± 7.5018.9 ± 0.028
      WR0.84 ± 0.114.43 ± 0.4644.1 ± 6.42
      p < 0.05.
      25.5 ± 0.0680.89 ± 0.05
      p < 0.01.
      5.01 ± 0.3152.5 ± 1.90
      p < 0.01.
      25.7 ± 0.03
      p < 0.001.
      140.49 ± 0.045.37 ± 0.3053.9 ± 1.6623.6 ± 0.02
      p < 0.01.
      8
      VI1.14 ± 0.08
      p < 0.001.
      6.39 ± 0.58
      p < 0.001.
      44.7 ± 2.99
      p < 0.01.
      21.2 ± 0.03121.14 ± 0.07
      p < 0.001.
      6.67 ± 0.47
      p < 0.001.
      50.4 ± 2.21
      p < 0.001.
      16.6 ± 0.03
      p < 0.05.
      150.48 ± 0.054.60 ± 0.4050.8 ± 2.3518.8 ± 0.0212
      a p < 0.05.
      b p < 0.001.
      c p < 0.01.
      Changes of inactivation parameters should also affect the availability of Cav2.1 channels during trains of frequent pulses. Delayed recovery from inactivation should allow less IBa to recover between pulses and promote accumulation of channels in inactivated states resulting in a cumulative decrease of IBa during the train. Fig. 6 illustrates that WT maximal current amplitude decreased during 15 1-Hz trains of 100-ms depolarizations by 26.8% (β1) to 32.5% (β4). All three mutants significantly enhanced the current decrease with co-expressed β1 and β3, but not with β4. Our data demonstrate that all three FHM1 mutants can potentially reduce Ca2+ influx during frequent depolarizations, at least if β1 and β3 form part of the channel complex.
      Figure thumbnail gr6
      Fig. 6Effects of FHM1 mutations on IBa decay during 1-Hz pulse trains. A, current traces illustrating the decay of IBa through WT and WR co-expressed with α2δ1 trains and β3-subunits during 1-Hz pulse of 15 100-ms depolarizations to Vmax from a holding potential of –60 mV. The 1st and 15th pulses of the train are indicated. B, statistics for the decay of maximal IBa during trains expressed as the percentage of IBa decrease between the 1st and 15th pulses of the train. Means ± S.E. for n = 9–16 are given. 1, p < 0.05; 2, p < 0.01; 3, p < 0.001, statistically significant differences to WT expressed with the same β-subunit as determined by one-way ANOVA followed by the Bonferroni post test.

