Functional impact of a congenital stationary night blindness type 2 mutation depends on subunit composition of Ca v 1.4 Ca 2 1 channels

Voltage-gated Ca v 1 and Ca v 2 Ca 2 1 channels are comprised of a pore-forming a 1 subunit (Ca v 1.1-1.4, Ca v 2.1-2.3) and auxiliary b ( b 1-4 ) and a 2 d ( a 2 d2 1 2 4) subunits. The properties of these channels vary with distinct combinations of Ca v subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Ca v subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Ca v 1.4 channels typical of those in human retina: Ca v 1.4 splice variants with or without exon 47 (Ca v 1.4 1 ex47 and Ca v 1.4 D ex47, respectively), and the auxiliary subunits, b 2X13 and a 2 d -4. We find that IT caused both Ca v 1.4 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 b 2X13 and a 2 d -4 but not in those with b 2A and a 2 d -1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Ca v 1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of b 2X13 and a 2 d -4. Our results reveal complex actions of IT in modifying the properties of Ca v 1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors. analysis; L. investigation; methodology; L. writing-origi-nal draft; J. J. and L. writing-review and pro-ject

Voltage-gated Ca v 1 and Ca v 2 Ca 21 channels are comprised of a pore-forming a 1 subunit (Ca v 1.1-1.4,Ca v 2.1-2.3) and auxiliary b (b 1-4 ) and a 2 d (a 2 d2124) subunits.The properties of these channels vary with distinct combinations of Ca v subunits and alternative splicing of the encoding transcripts.Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Ca v subunits and splice variants.Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Ca v 1.4 channels typical of those in human retina: Ca v 1.4 splice variants with or without exon 47 (Ca v 1.41ex47 and Ca v 1.4Dex47, respectively), and the auxiliary subunits, b 2X13 and a 2 d-4.We find that IT caused both Ca v 1.4 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 b 2X13 and a 2 d-4 but not in those with b 2A and a 2 d-1.We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Ca v 1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of b 2X13 and a 2 d-4.Our results reveal complex actions of IT in modifying the properties of Ca v 1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.
Voltage-gated Ca 21 (Ca v ) channels are comprised of a poreforming a 1 subunit and two auxiliary subunits, b and a 2 d (reviewed in Ref. 1).The a 1 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 (2).In contrast to the diverse complement of Ca v channels expressed in many neurons, the pore-forming a 1F subunit (referred to as Ca v 1.4 from here on), encoded by the Cacna1f gene, appears to be the major Ca v subtype localized in the syn-aptic terminals of photoreceptors in the retina (3,4) where it co-assembles with b 2 and a 2 d-4 subunits (5).Within photoreceptor synaptic terminals, Ca v 1.4 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 Ca v 1.4-dependent glutamate release by light stimuli enables disinhibition of a nonselective cation channel, initiating excitation of the ON pathway in the retina (6,7).Thus, the voltage-dependent properties of Ca v 1.4 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. 