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Functional impact of a congenital stationary night blindness type 2 mutation depends on subunit composition of Cav1.4 Ca2+ channels

  • Brittany Williams
    Affiliations
    Departments of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, Iowa Neuroscience Institute, Pappajohn Biomedical Institute, University of Iowa, Iowa City, Iowa USA

    Interdisciplinary Graduate Program in Neuroscience, University of Iowa, Iowa City, Iowa USA
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  • Josue A. Lopez
    Affiliations
    Departments of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, Iowa Neuroscience Institute, Pappajohn Biomedical Institute, University of Iowa, Iowa City, Iowa USA
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  • J. Wesley Maddox
    Affiliations
    Departments of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, Iowa Neuroscience Institute, Pappajohn Biomedical Institute, University of Iowa, Iowa City, Iowa USA
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  • Amy Lee
    Correspondence
    For correspondence: Amy Lee
    Affiliations
    Departments of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, Iowa Neuroscience Institute, Pappajohn Biomedical Institute, University of Iowa, Iowa City, Iowa USA
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Open AccessPublished:October 08, 2020DOI:https://doi.org/10.1074/jbc.RA120.014138
      Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming α1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ−1−4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.4+ex47 and Cav1.4Δex47, respectively), and the auxiliary subunits, β2X13 and α2δ-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β2X13 and α2δ-4 but not in those with β2A and α2δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β2X13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.
      Voltage-gated Ca2+ (Cav) channels are comprised of a pore-forming α1 subunit and two auxiliary subunits, β and α2δ (reviewed in Ref.
      • Dolphin A.C.
      • Lee A.
      Presynaptic calcium channels: specialized control of synaptic neurotransmitter release.
      ). The α1 subunit is comprised of 4 homologous domains (I-IV), each containing 6 alpha-helical transmembrane-spanning segments (S1–S6); the S1–S4 segments form a voltage-sensing domain, and S5–S6 forms the pore (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Qian X.
      • Lu S.
      • Dong M.
      • Zhou Q.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
      ). In contrast to the diverse complement of Cav channels expressed in many neurons, the pore-forming α1F subunit (referred to as Cav1.4 from here on), encoded by the Cacna1f gene, appears to be the major Cav subtype localized in the synaptic terminals of photoreceptors in the retina (
      • Mansergh F.
      • Orton N.C.
      • Vessey J.P.
      • Lalonde M.R.
      • Stell W.K.
      • Tremblay F.
      • Barnes S.
      • Rancourt D.E.
      • Bech-Hansen N.T.
      Mutation of the calcium channel gene Cacna1f disrupts calcium signaling, synaptic transmission and cellular organization in mouse retina.
      ,
      • Chang B.
      • Heckenlively J.R.
      • Bayley P.R.
      • Brecha N.C.
      • Davisson M.T.
      • Hawes N.L.
      • Hirano A.A.
      • Hurd R.E.
      • Ikeda A.
      • Johnson B.A.
      • McCall M.A.
      • Morgans C.W.
      • Nusinowitz S.
      • Peachey N.S.
      • Rice D.S.
      • et al.
      The nob2 mouse, a null mutation in Cacna1f: anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses.
      ) where it co-assembles with β2 and α2δ-4 subunits (
      • Lee A.
      • Wang S.
      • Williams B.
      • Hagen J.
      • Scheetz T.E.
      • Haeseleer F.
      Characterization of Cav1.4 complexes (α11.4, β2, and α2δ−4) in HEK293T cells and in the retina.
      ). Within photoreceptor synaptic terminals, Cav1.4 channels are activated at the relatively depolarized voltage of these cells in darkness, causing the tonic release of glutamate. At the sign-inverting synapse formed between photoreceptors and depolarizing bipolar cells, the termination of Cav1.4-dependent glutamate release by light stimuli enables disinhibition of a nonselective cation channel, initiating excitation of the ON pathway in the retina (
      • Shen Y.
      • Heimel J.A.
      • Kamermans M.
      • Peachey N.S.
      • Gregg R.G.
      • Nawy S.
      A transient receptor potential-like channel mediates synaptic transmission in rod bipolar cells.
      ,
      • Shiells R.A.
      • Falk G.
      • Naghshineh S.
      Action of glutamate and aspartate analogues on rod horizontal and bipolar cells.
      ). Thus, the voltage-dependent properties of Cav1.4 are critical parameters for controlling the dynamic range of visual signaling.
      More than 140 mutations in Cacna1f have been identified and are linked to vision disorders including congenital stationary night blindness type 2 (CSNB2) (reviewed in Ref.
      • Waldner D.M.
      • Bech-Hansen N.T.
      • Stell W.K.
      Channeling vision: CaV1.4-A critical link in retinal signal transmission.
      ). The sequelae of these mutations are not entirely clear because, when analyzed in heterologous expression systems, they can weaken, enhance, or have no impact on the function of Cav1.4 (
      • Hoda J.C.
      • Zaghetto F.
      • Singh A.
      • Koschak A.
      • Striessnig J.
      Effects of congenital stationary night blindness type 2 mutations R508Q and L1364H on Cav1.4 L-type Ca2+ channel function and expression.
      ,
      • Singh A.
      • Hamedinger D.
      • Hoda J.C.
      • Gebhart M.
      • Koschak A.
      • Romanin C.
      • Striessnig J.
      C-terminal modulator controls Ca2+-dependent gating of Cav1.4 L-type Ca2+ channels.
      ,
      • Hoda J.C.
      • Zaghetto F.
      • Koschak A.
      • Striessnig J.
      Congenital stationary night blindness type 2 mutations S229P, G369D, L1068P, and W1440X alter channel gating or functional expression of Cav1.4 L-type Ca2+ channels.
      ,
      • Hemara-Wahanui A.
      • Berjukow S.
      • Hope C.I.
      • Dearden P.K.
      • Wu S.B.
      • Wilson-Wheeler J.
      • Sharp D.M.
      • Lundon-Treweek P.
      • Clover G.M.
      • Hoda J.C.
      • Striessnig J.
      • Marksteiner R.
      • Hering S.
      • Maw M.A.
      A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.
      ,
      • Peloquin J.B.
      • Rehak R.
      • Doering C.J.
      • McRory J.E.
      Functional analysis of congenital stationary night blindness type-2 CACNA1F mutations F742C, G1007R, and R1049W.
      ). Understanding the pathological consequences of CSNB2 mutations is complicated by the functional diversity of retinal Cav1.4 conferred in part by alternative splicing of the pre-mRNAs corresponding to each subunit (
      • Lee A.
      • Wang S.
      • Williams B.
      • Hagen J.
      • Scheetz T.E.
      • Haeseleer F.
      Characterization of Cav1.4 complexes (α11.4, β2, and α2δ−4) in HEK293T cells and in the retina.
      ,
      • Bacchi N.
      • Messina A.
      • Burtscher V.
      • Dassi E.
      • Provenzano G.
