Calcium current modulation by the γ1 subunit depends on alternative splicing of CaV1.1

El Ghaleb et al. analyzed the effects of the γ1 subunit on current properties and expression of the adult (CaV1.1a) and embryonic (CaV1.1e) calcium channel splice variants, demonstrating that γ1 reduces the current amplitude in a splicing-dependent manner.


Introduction
Excitation-contraction (EC) coupling in skeletal muscle is initiated by action potentials that activate the voltage-gated calcium channel Ca V 1.1 located in the transverse tubules (T-tubules). In adult skeletal muscle, Ca V 1.1 functions as a voltage sensor that triggers the opening of the calcium release channel, the ryanodine receptor (RYR1), in the SR via protein-protein interactions, thus initiating muscle contraction (Rios and Brum, 1987;Schneider and Chandler, 1973). Because of the conformational coupling between Ca V 1.1 and RYR1, Ca V 1.1 currents are dispensable for skeletal muscle EC coupling (Armstrong et al., 1972;Dayal et al., 2017). Accordingly, in mammals, Ca V 1.1 channels activate only upon strong, non-physiological membrane depolarization and conduct small and slowly activating currents (Tanabe et al., 1988). This is strikingly different in the embryonic splice variant (Ca V 1.1e), which lacks 19 amino acids in the extracellular loop connecting segments S3 and S4 in the fourth homologous repeat, owing to alternative splicing excluding exon 29 (Tuluc et al., 2009). The embryonic Ca V 1.1e isoform activates upon physiological membrane depolarization and conducts currents that are substantially larger in amplitude than those of the adult Ca V 1.1a isoform.
Ca V 1.1 is a multiprotein complex consisting of a pore-forming α 1 subunit and several auxiliary proteins: the intracellular β 1a , the glycosylphosphatidylinositol-anchored extracellular α 2 δ-1, and the transmembrane γ 1 subunits (Curtis and Catterall, 1984;Zamponi et al., 2015). While the β 1a subunit was shown to be essential for the functional expression of Ca V 1.1 and for EC coupling (Gregg et al., 1996;Schredelseker et al., 2005), α 2 δ-1 and γ 1 are dispensable for functional expression of Ca V 1.1 in muscle cells but displayed an inhibitory effect on Ca V 1.1 currents (Freise et al., 2000;Obermair et al., 2005;Held et al., 2002;Ursu et al., 2001;Arikkath et al., 2003;Tuluc et al., 2009;Ahern et al.,   ). The α 2 δ-1 subunit slows down the kinetics of activation of Ca V 1.1 currents, whereas the γ 1 subunit reduces the current amplitude and shifts the voltage dependence of inactivation. However, neither the α 2 δ-1 nor the γ 1 subunit is essential for EC coupling. In their absence, the amplitude and voltage dependence of the depolarization-induced calcium transients are unchanged Ahern et al., 2001;Ursu et al., 2004).
All the cited studies were performed in skeletal muscle cells using a knockout or knockdown approach since Ca V 1.1 expresses poorly in mammalian non-muscle cells. Whereas coexpression of the auxiliary subunits β and α 2 δ is sufficient to support functional expression of all other high voltage activated calcium channels Lacerda et al., 1991;Zamponi et al., 2015), Ca V 1.1 coexpression with these subunits does not yield functional currents in heterologous cell systems. Only recently, it was demonstrated that the skeletal muscle-specific adaptor protein STAC3 is essential for membrane expression and robust currents of Ca V 1.1 in heterologous cells (Polster et al., 2015;Wu et al., 2018).