8).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 Ca v 1.4 (9)(10)(11)(12)(13).Understanding the pathological consequences of CSNB2 mutations is complicated by the functional diversity of retinal Ca v 1.4 conferred in part by alternative splicing of the pre-mRNAs corresponding to each subunit (5,(14)(15)(16).The b 2 variant that is most highly expressed in human retina contains an alternatively spliced exon 7B (b 2X13 ) and causes stronger voltage-dependent inactivation of Ca v 1.4 than b 2 variants with exon 7A (b 2A ) (5).The Ca v 1.4 a 1 pre-mRNA also undergoes alternative splicing, particularly in the sequence encoding the large cytoplasmic C-terminal domain (15,16).We previously characterized a Ca v 1.4 variant lacking exon 47 (Ca v 1.4Dex47) that is highly expressed in human retina (16).Exon 47 encodes a portion of a C-terminal modulatory domain (CTM) in Ca v 1.4 that suppresses Ca 21 -dependent inactivation (CDI) and causes depolarizing shifts in the voltage-dependence of activation (10,17).When expressed in a human embryonic kidney cell line (HEK293T), Ca v 1.4Dex47 exhibits more negative activation thresholds and stronger CDI than Ca v 1.4 variants containing exon 47 (Ca v 1.41ex47) (16,18).
Studies investigating the electrophysiological consequences of Cacna1f mutations have focused on the Ca v 1.41ex47 variant coexpressed with auxiliary subunits other than b 2X13 and a 2 d-4 (9-13).Alternative splicing can affect the severity of disease-causing mutations in Ca v channel genes (19).Thus, analysis of Cacna1f mutations in the context of Ca v 1.4 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 Ca v 1.41ex47 and Ca v 1.4Dex47 channels containing b 2X13 and a 2 d-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 Ca v 1.41ex47 coexpressed with a 2 d-1 and b 3 or b 2 , the IT mutation causes a large hyperpolarizing shift (.30 mV) in the voltage-dependence of activation (12).Our results indicate that, when coexpressed with b 2X13 and a 2 d-4, Ca v 1.41ex47 channels bearing the IT mutation (Ca v 1.41ex47 IT ) show hyperpolarized activation voltages compared with wild-type (WT) channels.The gain-of function effect is more severe for Ca v 1.4Dex47 channels with the IT mutation (Ca v 1.4Dex47 IT ), which showed more negative activation thresholds and slower deactivation kinetics than Ca v 1.41ex47 IT .An unexpected finding is that IT alters the ion selectivity of both Ca v 1.4 splice variants in a manner that varies with the identity of the a 2 d subunit.Our findings highlight the importance of splice variation and auxiliary subunit composition as potential modifiers of disease-causing mutations affecting Ca v channels.