      • Bozzi Y.
      • Demontis G.C.
      • Koschak A.
      • Denti M.A.
      • Casarosa S.
      A new splicing isoform of Cacna2d4 mimicking the effects of c.2451insC mutation in the retina: novel molecular and electrophysiological insights.
      ,
      • Tan G.M.
      • Yu D.
      • Wang J.
      • Soong T.W.
      Alternative splicing at C terminus of Cav1.4 calcium channel modulates calcium-dependent inactivation, activation potential, and current density.
      ,
      • Haeseleer F.
      • Williams B.
      • Lee A.
      Characterization of C-terminal splice variants of Cav1.4 Ca2+ channels in human retina.
      ). The β2 variant that is most highly expressed in human retina contains an alternatively spliced exon 7B (β2X13) and causes stronger voltage-dependent inactivation of Cav1.4 than β2 variants with exon 7A (β2A) (
      • Lee A.
      • Wang S.
      • Williams B.
      • Hagen J.
      • Scheetz T.E.
      • Haeseleer F.
      Characterization of Cav1.4 complexes (α11.4, β2, and α2δ−4) in HEK293T cells and in the retina.
      ). The Cav1.4 α1 pre-mRNA also undergoes alternative splicing, particularly in the sequence encoding the large cytoplasmic C-terminal domain (
      • Tan G.M.
      • Yu D.
      • Wang J.
      • Soong T.W.
      Alternative splicing at C terminus of Cav1.4 calcium channel modulates calcium-dependent inactivation, activation potential, and current density.
      ,
      • Haeseleer F.
      • Williams B.
      • Lee A.
      Characterization of C-terminal splice variants of Cav1.4 Ca2+ channels in human retina.
      ). We previously characterized a Cav1.4 variant lacking exon 47 (Cav1.4Δex47) that is highly expressed in human retina (
      • Haeseleer F.
      • Williams B.
      • Lee A.
      Characterization of C-terminal splice variants of Cav1.4 Ca2+ channels in human retina.
      ). Exon 47 encodes a portion of a C-terminal modulatory domain (CTM) in Cav1.4 that suppresses Ca2+-dependent inactivation (CDI) and causes depolarizing shifts in the voltage-dependence of activation (
      • Singh A.
      • Hamedinger D.
      • Hoda J.C.
      • Gebhart M.
      • Koschak A.
      • Romanin C.
      • Striessnig J.
      C-terminal modulator controls Ca2+-dependent gating of Cav1.4 L-type Ca2+ channels.
      ,
      • Wahl-Schott C.
      • Baumann L.
      • Cuny H.
      • Eckert C.
      • Griessmeier K.
      • Biel M.
      Switching off calcium-dependent inactivation in L-type calcium channels by an autoinhibitory domain.
      ). When expressed in a human embryonic kidney cell line (HEK293T), Cav1.4Δex47 exhibits more negative activation thresholds and stronger CDI than Cav1.4 variants containing exon 47 (Cav1.4+ex47) (
      • Haeseleer F.
      • Williams B.
      • Lee A.
      Characterization of C-terminal splice variants of Cav1.4 Ca2+ channels in human retina.
      ,
      • Williams B.
      • Haeseleer F.
      • Lee A.
      Splicing of an automodulatory domain in Cav1.4 Ca(2+) channels confers distinct regulation by calmodulin.
      ).
      Studies investigating the electrophysiological consequences of Cacna1f mutations have focused on the Cav1.4+ex47 variant coexpressed with auxiliary subunits other than β2X13 and α2δ-4 (
      • Hoda J.C.
      • Zaghetto F.
      • Singh A.
      • Koschak A.
      • Striessnig J.
      Effects of congenital stationary night blindness type 2 mutations R508Q and L1364H on Cav1.4 L-type Ca2+ channel function and expression.
      ,
      • Singh A.
      • Hamedinger D.
      • Hoda J.C.
      • Gebhart M.
      • Koschak A.
      • Romanin C.
      • Striessnig J.
      C-terminal modulator controls Ca2+-dependent gating of Cav1.4 L-type Ca2+ channels.
      ,
      • Hoda J.C.
      • Zaghetto F.
      • Koschak A.
      • Striessnig J.
      Congenital stationary night blindness type 2 mutations S229P, G369D, L1068P, and W1440X alter channel gating or functional expression of Cav1.4 L-type Ca2+ channels.
      ,
      • Hemara-Wahanui A.
      • Berjukow S.
      • Hope C.I.
      • Dearden P.K.
      • Wu S.B.
      • Wilson-Wheeler J.
      • Sharp D.M.
      • Lundon-Treweek P.
      • Clover G.M.
      • Hoda J.C.
      • Striessnig J.
      • Marksteiner R.
      • Hering S.
      • Maw M.A.
      A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.
      ,
      • Peloquin J.B.
      • Rehak R.
      • Doering C.J.
      • McRory J.E.
      Functional analysis of congenital stationary night blindness type-2 CACNA1F mutations F742C, G1007R, and R1049W.
      ). Alternative splicing can affect the severity of disease-causing mutations in Cav channel genes (
      • 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.
      ). Thus, analysis of Cacna1f mutations in the context of Cav1.4 variants expressed in photoreceptors in human retina is necessary for understanding the visual phenotypes associated with such mutations.
      Here, we investigated the effects of a CSNB2-causing mutation on the properties of Cav1.4+ex47 and Cav1.4Δex47 channels containing β2X13 and α2δ-4. The mutation results in the replacement of isoleucine 745 with a threonine (IT) in the S6 helix of domain 2 (IIS6, Fig. 1A). In Cav1.4+ex47 coexpressed with α2δ-1 and β3 or β2, the IT mutation causes a large hyperpolarizing shift (>30 mV) in the voltage-dependence of activation (
      • Hemara-Wahanui A.
      • Berjukow S.
      • Hope C.I.
      • Dearden P.K.
      • Wu S.B.
      • Wilson-Wheeler J.
      • Sharp D.M.
      • Lundon-Treweek P.
      • Clover G.M.
      • Hoda J.C.
      • Striessnig J.
      • Marksteiner R.
      • Hering S.
      • Maw M.A.
      A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.
      ). Our results indicate that, when coexpressed with β2X13 and α2δ-4, Cav1.4+ex47 channels bearing the IT mutation (Cav1.4+ex47IT) show hyperpolarized activation voltages compared with wild-type (WT) channels. The gain-of function effect is more severe for Cav1.4Δex47 channels with the IT mutation (Cav1.4Δex47IT), which showed more negative activation thresholds and slower deactivation kinetics than Cav1.4+ex47IT. An unexpected finding is that IT alters the ion selectivity of both Cav1.4 splice variants in a manner that varies with the identity of the α2δ subunit. Our findings highlight the importance of splice variation and auxiliary subunit composition as potential modifiers of disease-causing mutations affecting Cav channels.
      Figure thumbnail gr1
      Figure 1IT enhances voltage-dependence of activation of Cav1.4+ex47. A, schematic of Cav1.4 pore-forming α1 subunit with 4 transmembrane spanning domains (I-IV; blue), and β2X13 (tan) and α2δ-4 (green) subunits. The CTM of Cav1.4 (purple) contains exon 47 (orange). The red star illustrates the location of IT in domain II. B, representative IBa family of traces for Cav1.4+ex47 or Cav1.4+ex47IT. C, I-V plots for IBa current density (pA/pF) in cells transfected with Cav1.4+ex47 (black) or Cav1.4+ex47IT (red). IBa was evoked by 50-ms pulses from −100 mV to various voltages. Here and in all graphs of electrophysiological data, parentheses indicate number of cells, and symbols and error bars represent mean ± S.E., respectively.