In the present study, we generated two HEK cell lines stably expressing the three subunits (STAC3, β 3 , and α 2 δ-1) necessary to support functional membrane expression of Ca V 1.1. These cell lines provide a unique tool for analysis of wild type and mutant Ca V 1.1 channel currents and pharmacology in non-muscle cells. Interestingly, in contrast to what had been reported in myotubes, our current analysis of the adult and embryonic Ca V 1.1 splice variants in the STAC3-HEK cell lines revealed no difference in their current densities, but still displayed the typical differences in voltage dependence of activation. Because coexpression of γ 1 inhibits gating of Ca V 1.1a calcium currents in skeletal myotubes and tsA201 cells (Polster et al., 2016;Freise et al., 2000;Ahern et al., 2001), and because the recently resolved Ca V 1.1 structure revealed an interaction of γ 1 subunit with the IVS3-S4 loop of Ca V 1.1a (Wu et al., 2016;Wu et al., 2015), we hypothesized that regulation of the gating properties of Ca V 1.1 channels by the γ 1 subunit occurs in a splice variant-dependent manner. Indeed, we found that coexpressed γ 1 subunits selectively reduced the current density of the adult Ca V 1.1a isoform, and not that of the embryonic Ca V 1.1e isoform. In contrast, γ 1 similarly shifted the voltage dependence of steady-state inactivation to more negative voltages and increased Ca V 1.1 membrane expression of both isoforms. Molecular modeling predicted several ionic interactions between the γ 1 subunit and the IVS3-S4 linker of Ca V 1.1a. However, site-directed mutagenesis of the putative ion-pair partners did not abolish γ 1dependent inhibition of the Ca V 1.1a currents, suggesting an allosteric effect of exon 29 that is important for modulation of current density by the γ 1 subunit.

Materials and methods
Generation of stable cell lines Two HEK293 cell lines stably expressing mouse STAC3 were generated using the Flp-In T-Rex system (Invitrogen). Host cells, already expressing human α 2 δ-1 and β 3 subunits and containing a flippase recognition target (FRT) site, allowed the integration of STAC3 into the genome in a Flp recombinase-dependent manner. Briefly, the coding sequence of mouse STAC3 (Q8BZ71) was cloned into the pTO-HA-strepIII C GW FRT vector (containing an FRT site and a hygromycin resistance gene). To generate the cell line constitutively expressing STAC3 (HEK-STAC3), STAC3 expression was under the control of a CMV promoter. To generate the inducible STAC3 expression cell line (HEK-TetOn-STAC3), STAC3 expression was under the control of a CMV promoter with a tetracycline operator (TetOn) element. HEK293 host cells were transfected using the calcium phosphate method with either plasmid or a Flp recombinaseexpressing vector (pOG44). Subsequently cells were selected with hygromycin B (50 μg/ml; cat. #CP12.2; Lactan/Roth) and selection agents for the other subunits (see below), and singlepositive cell clones were propagated and characterized. The electrophysiological experiments for the characterization of the cell lines were carried out using the TetOn-STAC3 cell line (Figs. 3,4,6,and S1).
Although the cell lines contain the β 3 isoform, rather than the skeletal muscle-specific β 1a , no drawbacks are expected when expressing a non-muscle β subunit in non-muscle cells. Accordingly, the cell lines expressing β 3 efficiently supported robust Ca V 1.1 currents (Fig. 2). Also, because we compared differences due to splicing or γ 1 coexpression (mostly involving the transmembrane or extracellular part of the channel), the type of the intracellular β subunit is not expected to affect our current analysis.
The 13-residue bungarotoxin (BTX) binding site (BBS) was inserted in the IIS5-S6 loop of Ca V 1.1a or Ca V 1.1e at residue 593 by overlap extension PCR. Briefly, the cDNA sequence of Ca V 1.1 was amplified with overlapping primers in separate PCR reactions using GFP-Ca V 1.1a as template. Primers used for the first fragment were forward, 59-TACATGAGCTGGATCACG-39, and reverse, 59-GTAGGGCTCCAGGGAGCTCTCGTAGTATCTCCAGTG TCGCACTTCCGTGTCCTCGAAGTC-39. Primers used for the second fragment were forward, 59-TACGAGAGCTCCCTGGAGCCC TACCCTGACGTCACGTTCGAGGACACGGAAGTGCGACGC-39, and reverse, 59-GAACACGCACTGGACCACG-39. The two separate PCR products were then used as template for a final PCR reaction with flanking primers to connect the nucleotide sequences. The resulting PCR fragment was EcoRI/XhoI digested and inserted into EcoRI/XhoI-digested GFP-Ca V 1.1a or GFP-Ca V 1.1e, yielding GFP-Ca V 1.1a-BBS or GFP-Ca V 1.1e-BBS.