IT mutation enhances activation and slows deactivation of Ca v 1.41ex47
Exon 47 resides in the CTM of Ca v 1.4 (Fig. 1A); deletion of this exon, like the IT mutation, causes a large negative shift in the voltage dependence of channel activation (16,18).Thus, the effect of IT on Ca v 1.4 activation could be additive, or alternatively, could be occluded by exon 47 deletion.To distinguish between these possibilities, we compared the activation properties of Ba 21 currents (I Ba ) mediated by Ca v 1.41ex47 and Ca v 1.4Dex47, and the corresponding IT mutant channels, in transfected HEK293T cells.Ba 21 rather than Ca 21 was used as the charge carrier to minimize the complicating effects of CDI which, whereas negligible in Ca v 1.41ex47, is prominent in Ca v 1.4Dex47 (18).Because previous analyses of Ca v 1.41ex47 IT were performed primarily with b 3 or b 2a , and a 2 d-1 (12), we first characterized the effect of IT on Ca v 1.41ex47 coexpressed with auxiliary subunits representative of Ca v 1.4 complexes in the retina (i.e.b 2X13 and a 2 d-4 (5)).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 (V h ) of Ca v 1.41ex47 IT was significantly more negative than that of Ca v 1.41ex47 (Fig. 1, B and C, Table 1).
Exponential fits of the rising phase of the peak currents yielded time constants for activation (t act ) that were significantly longer (Table 2) and with weaker voltage dependence for Ca v 1.41ex47 IT (v = 250.3mV) than for Ca v 1.41ex47 (v = 226.9mV; F 2,7 = 16.4,p = 0.002; Fig. 2, A and B).To analyze rates of channel closure, the time constant for deactivation (t deact ) was obtained from exponential fits of the decay phase of the tail current evoked upon repolarization of the membrane voltage.t deact was significantly greater at the most positive repolarization voltage tested (-60 mV, Table 2) and the voltage-dependence of t deact was significantly steeper for Ca v 1.41ex47 IT (v = 43.1 mV) than for Ca v 1.41ex47 (v = 169.9mV; F 2,22 = 59.2, p , 0.0001; Fig. 2, C and D).Thus, as has been shown for Ca v 1.2 channels bearing the analogous IT mutation (20), IT slows the activation and deactivation of Ca v 1.4 containing exon 47 in a highly voltage-dependent manner.
Deletion of exon 47 augments effects of the IT mutation on voltage-dependent gating of Ca v 1.4 We next investigated how deletion of exon 47 affects the impact of the IT mutation (Fig. 3A).As for Ca v 1.41ex47 (Fig. 1C), IT caused a negative shift in V h for Ca v 1.4Dex47 (Fig. 3, B  and C, Table 1).The net hyperpolarizing effect of IT (DV h ) was not significantly different between Ca v 1.41ex47 (median DV h = 19.9mV, n = 11) and Ca v 1.4Dex47 (median DV h = 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 Ca v 1.4Dex47 IT (; 270 mV, Fig. 3C).Moreover, IT enhanced rather than      F 2,22 = 151.6,p , 0.0001; Fig. 4, C and D).However, IT increased t deact more than 10-fold for Ca v 1.4Dex47 versus ;4fold for Ca v 1.41ex47 upon repolarization to 260 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 Ca v 1.4Dex47
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 Ca v 1.4Dex47 protein.However, Western blots indicated similar levels of total channel protein in cells transfected with either Ca v 1.4Dex47 or Ca v 1.4Dex47 IT (Fig. 5A).Moreover, biotinylation and streptavidin pulldown of cell-surface proteins revealed no significant difference in the levels of Ca v 1.4Dex47 or Ca v 1.4Dex47 IT in the plasma mem-brane (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 (E rev ) (Figs. 1C  and 3C, Table 1).The outward currents and median change in E rev (DE rev ) were significantly larger for Ca v 1.4Dex47 IT (237.2 mV, n = 8) than for Ca v 1.41ex47 IT (216.6 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 Ca v 1.4Dex47.
The nature of the outward currents was mysterious considering that the major intracellular cation in our recording solutions was NMDG 1 (N-methyl-D-glucamine), a large organic cation that does not permeate most voltage-gated ion channels.However, Ca v 1.2 and Ca v 1.3 are permeable to NMDG 1 under some conditions (21,22).If IT enabled NMDG 1 efflux through  We further assessed the effect of IT on selectivity of Ca v 1.4Dex47 by measuring E rev and P Ba /P x under other bi-ionic conditions.With intracellular solutions containing Na 1 or K 1 , IT caused a negative shift in E rev and lowered P Ba /P x (Fig. 8, Table 3).Taken together, these results signified a reduction in the ionic selectivity of Ca v 1.4Dex47 IT compared with WT channels.
Although smaller for Ca v 1.41ex47 than for Ca v 1.4Dex47 (Fig. 1C, Table 1) the effects of IT on E rev were, nevertheless, not reported for Ca v 1.41ex47 in a previous study (12).A key difference was in the choice of auxiliary subunits (b 2X13 and a 2 d-4, this study) versus b 3 or b 2A and a 2 d-1 (12)).Therefore, we tested the impact of IT on the Ca v 1.4 variants containing b 2A and a 2 d-1.Consistent with the previous study, IT caused a large negative shift in V h in these experiments.Although the mutation strongly reduced current densities of Ca v 1.4Dex47 1 b 2A 1 a 2 d-1, IT did not affect E rev (Fig. 9, A-C, Table 1).Thus, the identity of the auxiliary b and a 2 d subunits critically determines the effects of IT on selectivity of Ca v 1.4.