      Results

      IT mutation enhances activation and slows deactivation of Cav1.4+ex47

      Exon 47 resides in the CTM of Cav1.4 (Fig. 1A); deletion of this exon, like the IT mutation, causes a large negative shift in the voltage dependence of channel activation (
      • Haeseleer F.
      • Williams B.
      • Lee A.
      Characterization of C-terminal splice variants of Cav1.4 Ca2+ channels in human retina.
      ,
      • Williams B.
      • Haeseleer F.
      • Lee A.
      Splicing of an automodulatory domain in Cav1.4 Ca(2+) channels confers distinct regulation by calmodulin.
      ). Thus, the effect of IT on Cav1.4 activation could be additive, or alternatively, could be occluded by exon 47 deletion. To distinguish between these possibilities, we compared the activation properties of Ba2+ currents (IBa) mediated by Cav1.4+ex47 and Cav1.4Δex47, and the corresponding IT mutant channels, in transfected HEK293T cells. Ba2+ rather than Ca2+ was used as the charge carrier to minimize the complicating effects of CDI which, whereas negligible in Cav1.4+ex47, is prominent in Cav1.4Δex47 (
      • Williams B.
      • Haeseleer F.
      • Lee A.
      Splicing of an automodulatory domain in Cav1.4 Ca(2+) channels confers distinct regulation by calmodulin.
      ). Because previous analyses of Cav1.4+ex47IT were performed primarily with β3 or β2a, and α2δ-1 (
      • Hemara-Wahanui A.
      • Berjukow S.
      • Hope C.I.
      • Dearden P.K.
      • Wu S.B.
      • Wilson-Wheeler J.
      • Sharp D.M.
      • Lundon-Treweek P.
      • Clover G.M.
      • Hoda J.C.
      • Striessnig J.
      • Marksteiner R.
      • Hering S.
      • Maw M.A.
      A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.
      ), we first characterized the effect of IT on Cav1.4+ex47 coexpressed with auxiliary subunits representative of Cav1.4 complexes in the retina (i.e. β2X13 and α2δ-4 (
      • Lee A.
      • Wang S.
      • Williams B.
      • Hagen J.
      • Scheetz T.E.
      • Haeseleer F.
      Characterization of Cav1.4 complexes (α11.4, β2, and α2δ−4) in HEK293T cells and in the retina.
      )). Although there was no effect of IT on the slope factor (k), Boltzmann fits of current-voltage (I-V) plots showed that the half-maximal voltage of activation (Vh) of Cav1.4+ex47IT was significantly more negative than that of Cav1.4+ex47 (Fig. 1, B and C, Table 1).
      Table 1Parameters from I–V relationships of Cav1.4+ex47 and Cav1.4Δex47 with or without IT mutation
      Peak IBa (pA/pF)p ValueVh (mV)p Valuekp ValueErev (mV)p Value
      Cav1.4+ex47 + β2x13 + α2δ-4−5.6 ± 1.4−8.6 ± 0.4−6.2 ± 1.658.3 ± 3.7
      Cav1.4+ex47IT + β2x13 + α2δ-4−5.3 ± 1.20.904
      Mann-Whitney test.
      −28.5 ± 2.0<0.0001
      Mann-Whitney test.
      −8.5 ± 1.20.253
      Student's t test.
      37.1 ± 3.7<0.0001
      Mann-Whitney test.
      Cav1.4Δex47 + β2x13 + α2δ-4−10.5 ± 1.9−24.8 ± 1.3−6.6 ± 0.454.3 ± 1.5v
      Cav1.4Δex47IT + β2x13 + α2δ-4−2.6 ± 0.40.002
      Student's t test.
      −43.7 ± 2.1<0.0001
      Student's t test.
      −11.2 ± 1.0<0.001
      Student's t test.
      16.7 ± 5.0<0.0001
      Mann-Whitney test.
      Cav1.4+ex47 + β2A + α2δ-1−11.2 ± 2.0−2.7 ± 1.2−7.1 ± 0.252.7 ± 0.8
      Cav1.4+ex47IT + β2A + α2δ-1−9.2 ± 3.00.581
      Student's t test.
      −28.3 ± 2.0<0.0001
      Student's t test.
      −7.4 ± 1.10.527
      Mann-Whitney test.
      50.1 ± 2.40.237
      Student's t test.
      Cav1.4Δex47 + β2A + α2δ-1−17.8 ± 0.9−19.2 ± 0.5−4.6 ± 0.850.4 ± 3.2
      Cav1.4Δex47IT + β2A + α2δ-1−0.6 ± 0.10.082
      Student's t test.
      −36.1 ± 2.50.003
      Student's t test.
      −7.7 ± 0.50.009
      Student's t test.
      41.0 ± 3.20.121
      Student's t test.
      a Mann-Whitney test.
      b Student's t test.
      Exponential fits of the rising phase of the peak currents yielded time constants for activation (τact) that were significantly longer (Table 2) and with weaker voltage dependence for Cav1.4+ex47IT (v = −50.3 mV) than for Cav1.4+ex47 (v = −26.9 mV; F2,7 = 16.4, p = 0.002; Fig. 2, A and B). To analyze rates of channel closure, the time constant for deactivation (τdeact) was obtained from exponential fits of the decay phase of the tail current evoked upon repolarization of the membrane voltage. τdeact was significantly greater at the most positive repolarization voltage tested (-60 mV, Table 2) and the voltage-dependence of τdeact was significantly steeper for Cav1.4+ex47IT (v = 43.1 mV) than for Cav1.4+ex47 (v = 169.9 mV; F2,22 = 59.2, p < 0.0001; Fig. 2, C and D). Thus, as has been shown for Cav1.2 channels bearing the analogous IT mutation (
      • Hohaus A.
      • Beyl S.
      • Kudrnac M.
      • Berjukow S.
      • Timin E.N.
      • Marksteiner R.
      • Maw M.A.
      • Hering S.
      Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder.
      ), IT slows the activation and deactivation of Cav1.4 containing exon 47 in a highly voltage-dependent manner.
      Table 2Time constants for activation and deactivation of Cav1.4+ex47 and Cav1.4Δex47 with or without IT mutation
      Channelτact (ms)p Valueτdeact (ms)p-Value
      Cav1.4+ex47 + β2x13 + α2δ-41.8 ± 0.2 (0 mV)4.4 ± 0.8 (0 mV)
      Cav1.4+ex47IT + β2x13 + α2δ-42.8 ± 0.2 (−20 mV)0.019
      Student's t test.
      14.2 ± 1.8 (−20 mV)<0.001
      Student's t test.
      Cav1.4Δex47 + β2x13 + α2δ-42.0 ± 0.2 (−10 mV)3.1 ± 0.3 (−10 mV)
      Cav1.4Δex47IT + β2x13 + α2δ-42.5 ± 0.4 (−30 mV)0.343
      Student's t test.
      37 ± 9.7 (−30 mV)0.004
      Mann-Whitney test.
      a Student's t test.
      b Mann-Whitney test.
      Figure thumbnail gr2
      Figure 2IT alters kinetics of activation and deactivation for Cav1.4+ex47. A, voltage protocol for measuring activation kinetics (left) and representative IBa family of traces (right). IBa was evoked by 50-ms pulses from −100 mV to various test voltages. Current traces are color-coded according to the depolarizations used to evoke them in voltage protocol. Exponential fits (black lines) are overlaid on corresponding current traces. B, activation time constants (τact) were obtained from exponential fits of IBa and plotted against test voltage in cells transfected with Cav1.4+ex47 (black symbols) or Cav1.4+ex47IT (red symbols). C, voltage protocol for measuring deactivation kinetics (left) and representative family of IBa traces (right). Tail IBa was evoked by 10-ms pulses to voltages evoking peak inward IBa (see ) followed by repolarizations to various voltages. Exponential fits of tail IBa are color-coded according to the repolarization voltage used to evoke them in voltage protocol. D, deactivation time constants (τdeact) were obtained from exponential fits of tail IBa and plotted against repolarization voltage in cells transfected with Cav1.4+ex47 (black symbols) or Cav1.4+ex47IT (red symbols). In B and D, solid lines represent exponential fits of the averaged data. Cav1.4 variants were co-expressed with β2X13 and α2δ-4.

      Deletion of exon 47 augments effects of the IT mutation on voltage-dependent gating of Cav1.4

      We next investigated how deletion of exon 47 affects the impact of the IT mutation (Fig. 3A). As for Cav1.4+ex47 (Fig. 1C), IT caused a negative shift in Vh for Cav1.4Δex47 (Fig. 3, B and C, Table 1). The net hyperpolarizing effect of IT (ΔVh) was not significantly different between Cav1.4+ex47 (median ΔVh= 19.9 mV, n = 11) and Cav1.4Δex47 (median ΔVh= 18.9 mV, n = 8; Mann-Whitney U = 44, p > 0.999). However, the additive effects of the IT mutation and deletion of exon 47 resulted in an extremely negative activation threshold of Cav1.4Δex47IT (∼ −70 mV, Fig. 3C). Moreover, IT enhanced rather than weakened the voltage-dependence of τact in the absence of exon 47 (v = −21.4 mV for Cav1.4Δex47IT versus v = −33.4 mV for Cav1.4Δex47; F2,8 = 5.3, p = 0.03; Fig. 4, A and B).
      Figure thumbnail gr3
      Figure 3IT enhances voltage-dependence of activation of Cav1.ex47 and decreases current density. A–C, same as described in the legend to , except for cells transfected with Cav1.4Δex47 or Cav1.4Δex47IT (black and red symbols, respectively in C).
      Figure thumbnail gr4
      Figure 4IT significantly alters the activation and deactivation kinetics of Cav1.4Δex47. A–D, same as described in the legend to except for cells transfected Cav1.4Δex47 or Cav1.4Δex47IT (black and red symbols, respectively in B and D).
      Similar to its effects in the presence of exon 47 (Fig. 2, C and D), IT strengthened the voltage-dependence of τdeact (v = 104.1 mV for Cav1.4Δex47 versus v = 27.6 mV for Cav1.4Δex47IT; F2,22 = 151.6, p < 0.0001; Fig. 4, C and D). However, IT increased τdeact more than 10-fold for Cav1.4Δex47 versus ∼4-fold for Cav1.4+ex47 upon repolarization to −60 mV (Table 2). These results indicate that deletion of exon 47 augments the gain-of-function effects of IT by modifying the kinetics and voltage-dependence of channel activation and deactivation.