The R160A mutation was introduced by overlap extension PCR. Briefly, the cDNA sequence of γ 1 was amplified with overlapping primers mutating R160 into an alanine in separate PCR reactions using pcDNA3-γ 1 as template. Primers used for the first fragment were forward, 59-ATATGGTACCATGTCACA GACCAAAACAGCGAAG-39, and reverse, 59-CACCGACTGCGC CATGACCTCCACGGAGACGATGAG-39. Primers used for the second fragment were forward, 59-GAGGTCATGGCGCAGTCG GTGAAGCGTATGATTGAC-39, and reverse, 59-ATATGTCGACG CTAGTGCTCTGGCTCAGCGTCCATGCA-39. The two separate PCR products were then used as template for a final PCR reaction with flanking primers to connect the nucleotide sequences. The resulting PCR fragment was KpnI/SalI digested and inserted into the KpnI/XhoI-digested pcDNA3 vector, yielding pcDNA3-γ 1 -R160A.
The K102A and E103A mutations were introduced by PCR. Briefly, the cDNA sequence of γ 1 (nt 288-672) was amplified by PCR with a forward primer introducing the K102A and the E103A mutations downstream of the EcoRI site and the reverse primer introducing an ApaI site after the stop codon. Primers used were forward, 59-TGAATTCACCACTCAAGCGGCGTACAG CATCTCAGCAGCGGCCATT-39, and reverse, 59-AGAATAGGG CCCCCCCTCGACGCT-39. After EcoRI/ApaI digestion, the PCR fragment obtained was inserted into the EcoRI/ApaI-digested pcDNA3-γ 1 vector, yielding pcDNA3-γ 1 -K102A-E103A. To combine the three mutations, we introduced the K102A and E103A mutations as described above, but using γ 1 -R160A as template for the PCR, yielding γ 1 -R160A-K102A-E103A (γ 1 -RKE AAA). Sequence integrity of all newly generated constructs was confirmed by sequencing (MWG Biotech).

RT-PCR
RNA was isolated from the three HEK293 cell lines after 48 h in culture using the RNeasy Protect Mini Kit (cat. #74124; Qiagen). After reverse transcription (Super-Script II reverse transcriptase, cat. #18064022; Invitrogen), the absolute number of transcripts in each sample was assessed by quantitative TaqMan PCR (Mm01159196_m1; Thermo Fisher Scientific), using a standard curve generated from known concentrations of a PCR product containing the target of the assay as described previously (Rufenach et al, 2020).

Western blotting
Proteins isolated from the three HEK cell lines were prepared as previously described (Campiglio and Flucher, 2017). Briefly, cells plated in 100-mm dishes were trypsinized after 48 h in culture. Cells were lysed in radioimmunoprecipitation assay buffer with a pestle and left on ice for 30 min. The lysates were then centrifuged for 10 min. The protein concentration was determined using a BCA assay (cat. #23250; Pierce). 20 µg of protein samples were loaded on a NuPage gel (4-12% polyacrylamide, cat. #NP0321; Invitrogen) and separated by SDS-PAGE at 160 V. The protein samples were then transferred to a PVDF membrane at 25 V and 100 mA for 3 h at 4°C with a semidry blotting system (Roth). The membrane was then cut and incubated with rabbit anti-STAC3 (1:2,000; cat. #20392-1; Proteintech; RRID:AB_10693618) or mouse anti-GAPDH (1:100,000; cat. #sc-32233, Santa Cruz Biotechnology; RRID:AB_627679) antibodies overnight at 4°C and then with HRP-conjugated secondary antibody (1:5,000; Pierce) for 1 h at room temperature. The chemiluminescent signal was developed with ECL Supersignal WestPico kit (cat. #34579; Thermo Fisher Scientific) and detected with ImageQuant LAS 4000.