      DISCUSSION

      The major novel finding of our study is that β-subunits critically determine the disturbances of channel gating found for all three FHM1 missense mutations, which we selected randomly from nine not yet functionally characterized ones. This implies that mutant phenotypes could vary between different neurons expressing different β-subunit isoforms. Another important finding is that two mutations, KE and WR, allow Cav2.1 channel activation at more negative voltages. This is in accordance with the hypothesis that enhanced activity of Cav2.1 channels at negative voltages serves as a pathogenetic mechanism in FHM1 (
      • Pietrobon D.
      • Striessnig J.
      ). Our study emphasizes that further analysis of FHM1 mutations on channel function requires the co-expression with those β-subunit isoforms most frequently associated Cav2.1 Ca2+ channels in mammalian brain.
      As shown in biochemical studies, brain Cav2.1 channel complexes predominantly contain either β3 (36%) or β4 (48%) and to a much smaller extent β1 (8.4%) and β2 (7.2%) subunits (
      • Liu H.
      • De Waard M.
      • Scott V.E.S.
      • Gurnett C.A.
      • Lennon V.A.
      • Campbell K.P.
      ). Similar findings were obtained for brain Cav2.2 (
      • Scott V.E.S.
      • De Waard M.
      • Liu H.
      • Gurnett C.A.
      • Venzke D.P.
      • Lennon V.A.
      • Campbell K.P.
      ) and Cav1 (L-type) (
      • Pichler M.
      • Cassidy T.N.
      • Reimer D.
      • Haase H.
      • Kraus R.
      • Ostler D.
      • Striessnig J.
      ) channels. Analysis of the β-subunit composition of Cav1, Cav2.2, and Cav2.1 channels in defined brain regions revealed that equally large fractions of channels associate with β3 or β4 in cerebral cortex, whereas β4-subunits predominate in channel complexes immunoprecipitated from the cerebellum (
      • Pichler M.
      • Cassidy T.N.
      • Reimer D.
      • Haase H.
      • Kraus R.
      • Ostler D.
      • Striessnig J.
      ). In the cerebellum, β4 and β3 mRNAs are both expressed in granule cells, whereas β4 predominates in the Purkinje cells (
      • Ludwig A.
      • Flockerzi V.
      • Hofmann F.
      ). β4, β3, and β1 mRNA was detected in cerebral cortex (
      • Ludwig A.
      • Flockerzi V.
      • Hofmann F.
      ). In the human hippocampus β1-, β2-, and β3-subunit immunoreactivity is mainly localized to somata, whereas β4 staining is intense at dendritic locations (
      • Lie A.A.
      • Blumcke I.
      • Volsen S.G.
      • Wiestler O.D.
      • Elger C.E.
      • Beck H.
      ,
      • Day N.C.
      • Volsen S.G.
      • McCormack A.L.
      • Craig P.J.
      • Smith W.
      • Beattie R.E.
      • Shaw P.J.
      • Ellis S.B.
      • Harpold M.M.
      • Ince P.G.
      ). Based on the differential localization of these subunits and our findings FHM1 mutations may induce different patterns of Cav2.1 dysfunction in different neurons and even between different subcellular compartments of a single neuron. For example, in the presence of β3-subunits, which appear to contribute more to presynaptic channels than β4, at least in hippocampal neurons (
      • Day N.C.
      • Volsen S.G.
      • McCormack A.L.
      • Craig P.J.
      • Smith W.
      • Beattie R.E.
      • Shaw P.J.
      • Ellis S.B.
      • Harpold M.M.
      • Ince P.G.
      ), all three mutations increase channel accumulation in inactivated states during frequent channel opening. This effect is mainly due to slowed recovery from voltage-dependent inactivation (Table III and Fig. 5). This is not seen with channels containing the β4 subunit, which appears to be the predominant form in dendritic compartments of hippocampal neurons (
      • Lie A.A.
      • Blumcke I.
      • Volsen S.G.
      • Wiestler O.D.
      • Elger C.E.
      • Beck H.
      ,
      • Day N.C.
      • Volsen S.G.
      • McCormack A.L.
      • Craig P.J.
      • Smith W.
      • Beattie R.E.
      • Shaw P.J.
      • Ellis S.B.
      • Harpold M.M.
      • Ince P.G.
      ). Cav2.1-current decrease during high neuronal firing rates may therefore be affected differently by the mutations at pre- and postsynaptic sites and/or in different neurons.
      A still unresolved, yet important question concerning the FHM1 genotype-phenotype relationship is why about half of the known FHM1 mutations (black-filled symbols in Fig. 1 (
      • Ducros A.
      • Denier C.
      • Joutel A.
      • Cecillon M.
      • Lescoat C.
      • Vahedi K.
      • Darcel F.
      • Vicaut E.
      • Bousser M.G.
      • Tournier-Lasserve E.
      )) cause permanent cerebellar signs (PCS), such as ataxia, nystagmus, or dysarthria. PCS likely reflect permanent neuronal damage of cerebellar neurons, which may present clinically as cerebellar atrophy (
      • Kors E.E.
      • Haan J.
      • Giffin N.J.
      • Pazdera L.
      • Schnittger C.
      • Lennox G.G.
      • Terwindt G.M.
      • Vermeulen F.L.
      • Van den Maagdenberg A.M.
      • Frants R.R.
      • Ferrari M.D.
      ). Due to the predominant expression of β4 subunits in cerebellar neurons, it is possible that only mutation-induced abnormalities seen with β4-subunits underlie PCS. From the mutations investigated here, only WR is associated with PCS (
      • Pietrobon D.
      • Striessnig J.