      Unique effects of IT on Cav1.4Δex47

      An effect of IT that was not reported previously was a reduction in current density, which was only seen in the absence of exon 47 (Figs. 1C and 3C, Table 1). We first tested the possibility that IT impaired the stability of the channel in ways that diminished overall levels of the Cav1.4Δex47 protein. However, Western blots indicated similar levels of total channel protein in cells transfected with either Cav1.4Δex47 or Cav1.4Δex47IT (Fig. 5A). Moreover, biotinylation and streptavidin pulldown of cell-surface proteins revealed no significant difference in the levels of Cav1.4Δex47 or Cav1.4Δex47IT in the plasma membrane (Fig. 5B). Thus, impaired trafficking of the mutant channels to the cell surface was unlikely to be the major cause of the decrease in current density. A second unexpected effect of IT was an apparent decrease in ion selectivity based on the development of large outward currents at positive voltages and hyperpolarizing shift in the reversal potential (Erev) (Figs. 1C and 3C, Table 1). The outward currents and median change in ErevErev) were significantly larger for Cav1.4Δex47IT (−37.2 mV, n = 8) than for Cav1.4+ex47IT (−16.6 mV, n = 11; Mann-Whitney U = 14, p = 0.01) relative to the corresponding WT channels. Therefore, we probed the underlying mechanism with an emphasis on Cav1.4Δex47.
      Figure thumbnail gr5
      Figure 5IT does not alter the expression levels or cell-surface density of Cav1.4Δex47. A, representative Western blotting images of lysates from HEK293T cells that were untransfected (Control) or transfected with either Cav1.4Δex47 (Δex47) or Cav1.4Δex47IT (Δex47IT) as well as β2X13 and α2δ−4. Blots were probed with antibodies against Cav1.4, Na/K-ATPase, or GAPDH. The percentage of lysates used for total protein (left 3 lanes) and biotinylated cell-surface proteins (right 3 lanes) were 10 and 90%, respectively. B, densitometric analysis of total and cell-surface Cav1.4 protein normalized to those for GAPDH and Na/K-ATPase, respectively. The use of these proteins as normalization controls was justified because there was no effect of transfection on their levels (p = 0.84 for GAPDH and p = 0.99 for Na/K-ATPase, both by analysis of variance). Each point represents result from an independent experiment. Bars represent mean; p values were determined by t test.
      The nature of the outward currents was mysterious considering that the major intracellular cation in our recording solutions was NMDG+ (N-methyl-d-glucamine), a large organic cation that does not permeate most voltage-gated ion channels. However, Cav1.2 and Cav1.3 are permeable to NMDG+ under some conditions (
      • Wang Y.
      • Tang S.
      • Harvey K.E.
      • Salyer A.E.
      • Li T.A.
      • Rantz E.K.
      • Lill M.A.
      • Hockerman G.H.
      Molecular determinants of the differential modulation of Cav1.2 and Cav1.3 by nifedipine and FPL 64176.
      ,
      • Hockerman G.H.
      • Johnson B.D.
      • Scheuer T.
      • Catterall W.A.
      Molecular determinants of high affinity phenylaklyamine block of L-type calcium channels.
      ). If IT enabled NMDG+ efflux through Cav1.4 channels, then the outward currents in cells transfected with Cav1.4Δex47IT should be reduced by known blockers of Cav channels such as Cd2+ (
      • Lansman J.B.
      • Hess P.
      • Tsien R.W.
      Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+: voltage and concentration dependence of calcium entry into the pore.
      ) and by decreasing the chemical gradient of NMDG+ across the membrane. Consistent with these predictions, Cd2+ abolished outward currents in cells transfected with Cav1.4Δex47IT as well as the inward currents in cells expressing either WT or mutant channels (Fig. 6). To test the effects of altering the NMDG+ concentration, we compared Erev using extracellular solutions containing 5 or 130 mm NMDG+ ([NMDG+]5 and [NMDG+]130, respectively, Fig. 7A). Although having no effect on Erev of Cav1.4Δex47 (66.5 ± 2.3 mV with [NMDG+]5, n = 4 versus 68.3 ± 1.4 mV with [NMDG+]130, n = 4, p = 0.532 by t test; Fig. 7, B and C), increasing extracellular NMDG+ caused a positive shift in Erev (31.2 ± 2.7 mV with [NMDG+]5, n = 4 versus 53.1 ± 3.6 mV with [NMDG+]130, n = 3, p = 0.004 by t test) and diminished outward currents in cells expressing Cav1.4Δex47IT (Fig. 7, B and C). In addition, increasing the extracellular [NMDG+] had no effect on the permeability of Ba2+ versus NMDG+ (PBa/PNMDG) for Cav1.4Δex47 (351.4 ± 53.4, n = 4, for [NMDG+]5 versus 391.7 ± 36.9, n = 4, for [NMDG+]130, p = 0.558 by t test) but significantly increased that for Cav1.4Δex47IT (27.4 ± 5.3, n = 4, for [NMDG+]5 versus 133.4 ± 37.0, n = 3, for [NMDG+]130, p = 0.020 by t test). We further assessed the effect of IT on selectivity of Cav1.4Δex47 by measuring Erev and PBa/Px under other bi-ionic conditions. With intracellular solutions containing Na+ or K+, IT caused a negative shift in Erev and lowered PBa/Px (Fig. 8, Table 3). Taken together, these results signified a reduction in the ionic selectivity of Cav1.4Δex47IT compared with WT channels.
      Figure thumbnail gr6
      Figure 6Cd2+ suppresses inward and outward currents in cells transfected with Cav1.4Δex47IT. A and B, representative traces (left) for IBa evoked by 50-ms pulses to the indicated voltages before (black) and after perfusion of extracellular solution containing Cd2+ (red; 100 μm). I-V plots (right) obtained before (Ba2+, black symbols) and after (Cd2+, red symbols) perfusion of Cd2+. Cav1.4 variants were co-expressed with β2X13 and α2δ-4.
      Figure thumbnail gr7
      Figure 7Increasing NMDG+ in the extracellular recording solution minimizes alterations in Erev and outward currents caused by IT mutation. A, schematic showing composition of [NMDG]5 and [NMDG]130 recording solutions. B and C, representative current traces evoked by 50-ms pulses from −100 mV to the indicated voltages (left) and I-V plots (right) in cells transfected with Cav1.4Δex47 (top) or Cav1.4Δex47IT (bottom). I/IMax represents IBa normalized to peak inward current amplitude. Cav1.4 variants were co-expressed with β2X13 and α2δ-4.
      Figure thumbnail gr8
      Figure 8IT impairs selectivity of Cav1.4Δex47. A, representative current traces evoked by 50-ms pulses from −100 mV to the indicated voltages. B, I-V plots in cells transfected with Cav1.4Δex47 or Cav1.4Δex47IT. I/Imax represents IBa normalized to peak inward current amplitude. C, expanded view of I-V plots in B with Erev indicated by arrows for Cav1.4Δex47 (black) or Cav1.4Δex47IT (red). Data were fit by linear regression. Intracellular solution contained 140 mm Na+ (top panels) or K+ (bottom panels). Cav1.4 variants were co-expressed with β2X13 and α2δ-4.
      Table 3Parameters for monovalent and Ba2+ permeability for Cav1.4Δex47 and Cav1.4Δex47IT
      Erev (mV)Cav1.4Δex47Cav1.4Δex47ITp Value
      NMDG+66.5 ± 2.331.2 ± 2.7<0.001
      Na+65.5 ± 1.651.0 ± 2.70.004
      K+60.9 ± 3.450.8 ± 3.10.064
      PBa/Px
      NMDG+351.4 ± 53.431.2 ± 2.70.0009
      Na+316.6 ± 34.6113.8 ± 19.10.0009
      K+243.7 ± 58.8116.2 ± 28.40.074
      Although smaller for Cav1.4+ex47 than for Cav1.4Δex47 (Fig. 1C, Table 1) the effects of IT on Erev were, nevertheless, not reported for Cav1.4+ex47 in a previous study (
      • Hemara-Wahanui A.
      • Berjukow S.
      • Hope C.I.
      • Dearden P.K.
      • Wu S.B.
      • Wilson-Wheeler J.
      • Sharp D.M.
      • Lundon-Treweek P.
      • Clover G.M.
      • Hoda J.C.
      • Striessnig J.
      • Marksteiner R.
      • Hering S.
      • Maw M.A.
      A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.
      ). A key difference was in the choice of auxiliary subunits (β2X13 and α2δ-4, this study) versus β3 or β2A and α2δ-1 (
      • Hemara-Wahanui A.
      • Berjukow S.
      • Hope C.I.
      • Dearden P.K.
      • Wu S.B.
      • Wilson-Wheeler J.
      • Sharp D.M.
      • Lundon-Treweek P.
      • Clover G.M.
      • Hoda J.C.
      • Striessnig J.
      • Marksteiner R.
      • Hering S.
      • Maw M.A.
      A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.
      )). Therefore, we tested the impact of IT on the Cav1.4 variants containing β2A and α2δ-1. Consistent with the previous study, IT caused a large negative shift in Vh in these experiments. Although the mutation strongly reduced current densities of Cav1.4Δex47 + β2A + α2δ-1, IT did not affect Erev (Fig. 9, A–C, Table 1). Thus, the identity of the auxiliary β and α2δ subunits critically determines the effects of IT on selectivity of Cav1.4.
      Figure thumbnail gr9
      Figure 9IT does not alter selectivity in Cav1.4 channels containing β2Aδ and α2δ-1. A, schematics of Cav1.4 (left). Representative current traces evoked by 50-ms pulses from −100 mV to the indicated voltages (right). B, I-V plots in cells transfected with Cav1.4+ex47, Cav1.4+ex47IT, Cav1.4Δex47, or Cav1.4Δex47IT. IBa (pA/pF) represents IBa normalized to peak inward current amplitude. C, expanded view of I-V plot in B. Data were fit by linear regression. Extracellular and intracellular solutions were the same as those used in Figure 2, Figure 3.