Immunocytochemistry
The three HEK cell lines were plated on poly-L-lysine-coated coverslips and fixed in paraformaldehyde at room temperature after 2 d in culture. Fixed cells were incubated in 5% normal goat serum in PBS/BSA/Triton for 30 min. The rabbit anti-STAC3 antibody (1:2,000) was applied overnight at 4°C and detected with Alexa Fluor 594-conjugated secondary antibody. During the last washing step, cells were incubated with Hoechst dye to stain nuclei. Preparations were analyzed on an Axioimager microscope (Carl Zeiss) using a 63×, 1.4-NA objective. Images were recorded with a cooled charge-coupled device camera (SPOT; Diagnostic Instruments) and Metamorph image processing software (Universal Imaging). Images were arranged in Adobe Photoshop CS6 (Adobe Systems), and linear adjustments were performed to correct black level and contrast. To quantify the fluorescence intensity of the STAC3 staining, 14-bit grayscale images of the red (STAC3) and blue (Hoechst) channels were acquired for each cell line. A region of interest was manually traced around each cell in the STAC3 staining image, and its intensity was recorded and background corrected using Metamorph. For each condition, between 15 and 31 cells were analyzed from each of three independent experiments.
Labeling of cell surface Ca V 1.1 channels with QD 655 For cell-surface labeling, a 13-amino acid high-affinity BBS was inserted into Ca V 1.1a and Ca V 1.1e as described (Yang et al., 2010) and expressed in HEK-293 cells. 48 h after transfection, cells were resuspended from 35-mm dishes with ice-cold PBS ++ containing calcium and magnesium (pH 7.4, 0.9 mM CaCl 2 , and 0.49 mM MgCl 2 ), washed, and incubated with 5 µM biotinylated α-BTX (cat. #B1196; Invitrogen) in PBS ++ /3% BSA in the dark for 1 h on ice. Cells were washed twice with PBS ++ /3% BSA and incubated with 10 nM streptavidin-conjugated quantum dots (QD 655 ; cat. #Q10121MP; Invitrogen) in the dark for 1 h on ice. Finally, cells were washed twice with PBS ++ /3% BSA and either assayed by flow cytometry or plated on poly-L-lysine-coated coverslips and imaged.

Microscopy
Cells were mounted in Tyrode's physiological solution and imaged using a 63×, 1.4-NA objective Axioimager microscope (Carl Zeiss). 14-bit images were recorded with a cooled chargecoupled device camera (SPOT; Diagnostic Instruments) and Metaview image processing software (Universal Imaging). Image composites were arranged in Adobe Photoshop CS6.
Multiparameter flow cytometry Labeled cells were counted by flow cytometry using a BD FACSVerse analyzer. For flow cytometric analyses, labeled cells were counted and analyzed using BD FACSuite v1.0.6 and BD FACS Diva v9.0 software. Cells expressing GFP were excited at 488 nm, and red signal was excited at 633 nm. Our gating strategy assured that the same cell population in terms of size and granularity was counted in each condition. In each set of experiments, untransfected or unlabeled cells, as well as singlecolor controls, were used to adjust threshold values, and these settings were then used when analyzing all samples.

Electrophysiology
Calcium currents in HEK cells were recorded with the wholecell patch-clamp technique in voltage-clamp mode using an Axopatch 200A amplifier (Axon Instruments). Patch pipettes (borosilicate glass; Science Products) had resistances between 1.8 and 4.0 MΩ when filled with (in mM) 135 CsCl, 1 MgCl 2 , 10 HEPES, 10 EGTA, and 4 ATP-Na 2 (pH 7.4 with CsOH). The extracellular bath solution contained (in mM) 15 CaCl 2 , 150 cholinechloride, 10 HEPES, and 1 Mg-Cl 2 (pH 7.4 with CsOH). Data acquisition and command potentials were controlled by pCLAMP software (Clampex version 10.2; Axon Instruments); analysis was performed using Clampfit version 10.7 (Axon Instruments) and SigmaPlot version 12.0 (SPSS Science) software. The current-voltage dependence of activation was determined using 300-or 500-ms-long square pulses to various test potentials (holding potential −80 mV), and curves were fitted according to where G max is the maximum conductance, V rev is the extrapolated reversal potential, V 1/2 is the potential for half-maximal activation, and k is the slope factor. The conductance was calculated using G = −I / (V rev − V), and its voltage dependence was fitted according to a Boltzmann distribution: Steady-state inactivation was calculated as the ratio between two current amplitudes elicited by 200-ms pulses to V max separated by a 45-s conditioning pulse to various test potentials (sweep start-to-start interval 30 s, time gap between the prepulse and the test pulse 10 ms; see Fig. 4 A, inset). Steady-state inactivation curves were fitted using a modified Boltzmann equation: where V 1/2 is the half-maximal inactivation voltage and k is the inactivation slope factor an I residual is the residual fractional current.