Discussion
Our study provides new insights about how IT affects the biophysical properties of Ca v 1.4.First, we show that IT produces a large negative shift in voltage-dependent activation of Ca v 1.4 channels containing the major auxiliary Ca v subunits in the retina, b 2X13 and a 2 d-4 (Figs. 1 and 3, Table 1), as well as Ca v 1.4 channels comprised of other auxiliary subunits (Fig. 9, Table 1, and see Ref. exacerbates the gain of function effects of IT: Ca v 1.4Dex47 IT activates at more negative voltages and exhibits stronger voltage-dependent alterations in the kinetics of activation and deactivation than Ca v 1.41ex47 IT (Figs. 1-4, Tables 1 and 2).Third, IT weakens the selectivity of Ca v 1.4 for Ba 21 in a manner that varies with the identity of the auxiliary b and a 2 d subunits (Figs. 1, 3, and 9, Tables 1 and 3).Our findings highlight the importance of splice variation and auxiliary subunit composition as potential modifiers of disease-causing mutations affecting Ca v channels.

Conserved role of Ile-745 in activation gating
The S5 and S6 pore-lining helices give rise to the selectivity filter ( 2 channel (2).Ile-745 of Ca v 1.4 corresponds to Ile-781 in IIS6 of Ca v 1.2, which lies in a cluster of hydrophobic residues (Leu-779-Ala-782, LAIA) in the S6 bundle-crossing region that are conserved among Ca v 1 and Ca v 2 channels (24).Disruptive mutations of these residues also cause hyperpolarizing shifts in activation and slowing of deactivation of Ca v 1.2 and Ca v 2.3 (20,25).Our study is the first to show that the IT mutation causes similar effects on Ca v 1.4.Based on the correlation of their hydrophobicity and the negative shift in V h (20,25), 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 Ca v 1.2 (26), contacts between Ile-745 with a corresponding hydrophobic residue in IIIS6 may stabilize helix-helix interactions, which support the closed conformation in Ca v 1.4, and are disrupted by the IT mutation.

Functions of exon 47 in regulating the impact of IT on Ca v 1.4 activation
Exon 47 encodes the initial 47 amino acids of the CTM, a modular domain present in both Ca v 1.4 and Ca v 1.3 that interacts with a region in the proximal C-terminal domain (10,17,27).The CTM nearly abolishes CDI of Ca v 1.4 by competing with calmodulin (CaM) for binding to the channel (10,17).Deletion of the CTM enables CDI by allowing CaM binding to the channel, but also causes a negative shift in V h (10).In Ca v 1.4, exon 47 is critical for the modulatory function of the CTM in that Ca v 1.4Dex47 exhibits similar alterations in V h and CDI as those caused by deletion of the entire CTM (16,18).Our findings that IT and deletion of exon 47 are additive with respect to hyperpolarizing V h (Table 1) suggest distinct mechanisms by which Ile-745 and the CTM facilitate activation.In Ca v 1.3, deletion of the CTM leads to stronger pairing of voltage sensor charge movement and channel opening (28).In Ca v 2.3, the IIS4-S5 loop and the cytoplasmic end of IIS6 are thought to functionally interact in the activation pathway (see Ref. 29).In Ca v 1.4, 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 K v channels (30).Similar approaches would be useful in dissecting the relationships of the corresponding regions, and of the CTM, with respect to activation gating of Ca v 1.4.
The effect of IT on hyperpolarizing V h , whereas decreasing the peak current density of Ca v 1.4Dex47 (Table 1), parallels the effect of the S218L migraine-causing mutation in Ca v 2.1 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 (31).Because IT did not affect the total or cellsurface levels of Ca v 1.4Dex47 protein (Fig. 5), the reduced current density of Ca v 1.4Dex47 IT 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 Ca v 1.4Dex47 IT could have compromised cell health such that outward leak currents compromised I Ba amplitudes and caused the negative shift in E rev .This scenario seems unlikely given that IT reduced current density but did not produce outward currents or alterations in E rev in Ca v 1.4Dex47 channels containing b 2A and a 2 d-1 (Fig. 9, Table 1).Single channel recordings will be necessary to fully uncover the impact of IT on the elementary properties of Ca v 1.4Dex47.