      Discussion

      Our study provides new insights about how IT affects the biophysical properties of Cav1.4. First, we show that IT produces a large negative shift in voltage-dependent activation of Cav1.4 channels containing the major auxiliary Cav subunits in the retina, β2X13 and α2δ-4 (Figure 1, Figure 2, Figure 3, Table 1), as well as Cav1.4 channels comprised of other auxiliary subunits (Fig. 9, Table 1, and see Ref.
      • Hemara-Wahanui A.
      • Berjukow S.
      • Hope C.I.
      • Dearden P.K.
      • Wu S.B.
      • Wilson-Wheeler J.
      • Sharp D.M.
      • Lundon-Treweek P.
      • Clover G.M.
      • Hoda J.C.
      • Striessnig J.
      • Marksteiner R.
      • Hering S.
      • Maw M.A.
      A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.
      ). Second, deletion of exon 47 exacerbates the gain of function effects of IT: Cav1.4Δex47IT activates at more negative voltages and exhibits stronger voltage-dependent alterations in the kinetics of activation and deactivation than Cav1.4+ex47IT (Figure 1, Figure 2, Figure 3, Figure 4, Table 1, Table 2). Third, IT weakens the selectivity of Cav1.4 for Ba2+ in a manner that varies with the identity of the auxiliary β and α2δ subunits (Figs. 1, 3, and 9, Table 1, Table 2, Table 3). Our findings highlight the importance of splice variation and auxiliary subunit composition as potential modifiers of disease-causing mutations affecting Cav channels.

      Conserved role of Ile-745 in activation gating

      The S5 and S6 pore-lining helices give rise to the selectivity filter (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Qian X.
      • Lu S.
      • Dong M.
      • Zhou Q.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
      ), with the four S6 helices (IS6–IVS6) converging at the intracellular side of the membrane in the closed state of the channel (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Qian X.
      • Lu S.
      • Dong M.
      • Zhou Q.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
      ). Ile-745 of Cav1.4 corresponds to Ile-781 in IIS6 of Cav1.2, which lies in a cluster of hydrophobic residues (Leu-779–Ala-782, LAIA) in the S6 bundle-crossing region that are conserved among Cav1 and Cav2 channels (
      • Hering S.
      • Zangerl-Plessl E.M.
      • Beyl S.
      • Hohaus A.
      • Andranovits S.
      • Timin E.N.
      Calcium channel gating.
      ). Disruptive mutations of these residues also cause hyperpolarizing shifts in activation and slowing of deactivation of Cav1.2 and Cav2.3 (
      • Hohaus A.
      • Beyl S.
      • Kudrnac M.
      • Berjukow S.
      • Timin E.N.
      • Marksteiner R.
      • Maw M.A.
      • Hering S.
      Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder.
      ,
      • Raybaud A.
      • Baspinar E.E.
      • Dionne F.
      • Dodier Y.
      • Sauve R.
      • Parent L.
      The role of distal S6 hydrophobic residues in the voltage-dependent gating of CaV2.3 channels.
      ). Our study is the first to show that the IT mutation causes similar effects on Cav1.4. Based on the correlation of their hydrophobicity and the negative shift in Vh (
      • Hohaus A.
      • Beyl S.
      • Kudrnac M.
      • Berjukow S.
      • Timin E.N.
      • Marksteiner R.
      • Maw M.A.
      • Hering S.
      Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder.
      ,
      • Raybaud A.
      • Baspinar E.E.
      • Dionne F.
      • Dodier Y.
      • Sauve R.
      • Parent L.
      The role of distal S6 hydrophobic residues in the voltage-dependent gating of CaV2.3 channels.
      ), the distal S6 residues are likely buried within a hydrophobic environment in the closed channel and become exposed to the aqueous milieu upon pore opening. By analogy to the model of Cav1.2 (
      • Hering S.
      • Beyl S.
      • Stary A.
      • Kudrnac M.
      • Hohaus A.
      • Guy H.R.
      • Timin E.
      Pore stability and gating in voltage-activated calcium channels.
      ), contacts between Ile-745 with a corresponding hydrophobic residue in IIIS6 may stabilize helix-helix interactions, which support the closed conformation in Cav1.4, and are disrupted by the IT mutation.