Statistical analysis
All experimental groups were analyzed in transiently transfected cells from at least three independent cell passages/ transfections. The means, SEM, and P values were calculated using Student's t test, two-tailed, with significance criteria as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. P values of the experiments in which more than two groups were compared to each other were calculated using ANOVA and Tukey's or Sidak's post-hoc test. The software used for statistical analysis was GraphPad Prism v9.

Structure modeling
The complex structures of both splice variants of the human α1subunit (Ca V 1.1e and Ca V 1.1a) and the γ 1 -subunit were modeled based on the rabbit cryo-EM structure of Ca V 1.1 in the inactivated state, with voltage sensors in the "up" conformation and a closed intracellular gate (PDB accession no. 5GJV; Wu et al., 2016). Homology modeling has been performed using MOE (Molecular Operating Environment, version 2018.08; Molecular Computing Group). Additionally, ab initio Rosetta modeling was used to generate structures for loops that were not resolved in the original Ca V 1.1 α1-subunit and γ 1 -subunit template (Rohl et al., 2004). The structures for the putative mutants were derived from both WT splice variant models by replacing the mutated residue and carrying out a local energy minimization using MOE. The C-terminal and N-terminal parts of each domain were capped with acetylamide and N-methylamide to avoid perturbations by free charged functional groups. The structure model was embedded in a plasma membrane consisting of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol in a 3:1 ratio, using the CHARMM-GUI Membrane Builder (Lee et al., 2019;Jo et al., 2009). Water molecules and 0.15 M KCl were included in the simulation box. Energy minimizations of Ca V 1.1e and Ca V 1.1a WT and mutant structures in the membrane environment were performed. The topology was generated with the LEaP tool of AmberTools18 (Case et al., 2008), using force fields for proteins (ff14SBonlysc) and lipids (Lipid14; Dickson et al., 2014). The structure models were heated from 0 to 300°K in two steps, keeping the lipids fixed, and then equilibrated over 1 ns. Molecular dynamics simulations were performed for 10 ns, with time steps of 2 fs, at 300°K and in anisotropic pressure scaling conditions. Van der Waals and short-range electrostatic interactions were cut off at 10Å, whereas long-range electrostatics were calculated by the particle mesh Ewald method (Salomon-Ferrer et al., 2013). As extracellular loop 1 was not resolved in the cryo-EM structure, we modeled 100 loop structures with Rosetta ab initio modeling (Rohl et al., 2004). By clustering on the loops using an RMSD distance criterion of 2Å, we obtained 10 clusters. These 10 clusters were carefully evaluated, and the two energetically most favorable cluster representatives, which formed interactions with the S3-S4 loop of VSD IV (exon 29), were considered for further minimizations in the membrane environment. MOE and Pymol were used to visualize the key interactions and point out differences in structure models (The PyMOL Molecular Graphics System; version 2.0, Schrödinger, LLC).
Online supplemental material Fig. S1 shows the activation and inactivation kinetics analysis pertaining to Fig. 3 (activation) and Fig. 4 (inactivation).