Effects of IT on the ion selectivity of Ca v 1.4Dex47
The exquisite selectivity of Ca v channels is largely determined by Ca 21 binding with high affinity to the selectivity filter (32, 33).Thus, the increased permeability of Na 1 , K 1 , and particularly NMDG 1 caused by a mutation outside of the selectivity filter was unexpected.However, in the absence of Ca 21 , Na 1 and large organic cations such as tetramethylammonium are capable of permeating Ca v 1 channels (34).These results suggest that the pore of Ca v channels is at least 6 Å in diameter, an interpretation that has been verified in structural analyses (2,35).Indeed, despite being a relatively large cation (;6.4 Å wide 3 12 Å long; ;7.3 Å mean diameter (36)), NMDG 1 can permeate Ca v 1.2 channels containing pore mutations (37) and Ca v 1.3 channels exposed to the dihydropyridine agonist FPL 64176 (FPL) (21).Functional interactions between the selectivity filter and the inner S6 helix bundle are involved in K v channel gating transitions (38) and may be conserved among Ca v channels.For example, CaM binding to the cytoplasmic domain promotes conformational changes in the selectivity filter of Ca v 1 channels that lead to CDI (39).Thus, IT could alter positioning of IIS6 and its contributions to the Ca 21 (or Ba 21 ) binding affinity within the selectivity filter, allowing monovalent ions including NMDG 1 and Na 1 to permeate even in the presence of significant extracellular concentrations of Ba 21 .
Our findings that impaired selectivity was specific to IT mutant channels containing b 2X13 and a 2 d-4 explain why previous analyses did not uncover any alteration in selectivity in these channels containing b 2A and a 2 d-1 (12).Unlike b 2A, b 2X13 lacks exon 7B, which causes increased voltage-dependent inactivation of Ca v 1.4 (5).Although it is unclear how this difference could affect ion selectivity of the IT mutant channels, there is evidence that structural alterations in a 2 d could affect the permeation properties of Ca v channels.For example, CACHD1 is Effect of CSNB2 mutations on Ca v 1.4 an a 2 d-like protein that has a disrupted metal-ion adhesion site that is critical for structural and functional interactions of a 2 d with the channel (2,40,41).When co-expressed with Ca v 2.2, CACHD1 impairs the ion selectivity of Ca v 2.2 (42).In the cryo-EM structure, a 2 d-1 forms multiple extracellular contacts with Ca v 1.1 including the extended loops between S5 and P1 helices in domains II and III (2).The L5 loops of each of the 4 domains form a domed window above the selectivity filter that direct Ca 21 ions into the pore (2).Differences in how a 2 d variants may interact with these extracellular sites, in concert with those produced by b subunits at intracellular sites, could determine the impact of IT on selectivity in the context of Ca v 1.4.

Significance for visual phenotypes of Ca v 1.4 channelopathies
CSNB2 is a nonprogressive retinal disorder with variable clinical features including reduced visual acuity, myopia, and nystagmus (43).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 (43,44).Of the numerous CSNB2 mutations affecting Cacna1f, the IT mutation causes the most severe form of visual impairment (45).Despite the reduced current density of Ca v 1.4Dex47 IT in our experiments, the mutation enabled significant inward I Ba at voltages negative to the activation thresholds of WT channels (Fig. 3C).Due to charge screening effects (46), our use of 20 mM Ba 21 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 V h and slow deactivation of Ca v 1.4Dex47 IT would lead to aberrant Ca 21 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 Ca v 1.4Dex47 IT 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 (47)(48)(49)(50).However, Ca v 1.4 splice variants lacking exon 47, although abundant in human and monkey retina, are conspicuously absent from mouse retina (16).An understanding of the pathological consequences of Ca v 1.4Dex47 IT could therefore benefit from analyses of the mutant channels in human stem-cell derived photoreceptors in the context of retinal organoids (51).