      Functions of exon 47 in regulating the impact of IT on Cav1.4 activation

      Exon 47 encodes the initial 47 amino acids of the CTM, a modular domain present in both Cav1.4 and Cav1.3 that interacts with a region in the proximal C-terminal domain (
      • Singh A.
      • Hamedinger D.
      • Hoda J.C.
      • Gebhart M.
      • Koschak A.
      • Romanin C.
      • Striessnig J.
      C-terminal modulator controls Ca2+-dependent gating of Cav1.4 L-type Ca2+ channels.
      ,
      • Wahl-Schott C.
      • Baumann L.
      • Cuny H.
      • Eckert C.
      • Griessmeier K.
      • Biel M.
      Switching off calcium-dependent inactivation in L-type calcium channels by an autoinhibitory domain.
      ,
      • Singh A.
      • Gebhart M.
      • Fritsch R.
      • Sinnegger-Brauns M.J.
      • Poggiani C.
      • Hoda J.C.
      • Engel J.
      • Romanin C.
      • Striessnig J.
      • Koschak A.
      Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain.
      ). The CTM nearly abolishes CDI of Cav1.4 by competing with calmodulin (CaM) for binding to the channel (
      • Singh A.
      • Hamedinger D.
      • Hoda J.C.
      • Gebhart M.
      • Koschak A.
      • Romanin C.
      • Striessnig J.
      C-terminal modulator controls Ca2+-dependent gating of Cav1.4 L-type Ca2+ channels.
      ,
      • Wahl-Schott C.
      • Baumann L.
      • Cuny H.
      • Eckert C.
      • Griessmeier K.
      • Biel M.
      Switching off calcium-dependent inactivation in L-type calcium channels by an autoinhibitory domain.
      ). Deletion of the CTM enables CDI by allowing CaM binding to the channel, but also causes a negative shift in Vh (
      • Singh A.
      • Hamedinger D.
      • Hoda J.C.
      • Gebhart M.
      • Koschak A.
      • Romanin C.
      • Striessnig J.
      C-terminal modulator controls Ca2+-dependent gating of Cav1.4 L-type Ca2+ channels.
      ). In Cav1.4, exon 47 is critical for the modulatory function of the CTM in that Cav1.4Δex47 exhibits similar alterations in Vh and CDI as those caused by deletion of the entire CTM (
      • Haeseleer F.
      • Williams B.
      • Lee A.
      Characterization of C-terminal splice variants of Cav1.4 Ca2+ channels in human retina.
      ,
      • Williams B.
      • Haeseleer F.
      • Lee A.
      Splicing of an automodulatory domain in Cav1.4 Ca(2+) channels confers distinct regulation by calmodulin.
      ). Our findings that IT and deletion of exon 47 are additive with respect to hyperpolarizing Vh (Table 1) suggest distinct mechanisms by which Ile-745 and the CTM facilitate activation. In Cav1.3, deletion of the CTM leads to stronger pairing of voltage sensor charge movement and channel opening (
      • Lieb A.
      • Ortner N.
      • Striessnig J.
      C-terminal modulatory domain controls coupling of voltage-sensing to pore opening in Cav1.3 L-type Ca2+ channels.
      ). In Cav2.3, the IIS4-S5 loop and the cytoplasmic end of IIS6 are thought to functionally interact in the activation pathway (see Ref.
      • Wall-Lacelle S.
      • Hossain M.I.
      • Sauve R.
      • Blunck R.
      • Parent L.
      Double mutant cycle analysis identified a critical leucine residue in the IIS4S5 linker for the activation of the Ca(V)2.3 calcium channel.
      ). In Cav1.4, partial deletion of exon 47 might disinhibit such intramolecular interactions, allowing IT to more freely destabilize closed channels and promote channel opening at more negative voltages than in channels with a complete CTM. Interactions of S4–S5 with S6 have been studied by homology modeling and molecular dynamics simulations of Kv channels (
      • Labro A.J.
      • Raes A.L.
      • Grottesi A.
      • Van Hoorick D.
      • Sansom M.S.
      • Snyders D.J.
      Kv channel gating requires a compatible S4-S5 linker and bottom part of S6, constrained by non-interacting residues.
      ). Similar approaches would be useful in dissecting the relationships of the corresponding regions, and of the CTM, with respect to activation gating of Cav1.4.
      The effect of IT on hyperpolarizing Vh, whereas decreasing the peak current density of Cav1.4Δex47 (Table 1), parallels the effect of the S218L migraine-causing mutation in Cav2.1 expressed in HEK293 cells. In the latter case, the reduction in current density was determined to be an artifact of overexpression and related to a reduction in the number of functional channels in the membrane rather than changes in unitary current amplitudes (
      • Tottene A.
      • Pivotto F.
      • Fellin T.
      • Cesetti T.
      • van den Maagdenberg A.M.
      • Pietrobon D.
      Specific kinetic alterations of human CaV2.1 calcium channels produced by mutation S218L causing familial hemiplegic migraine and delayed cerebral edema and coma after minor head trauma.
      ). Because IT did not affect the total or cell-surface levels of Cav1.4Δex47 protein (Fig. 5), the reduced current density of Cav1.4Δex47IT could result from a decrease in single channel conductance, and/or the functionality of the mutant channels within the membrane. Alternatively, the extremely negative activation properties of Cav1.4Δex47IT could have compromised cell health such that outward leak currents compromised IBa amplitudes and caused the negative shift in Erev. This scenario seems unlikely given that IT reduced current density but did not produce outward currents or alterations in Erev in Cav1.4Δex47 channels containing β2A and α2δ-1 (Fig. 9, Table 1). Single channel recordings will be necessary to fully uncover the impact of IT on the elementary properties of Cav1.4Δex47.

      Effects of IT on the ion selectivity of Cav1.4Δex47

      The exquisite selectivity of Cav channels is largely determined by Ca2+ binding with high affinity to the selectivity filter (
      • Ellinor P.T.
      • Yang J.
      • Sather W.A.
      • Zhang J.F.
      • Tsien R.W.
      Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions.
      ,
      • Yang J.
      • Ellinor P.T.
      • Sather W.A.
      • Zhang J.F.
      • Tsien R.W.
      Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels.
      ). Thus, the increased permeability of Na+, K+, and particularly NMDG+ caused by a mutation outside of the selectivity filter was unexpected. However, in the absence of Ca2+, Na+ and large organic cations such as tetramethylammonium are capable of permeating Cav1 channels (
      • McCleskey E.W.
      • Almers W.
      The Ca channel in skeletal muscle is a large pore.
      ). These results suggest that the pore of Cav channels is at least 6 Å in diameter, an interpretation that has been verified in structural analyses (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Qian X.
      • Lu S.
      • Dong M.
      • Zhou Q.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
      ,
      • Tang L.
      • Gamal El-Din T.M.
      • Payandeh J.
      • Martinez G.Q.
      • Heard T.M.
      • Scheuer T.
      • Zheng N.
      • Catterall W.A.
      Structural basis for Ca2+ selectivity of a voltage-gated calcium channel.
      ). Indeed, despite being a relatively large cation (∼6.4 Å wide × 12 Å long; ∼7.3 Å mean diameter (
      • Villarroel A.
      • Burnashev N.
      • Sakmann B.
      Dimensions of the narrow portion of a recombinant NMDA receptor channel.
      )), NMDG+ can permeate Cav1.2 channels containing pore mutations (
      • Hockerman G.H.
      • Johnson B.D.
      • Abbott M.R.
      • Scheuer T.
      • Catterall W.A.
      Molecular determinants of high affinity phenylalkylamine block of L-type calcium channels in transmembrane segment IIIS6 and the pore region of the alpha1 subunit.
      ) and Cav1.3 channels exposed to the dihydropyridine agonist FPL 64176 (FPL) (
      • Wang Y.
      • Tang S.
      • Harvey K.E.
      • Salyer A.E.
      • Li T.A.
      • Rantz E.K.
      • Lill M.A.
      • Hockerman G.H.
      Molecular determinants of the differential modulation of Cav1.2 and Cav1.3 by nifedipine and FPL 64176.
      ). Functional interactions between the selectivity filter and the inner S6 helix bundle are involved in Kv channel gating transitions (
      • Cuello L.G.
      • Cortes D.M.
      • Perozo E.
      The gating cycle of a K+ channel at atomic resolution.
      ) and may be conserved among Cav channels. For example, CaM binding to the cytoplasmic domain promotes conformational changes in the selectivity filter of Cav1 channels that lead to CDI (
      • Abderemane-Ali F.
      • Findeisen F.
      • Rossen N.D.
      • Minor Jr., D.L.
      A selectivity filter gate controls voltage-gated calcium channel calcium-dependent inactivation.
      ). Thus, IT could alter positioning of IIS6 and its contributions to the Ca2+ (or Ba2+) binding affinity within the selectivity filter, allowing monovalent ions including NMDG+ and Na+ to permeate even in the presence of significant extracellular concentrations of Ba2+.
      Our findings that impaired selectivity was specific to IT mutant channels containing β2X13 and α2δ-4 explain why previous analyses did not uncover any alteration in selectivity in these channels containing β2A and α2δ-1 (
      • Hemara-Wahanui A.
      • Berjukow S.
      • Hope C.I.
      • Dearden P.K.
      • Wu S.B.
      • Wilson-Wheeler J.
      • Sharp D.M.
      • Lundon-Treweek P.
      • Clover G.M.
      • Hoda J.C.
      • Striessnig J.
      • Marksteiner R.
      • Hering S.
      • Maw M.A.
      A CACNA1F mutation identified in an X-linked retinal disorder shifts the voltage dependence of Cav1.4 channel activation.
      ). Unlike β2A, β2X13 lacks exon 7B, which causes increased voltage-dependent inactivation of Cav1.4 (
      • Lee A.
      • Wang S.
      • Williams B.
      • Hagen J.
      • Scheetz T.E.
      • Haeseleer F.
      Characterization of Cav1.4 complexes (α11.4, β2, and α2δ−4) in HEK293T cells and in the retina.
      ). Although it is unclear how this difference could affect ion selectivity of the IT mutant channels, there is evidence that structural alterations in α2δ could affect the permeation properties of Cav channels. For example, CACHD1 is an α2δ-like protein that has a disrupted metal-ion adhesion site that is critical for structural and functional interactions of α2δ with the channel (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Qian X.
      • Lu S.
      • Dong M.
      • Zhou Q.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
      ,
      • Canti C.
      • Nieto-Rostro M.
      • Foucault I.
      • Heblich F.
      • Wratten J.
      • Richards M.W.
      • Hendrich J.
      • Douglas L.
      • Page K.M.
      • Davies A.
      • Dolphin A.C.
      The metal-ion-dependent adhesion site in the Von Willebrand factor: a domain of α2δ subunits is key to trafficking voltage-gated Ca2+ channels.
      ,
      • Cassidy J.S.
      • Ferron L.
      • Kadurin I.
      • Pratt W.S.
      • Dolphin A.C.
      Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α2δ-1 subunits.
      ). When co-expressed with Cav2.2, CACHD1 impairs the ion selectivity of Cav2.2 (
      • Dahimene S.
      • Page K.M.
      • Kadurin I.
      • Ferron L.
      • Ho D.Y.
      • Powell G.T.
      • Pratt W.S.
      • Wilson S.W.
      • Dolphin A.C.
      The α2δ-like protein Cachd1 increases N-type calcium currents and cell surface expression and competes with α2δ-1.
      ). In the cryo-EM structure, α2δ-1 forms multiple extracellular contacts with Cav1.1 including the extended loops between S5 and P1 helices in domains II and III (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Qian X.
      • Lu S.
      • Dong M.
      • Zhou Q.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
      ). The L5 loops of each of the 4 domains form a domed window above the selectivity filter that direct Ca2+ ions into the pore (
      • Wu J.
      • Yan Z.
      • Li Z.
      • Qian X.
      • Lu S.
      • Dong M.
      • Zhou Q.
      • Yan N.
      Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
      ). Differences in how α2δ variants may interact with these extracellular sites, in concert with those produced by β subunits at intracellular sites, could determine the impact of IT on selectivity in the context of Cav1.4.