Results
Generation of two HEK cell lines expressing β 3 , α 2 δ-1, and STAC3 In order to generate HEK293 cell lines that could reliably support Ca V 1.1 expression, we inserted STAC3 into the genome of a host cell line already available, stably expressing α 2 δ-1 and β 3 , using the Flp-In T-Rex system. We generated two cell lines: one in which the expression of STAC3 was constitutive (HEK-STAC3) and one in which the expression of STAC3 was DOX inducible (HEK-TetOn-STAC3). While the parental HEK293 cell line showed neither STAC3 mRNA nor protein expression, the selected clone of the constitutive HEK-STAC3 cell line strongly expressed STAC3 (Fig. 1). As expected, without DOX induction, the selected clone of the inducible HEK-TetOn-STAC3 cell line showed only weak basal STAC3 mRNA and protein expression. However, 24 h after the beginning of DOX induction, STAC3 expression levels were strongly increased and comparable to those of the constitutive HEK-STAC3 cell line (Fig. 1). We then analyzed the ability of the cell lines to support the expression of functional Ca V 1.1 currents by transient transfection with the adult Ca V 1.1a or the embryonic Ca V 1.1e isoforms. The two Ca V 1.1 isoforms differ in the length of the linker connecting helices S3 and S4 of the fourth homologous repeat, with the embryonic isoform skipping exon 29 and lacking 19 amino acids. Although both isoforms support skeletal muscle EC coupling, they display very different current properties when expressed in dysgenic (Ca V 1.1-null) myotubes. In contrast to the adult Ca V 1.1a isoform, the embryonic Ca V 1.1e splice variant activates at more hyperpolarizing potentials and conducts calcium currents that are several-fold larger than those of Ca V 1.1a (Tuluc et al., 2009). Our experiments show that both the constitutive (HEK-STAC3) and the inducible (HEK-TetOn-STAC3) cell lines efficiently supported functional expression of both the adult and the embryonic Ca V 1.1 variants (Fig. 2, A, B, E, and F; and Table 1). More interestingly, while the two Ca V 1.1 splice variants displayed the expected difference in the V 1/2 of activation ( Fig. 2, C, D, G, and H; and Table 1), the expected smaller current density in Ca V 1.1a was not observed in the two STAC3-HEK cell lines (Fig. 2, A, B, E, and F; and Table 1).
We reasoned that some factor is missing in HEK cells that specifically mediates the splicing-dependent effect on the current amplitude in muscle cells. In muscle, the specific function of exon 29 is to curtail the calcium currents, and in our STAC3-HEK cells the currents were equally large, so the missing factor might be a muscle-specific protein capable of diminishing Ca V 1.1 currents specifically in the adult splice variant. The only Ca V 1.1 subunit not present in our expression system is the γ 1 subunit. Moreover, the γ 1 subunit acts as a negative regulator of Ca V 1.1 currents both in skeletal muscle and in tsA201 cells (Freise et al., 2000;Ahern et al., 2001;Andronache et al., 2007;Polster et al., 2016), and its expression is restricted to skeletal muscle Jay et al., 1990). Therefore, we inferred that the γ 1 subunit may be the missing factor selectively reducing the currents of Ca V 1.1a and not those of Ca V 1.1e. This notion was further supported by the fact that cryo-EM structures of Ca V 1.1 predicted an interaction of the γ 1 subunit with the Ca V 1.1 IVS3-S4 region, exactly the site containing the alternatively spliced exon 29 (Wu et al., 2015(Wu et al., , 2016.
The γ 1 subunit selectively reduces the current density of Ca V 1.1a but not that of Ca V 1.1e To test this hypothesis, we measured the calcium current density of Ca V 1.1a and Ca V 1.1e in the presence and the absence of γ 1 in one of the newly established cell lines (HEK-TetOn-STAC3). As previously reported (Polster et al., 2016;Freise et al., 2000), the presence of γ 1 significantly reduced Ca V 1.1a current amplitudes, with no significant effect on the voltage dependence of  Table 2). The activation kinetics were unaltered by coexpression of the γ 1 subunit (Fig. S1, A-D; and Table 2), in agreement with what had been observed in myotubes (Freise et al., 2000) but contrary to what was previously reported in tsA201 cells (Polster et al., 2016). More importantly, in contrast to Ca V 1.1a, coexpression of γ 1 had no effect on the current density of Ca V 1.1e (Fig. 3, E-H; and Table 2), suggesting that the inclusion of the 19 amino acids encoded in exon 29 is essential for suppression of the Ca V 1.1 current by the γ 1 subunit.