Experimental procedures cDNAs and molecular biology
The following cDNAs were used: Ca (GenBank M86621), and a 2 d-4 (GenBank NM_172364) in pcDNA3.1.The construct encoding Ca v 1.4Dex47 was described previously (16).To incorporate the IT mutation into Ca v 1.4 (Ca v 1.41ex47 IT and Ca v 1.4Dex47 IT ), the upstream and downstream cDNA regions flanking the codon corresponding to I756 were amplified with Q5 High-Fidelity DNA polymerase (New England Biolabs) using Ca v 1.41ex47 as the template and primers incorporating the mutation.PCR products were digested with DpnI, column purified, and cloned into Ca v 1.41ex47 and Ca v 1.4Dex47 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% CO 2 .Cells were not used after they were passaged 15 times.At 70-80% confluence, the cells were co-transfected with cDNAs encoding human Ca v 1.4 a 1 (1.8 mg; Ca v 1.41ex47, Ca v 1.41ex47 IT , Ca v 1.4Dex47, or Ca v 1.4Dex47 IT ), b 2A or b 2X13 (0.6 mg), a 2 d-4 or a 2 d-1 (0.6 mg), and enhanced GFP in pEGFP-C1 (0.1 mg) 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 Ca 21 -activated K 1 channel (0.1 mg) 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.

Electrophysiology
To measure current density, I Ba was evoked by 50-ms pulses from a holding voltage of 2100 mV to various voltages and normalized to the cell capacitance.I-V data were fitted with the Boltzmann equation: , where I is the measured current at each test voltage (V m ), V h is the voltage of half-maximal activation, k is the slope factor, and G max is the maximal conductance.Peak current density was determined by dividing the maximal I Ba by the cell capacitance.Kinetic parameters for I Ba activation (t act ) and deactivation (t deact ) were obtained by fitting the test current and tail current, respectively, with a single exponential function (y 0 1 A (exp(2t/t)), where y 0 is the offset (asymptote), t is time, t is the time constant, and A is the amplitude.The voltage-dependence of t act and t deact was described by: y 0 1 A (exp(2v/ v)), where y 0 is the asymptote, v is voltage, v is the voltage constant, and A is the amplitude.Relative permeability of Ba 21 versus different monovalent cations (x) was calculated as: P Ba /P x = [x] i /4[Ba 21 ] o 3 exp (E rev F/RT){1 1 exp (E rev F/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 t act and t deact 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 6 S.E.from at least 3 independent transfections.

Figure 1 .
Figure 1.IT enhances voltage-dependence of activation of Ca v 1.41ex47.A, schematic of Ca v 1.4 pore-forming a 1 subunit with 4 transmembrane spanning domains (I-IV; blue), and b 2X13 (tan) and a 2 d-4 (green) subunits.The CTM of Ca v 1.4 (purple) contains exon 47 (orange).The red star illustrates the location of IT in domain II.B, representative I Ba family of traces for Ca v 1.41ex47 or Ca v 1.41ex47 IT .C, I-V plots for I Ba current density (pA/pF) in cells transfected with Ca v 1.41ex47 (black) or Ca v 1.41ex47 IT (red).I Ba was evoked by 50-ms pulses from 2100 mV to various voltages.Here and in all graphs of electrophysiological data, parentheses indicate number of cells, and symbols and error bars represent mean 6 S.E., respectively.

Figure 2 .
Figure 2. IT alters kinetics of activation and deactivation for Ca v 1.41ex47.A, voltage protocol for measuring activation kinetics (left) and representative I Ba family of traces (right).I Ba was evoked by 50-ms pulses from 2100 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 (t act ) were obtained from exponential fits of I Ba and plotted against test voltage in cells transfected with Ca v 1.41ex47 (black symbols) or Ca v 1.41ex47 IT (red symbols).C, voltage protocol for measuring deactivation kinetics (left) and representative family of I Ba traces (right).Tail I Ba was evoked by 10-ms pulses to voltages evoking peak inward I Ba (see Table 2) followed by repolarizations to various voltages.Exponential fits of tail I Ba are color-coded according to the repolarization voltage used to evoke them in voltage protocol.D, deactivation time constants (t deact ) were obtained from exponential fits of tail I Ba and plotted against repolarization voltage in cells transfected with Ca v 1.41ex47 (black symbols) or Ca v 1.41ex47 IT (red symbols).In B and D, solid lines represent exponential fits of the averaged data.Ca v 1.4 variants were co-expressed with b 2X13 and a 2 d-4.
Figure 2. IT alters kinetics of activation and deactivation for Ca v 1.41ex47.A, voltage protocol for measuring activation kinetics (left) and representative I Ba family of traces (right).I Ba was evoked by 50-ms pulses from 2100 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 (t act ) were obtained from exponential fits of I Ba and plotted against test voltage in cells transfected with Ca v 1.41ex47 (black symbols) or Ca v 1.41ex47 IT (red symbols).C, voltage protocol for measuring deactivation kinetics (left) and representative family of I Ba traces (right).Tail I Ba was evoked by 10-ms pulses to voltages evoking peak inward I Ba (see Table 2) followed by repolarizations to various voltages.Exponential fits of tail I Ba are color-coded according to the repolarization voltage used to evoke them in voltage protocol.D, deactivation time constants (t deact ) were obtained from exponential fits of tail I Ba and plotted against repolarization voltage in cells transfected with Ca v 1.41ex47 (black symbols) or Ca v 1.41ex47 IT (red symbols).In B and D, solid lines represent exponential fits of the averaged data.Ca v 1.4 variants were co-expressed with b 2X13 and a 2 d-4.