      Significance for visual phenotypes of Cav1.4 channelopathies

      CSNB2 is a nonprogressive retinal disorder with variable clinical features including reduced visual acuity, myopia, and nystagmus (
      • Boycott K.M.
      • Pearce W.G.
      • Bech-Hansen N.T.
      Clinical variability among patients with incomplete X-linked congenital stationary night blindness and a founder mutation in CACNA1F.
      ). A hallmark feature of this disorder is a reduced b-wave in electroretinograms, which is consistent with a defect in transmission from photoreceptors to second-order bipolar neurons (
      • Boycott K.M.
      • Pearce W.G.
      • Bech-Hansen N.T.
      Clinical variability among patients with incomplete X-linked congenital stationary night blindness and a founder mutation in CACNA1F.
      ,
      • Miyake Y.
      • Yagasaki K.
      • Horiguchi M.
      • Kawase Y.
      • Kanda T.
      Congenital stationary night blindness with negative electroretinogram: a new classification.
      ). Of the numerous CSNB2 mutations affecting Cacna1f, the IT mutation causes the most severe form of visual impairment (
      • Hope C.I.
      • Sharp D.M.
      • Hemara-Wahanui A.
      • Sissingh J.I.
      • Lundon P.
      • Mitchell E.A.
      • Maw M.A.
      • Clover G.M.
      Clinical manifestations of a unique X-linked retinal disorder in a large New Zealand family with a novel mutation in CACNA1F, the gene responsible for CSNB2.
      ). Despite the reduced current density of Cav1.4Δex47IT in our experiments, the mutation enabled significant inward IBa at voltages negative to the activation thresholds of WT channels (Fig. 3C). Due to charge screening effects (
      • Frankenhaeuser B.
      • Hodgkin A.L.
      The action of calcium on the electrical properties of squid axons.
      ), our use of 20 mm Ba2+ in the external recording solutions would cause activation voltages ∼20 mV more positive than those expected in the retina; however, the relative differences in the voltage-dependent properties of the WT and IT mutant channels should be preserved under our recording conditions. Even in the presence of reduced current density, the negative shift in Vh and slow deactivation of Cav1.4Δex47IT would lead to aberrant Ca2+ influx during light-dependent hyperpolarization of photoreceptors, thus degrading the fidelity of visual transmission to second-order neurons. However, our study also raises the possibility that the aberrant conductance of monovalent cations by Cav1.4Δex47IT could lead to alterations in the excitability of photoreceptors that could lead to degenerative changes. Photoreceptor degeneration, as well as altered retinal ganglion cell activity and morphological and functional defects in photoreceptor synapses, are characteristic of an IT knock-in mouse line (
      • Regus-Leidig H.
      • Atorf J.
      • Feigenspan A.
      • Kremers J.
      • Maw M.A.
      • Brandstätter J.H.
      Photoreceptor degeneration in two mouse models for congenital stationary night blindness type 2.
      ,
      • Liu X.
      • Kerov V.
      • Haeseleer F.
      • Majumder A.
      • Artemyev N.
      • Baker S.A.
      • Lee A.
      Dysregulation of Cav1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2.
      ,
      • Knoflach D.
      • Kerov V.
      • Sartori S.B.
      • Obermair G.J.
      • Schmuckermair C.
      • Liu X.
      • Sothilingam V.
      • Garcia Garrido M.
      • Baker S.A.
      • Glosmann M.
      • Schicker K.
      • Seeliger M.
      • Lee A.
      • Koschak A.
      Cav1.4 IT mouse as model for vision impairment in human congenital stationary night blindness type 2.
      ,
      • Knoflach D.
      • Schicker K.
      • Glösmann M.
      • Koschak A.
      Gain-of-function nature of Cav1.4 L-type calcium channels alters firing properties of mouse retinal ganglion cells.
      ). However, Cav1.4 splice variants lacking exon 47, although abundant in human and monkey retina, are conspicuously absent from mouse retina (
      • Haeseleer F.
      • Williams B.
      • Lee A.
      Characterization of C-terminal splice variants of Cav1.4 Ca2+ channels in human retina.
      ). An understanding of the pathological consequences of Cav1.4Δex47IT could therefore benefit from analyses of the mutant channels in human stem-cell derived photoreceptors in the context of retinal organoids (
      • Clevers H.
      Modeling development and disease with organoids.
      ).

      Experimental procedures

      cDNAs and molecular biology

      The following cDNAs were used: Cav1.4 (GenBank AF201304), β2A (GenBank AF465485), β2X13 (GenBank NM_053851), α2δ-1 (GenBank M86621), and α2δ-4 (GenBank NM_172364) in pcDNA3.1. The construct encoding Cav1.4Δex47 was described previously (
      • Haeseleer F.
      • Williams B.
      • Lee A.
      Characterization of C-terminal splice variants of Cav1.4 Ca2+ channels in human retina.
      ). To incorporate the IT mutation into Cav1.4 (Cav1.4+ex47IT and Cav1.4Δex47IT), the upstream and downstream cDNA regions flanking the codon corresponding to I756 were amplified with Q5 High-Fidelity DNA polymerase (New England Biolabs) using Cav1.4+ex47 as the template and primers incorporating the mutation. PCR products were digested with DpnI, column purified, and cloned into Cav1.4+ex47 and Cav1.4Δex47 between AgeI and ClaI with the NEBuilder HiFi DNA Assembly kit (New England Biolabs) following the manufacturer's protocol. All constructs were verified by DNA sequencing before use.