The γ 1 subunit shifts the steady-state inactivation to more negative potentials in both Ca V 1.1 isoforms The γ 1 subunit inhibits Ca V 1.1 currents not only by decreasing the current amplitude, but also by promoting inactivation. In fact, previous studies demonstrated that, in the presence of γ 1 , the voltage dependence of inactivation shifted toward more negative potentials, while the voltage dependence of activation remained unaltered (Ahern et al., 2001;Freise et al., 2000;Held et al., 2002;Ursu et al., 2004).
To determine whether this γ 1 effect on Ca V 1.1 currents is also restricted to the adult Ca V 1.1a isoform, we performed a steadystate inactivation protocol comparing the current size of test pulses before and after 45-s conditioning prepulses at incrementally increasing potentials (Fig. 4 A, inset). The normalized steady-state inactivation was plotted as a function of the prepulse potential. As previously demonstrated, coexpression of the γ 1 subunit resulted in a robust left shift in the voltage dependence of inactivation of the adult Ca V 1.1a isoform (Fig. 4 A). In the presence of γ 1 , the half-maximal inactivation potential was  Table 1 for parameters and statistics). shifted by 14.1 mV toward more hyperpolarizing potentials (Fig. 4 B and Table 2). Surprisingly, these effects were recapitulated with the embryonic Ca V 1.1e isoform. In the presence of the γ 1 subunit, the half-maximal inactivation potential was shifted to hyperpolarizing potentials by 13.7 mV (Fig. 4, C and D; and Table 2). These results suggest that, although the γ 1 subunit fails to suppress the current of the embryonic Ca V 1.1e splice variant by ) compared with Ca V 1.1a coexpressed with γ 1 (Ca V 1.1a + γ 1 , dark blue, n = 10). (E-H) Current properties of the embryonic splice variant Ca V 1.1e (red, n = 12) compared with Ca V 1.1e + γ 1 (dark red, n = 13). (A-H) Exemplary current traces at V max (A) and the scatter plot of the peak current density (I peak ; B) show a significant reduction (P = 0.012) when coexpressing γ 1 with Ca V 1.1a. In contrast, when coexpressing γ 1 with Ca V 1.1e (E and F), no difference in current density was observed (P = 0.69). The current-voltage relationship (C and G) and the fractional steady-state activation curves (D and H) show no effect of γ 1 on the voltage dependence of activation when coexpressed with Ca V 1.1a or Ca V 1.1e. Mean ± SEM; P values calculated with Student's t test. *, P < 0.05 (for parameters and statistics, see Table 2). reducing its amplitude (Fig. 3, E-G), it still inhibits Ca V 1.1e currents by left-shifting the steady-state inactivation. The γ 1 subunit was also reported to accelerate the inactivation kinetics of Ca V 1.1 (Ahern et al., 2001;Freise et al., 2000). Accordingly, the time constant of inactivation was reduced to 67% in the presence of γ 1 for both Ca V 1.1a and Ca V 1.1e, although not to a statistically significant extent (Fig. S1, E-H; and Table 2).
The γ 1 subunit increases membrane expression of both Ca V 1.1 isoforms Ca V 1.1 is the only 1 out of the 10 voltage-gated calcium channels that expresses poorly in non-muscle cells, unless the adaptor protein STAC3 is coexpressed (Polster et al., 2015). Recently it was shown that the γ 1 subunit also supports robust membrane expression of Ca V 1.1a in tsA201 cells; although in the absence of STAC3, these channels produce only very small calcium currents (Polster et al., 2016). To examine whether the γ 1 subunit supports only the membrane targeting of the adult Ca V 1.1a isoform or also of the embryonic Ca V 1.1e, we established a dual-labeling approach, originally developed by the lab of Henry Colecraft (Fang and Colecraft, 2011;Yang et al., 2010), to identify and quantify membrane-inserted Ca V 1.1 channels. To this end, a 13 amino acid high-affinity BBS was introduced into the extracellular IIS5-IIS6 domain of GFP-Ca V 1.1a and GFP-Ca V 1.1e. Then, the channels expressed on the cell surface of HEK cells (expressing β 3 and α 2 δ-1) were labeled by exposing unpermeabilized living cells to biotinylated BTX and subsequently to streptavidincoated quantum dots (QD 655 ; Fig. 5 A). Hence, the GFP fluorescence of a cell measures the total Ca V 1.1 expression, and the QD 655 fluorescence quantifies the fraction of surface-expressed Ca V 1.1 channels.