10 IFigure 3 .Figure 4 .
Figure 3. IT enhances voltage-dependence of activation of Ca v 1.4Dex47 and decreases current density.A-C, same as described in the legend to Fig. 1, except for cells transfected with Ca v 1.4Dex47 or Ca v 1.4Dex47 IT (black and red symbols, respectively in C).

Figure 5 .Figure 6 .
Figure 5. IT does not alter the expression levels or cell-surface density of Ca v 1.4Dex47.A, representative Western blotting images of lysates from HEK293T cells that were untransfected (Control) or transfected with either Ca v 1.4Dex47 (Dex47) or Ca v 1.4Dex47 IT (Dex47 IT ) as well as b 2X13 and a 2 d24.Blots were probed with antibodies against Ca v 1.4, 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 Ca v 1.4 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.

Figure 7 .Figure 8 .
Figure 7. Increasing NMDG 1 in the extracellular recording solution minimizes alterations in E rev 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 2100 mV to the indicated voltages (left) and I-V plots (right) in cells transfected with Ca v 1.4Dex47 (top) or Ca v 1.4Dex47 IT (bottom).I/I Max represents I Ba normalized to peak inward current amplitude.Ca v 1.4 variants were co-expressed with b 2X13 and a 2 d-4.

8 BFigure 9 .
Figure 9. IT does not alter selectivity in Ca v 1.4 channels containing b 2A d and a 2 d-1.A, schematics of Ca v 1.4 (left).Representative current traces evoked by 50-ms pulses from 2100 mV to the indicated voltages (right).B, I-V plots in cells transfected with Ca v 1.41ex47, Ca v 1.41ex47 IT , Ca v 1.4Dex47, or Ca v 1.4Dex47 IT .I Ba (pA/pF) represents I Ba 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 Figs.1-4.

Table 1
Parameters from I-V relationships of Ca v 1.41ex47 and Ca v 1.4Dex47 with or without IT mutation V h , k, E rev , and peak I Ba values (mean 6 S.E.) were determined from Boltzmann fits of the I-V data in Figs.1, 3, and 9 and as described under "Experimental procedures." a Mann-Whitney test.b Student's t test.

Table 2
Time constants for activation and deactivation of Ca v 1.41ex47 and Ca v 1.4Dex47 with or without IT mutation t act was obtained from single exponential fits of the rising phase of currents evoked by the voltages indicated in parentheses.t deact was obtained from single exponential fits of the rising phase of currents evoked upon depolarization from 10-ms steps to voltages evoking peak inward I Ba indicated in parentheses and repolarization to 260 mV.

Table 3
Parameters for monovalent and Ba 21 permeability for Ca v 1.4Dex47 and Ca v 1.4Dex47 IT E rev and P Ba /P x were determined from data shown in Figs.7 and 8and from equations described under "Experimental procedures," p values were determined by unpaired t tests.Intracellular solutions contained 140 mM NMDG 1 , Na 1 , or K 1 .Significance was determined by Student's t test.