      Cell culture and transfection

      Human embryonic kidney (HEK) 293 cells transformed with SV40 T antigen (HEK293T, CRL-3216, RRID:CVCL_0063; ATCC) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Grand Island, NY) with 10% fetal bovine serum (Atlantic Biologicals) at 37 °C in 5% CO2. Cells were not used after they were passaged 15 times. At 70–80% confluence, the cells were co-transfected with cDNAs encoding human Cav1.4 α1 (1.8 μg; Cav1.4+ex47, Cav1.4+ex47IT, Cav1.4Δex47, or Cav1.4Δex47IT), β2A or β2X13 (0.6 μg), α2δ-4 or α2δ-1 (0.6 μg), and enhanced GFP in pEGFP-C1 (0.1 μg) using FuGENE 6 transfection reagent (Promega) according to the manufacturer's protocol. In some experiments, cells were co-transfected with a plasmid encoding SK-1 Ca2+-activated K+ channel (0.1 μg) in an effort to reduce toxicity (there were no differences in results obtained in cells transfected with or without SK-1 and thus data were combined). Cells treated with the transfection mixture were incubated at 37 °C for 24 h. After 24 h, cells were incubated at 30 °C for at least 24 h prior to whole-cell patch clamp recordings.
      For Western blotting and cell-surface biotinylation assays, HEK293T cells were transfected using Lipofectamine 3000 reagent (Life Technologies). Plasmid DNA (Cav1.4Δex47 or Cav1.4Δex47IT (1.8 μg), β2X13, and α2δ-4 (0.6 μg each)) was diluted in Opti-MEM (50 μl, Life Technologies) and 4 μl of P3000 reagent. This was added to a mixture of Opti-MEM (50 μl) and Lipofectamine 3000 reagent (3 μl) and incubated for 10 min at room temperature. The DNA mixture was added incubated with the cells for 24 h at 37 °C in 5% CO2 after which the cell culture medium was replaced with fresh medium.

      Electrophysiology

      Whole-cell patch clamp recordings were performed at room temperature between 48 and 72 h after transfection. Data were obtained under voltage-clamp with an EPC-9 patch clamp amplifier operated by Patchmaster software (HEKA Elektronik). The composition of recording solutions contained as follows (in mm): for Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, external solution contained NMDG (140), BaCl2 (20), and MgCl2 (1); internal solution contained NMDG (140), HEPES (10), MgCl2 (2), Mg-ATP (2), and EGTA (5). For Fig. 6, Cd2+ (100 μm) was added to the external solution; pH was adjusted to 7.3 with methanesulfonic acid. For Fig. 7, the external solution contained Tris (130), NMDG (5 or 130), and BaCl2 (20); internal solution contained NMDG (140), EGTA (10), HEPES (5), Tris (5); pH was adjusted to 7.3 with methanesulfonic acid. For Fig. 8, the external solution contained TEA-Cl (130), BaCl2 (20), HEPES (5), pH 7.3, with TEA-OH); internal solution contained KCl or NaCl (140 mm), EGTA (5), HEPES (5), Tris (5), pH 7.3, with KOH or NaOH. Pipette resistances were typically 2-6 megaohms in the bath solution, and series resistance compensated up to 70%. Leak subtraction was conducted using a P/-4 protocol.
      To measure current density, IBa was evoked by 50-ms pulses from a holding voltage of −100 mV to various voltages and normalized to the cell capacitance. I-V data were fitted with the Boltzmann equation: I = Gmax × (Vm – Erev)/(1 + exp(Vh – Vm)/k, where I is the measured current at each test voltage (Vm), Vh is the voltage of half-maximal activation, k is the slope factor, and Gmax is the maximal conductance. Peak current density was determined by dividing the maximal IBa by the cell capacitance. Kinetic parameters for IBa activation (τact) and deactivation (τdeact) were obtained by fitting the test current and tail current, respectively, with a single exponential function (y0 + A (exp(−t/τ)), where y0 is the offset (asymptote), t is time, τ is the time constant, and A is the amplitude. The voltage-dependence of τact and τdeact was described by: y0 + A (exp(−v/v)), where y0 is the asymptote, v is voltage, v is the voltage constant, and A is the amplitude. Relative permeability of Ba2+ versus different monovalent cations (x) was calculated as: PBa/Px = [x]i/4[Ba2+]o × exp (ErevF/RT){1 + exp (ErevF/RT)}. Data were analyzed offline with Igor Pro (Wavemetrics) or Origin Pro (OriginLab Corporation) software. Statistical analysis and preparation of graphs were performed using GraphPad Prism software. The data were initially analyzed for normality using the Shapiro–Wilk or D'Agostino-Pearson omnibus test. For parametric data, significant differences were determined by Student's t test. For nonparametric data, Mann-Whitney test was used. Significant differences in the curve fits of τact and τdeact versus voltage relationships were determined by F tests. Data were incorporated into figures using GraphPad and Adobe Illustrator software. Unless otherwise indicated, averaged data represent mean ± S.E. from at least 3 independent transfections.

      Biochemical analysis of cell-surface CaV1.4 protein

      Transfected HEK293T cells were subject to cell-surface biotinylation and Western blotting as described previously (
      • Garza-Lopez E.
      • Lopez J.A.
      • Hagen J.
      • Sheffer R.
      • Meiner V.
      • Lee A.
      Role of a conserved glutamine in the function of voltage-gated Ca2+ channels revealed by a mutation in human CACNA1D.
      ). Cell-surface proteins were biotinylated according to the manufacturer's protocol. Briefly, cells were washed with ice-cold PBS (PBS, in mm: 2.5 KCl, 136 NaCl, 1.5 KH2PO4-Na2HPO4 6.5, pH 7.4), prior to incubation with sulfo-NHS-SS-biotin (Thermo Scientific) for 30 min at 4 °C. The cells were then incubated with biotin quenching solution (in mm: 50 glycine, 2.5 CaCl2, 1 MgCl2, pH 7.4), scraped off the plate in PBS, pelleted by centrifugation, and resuspended in lysis buffer containing in mm: 150 NaCl, 25 Tris–HCl, pH 7.6, with 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, and 0.5 phenylmethylsulfonyl fluoride and other protease inhibitors. After 10 min on ice, cell lysates were subject to centrifugation (16,000 × g for 10 min at 4 °C) and biotinylated proteins recovered with NeutrAvidin gel. The bound proteins were eluted in SDS-PAGE sample buffer (in mm: 58 Tris-Cl, 50 DTT, with 1.7% SDS, 5% glycerol, 0.002% bromphenol blue, pH 6.8) and subject to electrophoresis using NovexTM WedgeWellTM 4–20% Tris glycine gel (Invitrogen) and transfer to nitrocellulose blotting membranes.
      For Western blotting, the membranes were incubated in blocking buffer containing milk (5%) in TBS-T (100 mm Tris–HCl, 0.15 m NaCl, 0.05% Tween 20) followed by incubation with the following antibodies diluted in blocking buffer: CaV1.4 (1:4,000 (
      • Liu X.
      • Kerov V.
      • Haeseleer F.
      • Majumder A.
      • Artemyev N.
      • Baker S.A.
      • Lee A.
      Dysregulation of Cav1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2.
      )); Na+/K+ ATPase (1:700, Developmental Studies Hybridoma Bank, University of Iowa, RRID:AB_2314847), GAPDH (1:10,000; Cell Signaling catalog number 14C10). Horseradish peroxidase (HRP)-conjugated secondary antibodies used were anti-rabbit HRP (1:3000; GE Healthcare catalog number NA934-1ML) and anti-mouse HRP (1:3000; GE Healthcare catalog number NA931V) followed by chemiluminescent detection (Thermo Scientific; SuperSignal West Pico catalog number 34080). The Western blotting signals were visualized with the Odyssey Fc Imaging System (LI-COR). The results shown were obtained from at least 3 independent experiments. Densitometric analysis was performed with Image Studio Lite software (LI-COR).

      Data availability

      All data relevant to this work are contained within this manuscript or available upon request.

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