      ). Interestingly, only WR caused a shift of V0.5,inact to more negative potentials when co-expressed with β4. This could decrease channel availability in cerebellar neurons, which may result in neuronal damage. Note that a loss of Cav2.1-channel function also occurs in EA2 (
      • Wappl E.
      • Koschak A.
      • Poteser M.
      • Sinnegger M.J.
      • Walter D.
      • Eberhart A.
      • Groschner K.
      • Glossmann H.
      • Kraus R.L.
      • Grabner M.
      • Striessnig J.
      ,
      • Jen J.
      • Wan J.
      • Graves M.
      • Yu H.
      • Mock A.F.
      • Coulin C.J.
      • Kim G.
      • Yue Q.
      • Papazian D.M.
      • Baloh R.W.
      ,
      • Guida S.
      • Trettel F.
      • Pagnutti S.
      • Mantuano E.
      • Tottene A.
      • Veneziano L.
      • Fellin T.
      • Spadaro M.
      • Stauderman K.
      • Williams M.
      • Volsen S.
      • Ophoff R.
      • Frants R.
      • Jodice C.
      • Frontali M.
      • Pietrobon D.
      ), which leads to PCS and cerebellar atrophy of variable intensity (
      • Pietrobon D.
      ). Our results therefore prompt further studies investigating the correlation of β4-induced biophysical changes with PCS.
      Our data support the hypothesis that FHM1 mutants can activate at more hyperpolarized potentials. This was unequivocally demonstrated for WR and KE. For VI a smaller shift was observed when co-expressed with β1, which, however, did not reach statistical significance. Note that this does not rule the possibility of mutation-induced changes in channel activity: FHM1 mutation R192Q also caused only a minor shift of V0.5,act in whole cell experiments after expression in Xenopus oocytes or tsA-201 cells, but an increased open probability over a broad voltage range was revealed in single-channel recordings (
      • Kraus R.L.
      • Sinnegger M.J.
      • Glossmann H.
      • Hering S.
      • Striessnig J.
      ,
      • Hans M.
      • Luvisetto S.
      • Williams M.E.
      • Spagnolo M.
      • Urrutia A.
      • Tottene A.
      • Brust P.F.
      • Johnson E.C.
      • Harpold M.M.
      • Stauderman K.A.
      • Pietrobon D.
      ). Further detailed single channel analysis, clearly beyond the scope of the present study, employing these three FHM1 mutants co-expressed with different β-subunit isoforms, must address this question. Moreover, we cannot rule out the possibility that other factors not accounted for in our heterologous expression system (such as the association of Cav2.1 channels with SNARE proteins (
      • Spafford J.D.
      • Zamponi G.W.
      )) also affect the functional consequences of FHM1 mutations. Thus it will be interesting to see whether the mutant phenotypes of Cav2.1 channel currents vary in cultured neurons isolated from different brain regions of FHM1 knock-in mice (
      • van den Maagdenberg A.M.
      • Pietrobon D.
      • Pizzorusso T.
      • Kaja S.
      • Broos L.A.
      • Cesetti T.
      • van de Ven R.C.
      • Tottene A.
      • van der Kaa J.
      • Plomp J.J.
      • Frants R.R.
      • Ferrari M.D.
      ).
      In this study we have not systematically addressed the question of mutant-induced changes in IBa density and/or effects on α1-subunit plasma membrane targeting, because such data would be difficult to interpret based on the results of previous studies. Although heterologous expression nicely revealed changes in mutant channel gating that were confirmed in mutant mice in vivo (cf. Refs.
      • van den Maagdenberg A.M.
      • Pietrobon D.
      • Pizzorusso T.
      • Kaja S.
      • Broos L.A.
      • Cesetti T.
      • van de Ven R.C.
      • Tottene A.
      • van der Kaa J.
      • Plomp J.J.
      • Frants R.R.
      • Ferrari M.D.
      and
      • Hans M.
      • Luvisetto S.
      • Williams M.E.
      • Spagnolo M.
      • Urrutia A.
      • Tottene A.
      • Brust P.F.
      • Johnson E.C.
      • Harpold M.M.
      • Stauderman K.A.
      • Pietrobon D.
      ), their role for determining effects on current density remain controversial, because increased (
      • Hans M.
      • Luvisetto S.
      • Williams M.E.
      • Spagnolo M.
      • Urrutia A.
      • Tottene A.
      • Brust P.F.
      • Johnson E.C.
      • Harpold M.M.
      • Stauderman K.A.
      • Pietrobon D.
      ), decreased (
      • Tottene A.
      • Fellin T.
      • Pagnutti S.
      • Luvisetto S.
      • Striessnig J.
      • Fletcher C.
      • Pietrobon D.
      ), and unaltered (
      • van den Maagdenberg A.M.
      • Pietrobon D.
      • Pizzorusso T.
      • Kaja S.
      • Broos L.A.
      • Cesetti T.
      • van de Ven R.C.
      • Tottene A.
      • van der Kaa J.
      • Plomp J.J.
      • Frants R.R.
      • Ferrari M.D.
      ) Cav2.1 current densities were for example found for R192Q in tsA-201 cells, transfected Cav2.1-deficient neurons, and mutant mice, respectively.
      Taken together, our data clearly indicate that FHM1 mutation-induced changes of Cav2.1 channel function critically depends on the β-subunit composition of the channel. Obviously, our studies also prompt an even more detailed analysis of the expression of different Ca2+ channel β-subunit isoforms in neuronal circuits relevant to migraine pathophysiology.

      Acknowledgments

      We thank G. Pelster and J. Aldrian for excellent technical support and M. J. Sinnegger-Brauns and B. E. Flucher for helpful comments on the manuscript.

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