In cells expressing Ca V 1.1a alone, we detected minimal QD 655 fluorescence in the plasma membrane. By contrast, coexpression of STAC3 or γ 1 , individually or together, resulted in robust Ca V 1.1a membrane targeting (Fig. 5 B). To quantify membraneinserted Ca V 1.1 channels, we used flow cytometry analysis, which measures the fluorescence signals of a multitude of individual cells (Fig. 5 D). This analysis confirmed the lack of a robust QD 655 fluorescence signal in cells expressing only GFP-Ca V 1.1a but the presence of a strong QD 655 fluorescence in cells coexpressing GFP-Ca V 1.1a together with STAC3, γ 1 , or both. In four independent experiments, cells coexpressing STAC3 on average displayed a 140% increase of surface-expressed Ca V 1.1a, cells coexpressing γ 1 displayed an 80% increase, and cells expressing both STAC3 and γ 1 subunits displayed a 180% increase compared with cells expressing Ca V 1.1a alone (Fig. 5 F). These results corroborate the importance of STAC3 and γ 1 for Ca V 1.1a plasma membrane expression (Niu et al., 2018;Polster et al., 2016;Polster et al., 2015).
We then analyzed the effect of the STAC3 and γ 1 subunits on membrane expression of the embryonic Ca V 1.1e isoform. In contrast to the adult isoform, the embryonic Ca V 1.1e channel showed substantial membrane staining even when expressed alone (Fig. 5

, C [top] and E [left]
). Nevertheless, coexpression of STAC3 and γ 1 , individually or together, further increased the amount of QD 655 fluorescence (Fig. 5, C and E). In four independent experiments, cells coexpressing STAC3 displayed a 70% increase of surface-expressed Ca V 1.1e, cells coexpressing γ 1 displayed a 50% increase, and the ones expressing both STAC3 and γ 1 subunits displayed an 80% increase compared with cells expressing Ca V 1.1e alone (Fig. 5 G).
Altogether, these results demonstrate that, although the γ 1 subunit fails to modulate the current amplitude of the embryonic Ca V 1.1e isoform, it still modulates its steady-state inactivation and surface trafficking. Moreover, the reduction of current density induced by γ 1 cannot be explained by reduced channel availability at the cell surface.
Ca V 1.1-γ 1 ion-pair partners predicted by structure modeling are not essential for Ca V 1.1a-specific current reduction by γ 1 Because the recent cryo-EM structure of Ca V 1.1 revealed that the γ 1 subunit interacts with IVS3-S4 (Wu et al. 2016;Wu et al. 2015), and because we found that γ 1 fails to inhibit the current amplitude of the embryonic Ca V 1.1e isoform (Fig. 2 E), which lacks 19 amino acids in the IVS3-S4 linker, we hypothesized that γ 1 and the IVS3-S4 linker of Ca V 1.1a may establish an interaction responsible for the current inhibition in Ca V 1.1a. To identify putative interaction partners between the IVS3-S4 linker and γ 1 , we generated a structural model of the Ca V 1.1 channel based on the published cryo-EM structure (Wu et al., 2016;Fig. 6). We  1a (A and B) and Ca V 1.1e (C and D) currents in the presence or in the absence of γ 1 . The inset in A shows the steady-state inactivation protocol. Fractional inactivation curves and scatter plot of V 1/2 of Ca V 1.1a currents (blue, n = 6) compared with Ca V 1.1a + γ 1 currents (dark blue, n = 6; A and B); the same for Ca V 1.1e (red, n = 7) and Ca V 1.1e + γ 1 currents (dark red, n = 6; C and D). The voltage dependence of inactivation is left shifted in Ca V 1.1a + γ 1 (14.1 mV, P = 0.04) and Ca V 1.1e + γ 1 (13.7 mV, P < 0.001). Mean ± SEM; P values calculated with Student's t test. *, P < 0.05; ***, P < 0.001. used Rosetta computational modeling software (Bender et al., 2016;Rohl et al., 2004) to model the structure of the IVS3-S4 linker of Ca V 1.1a. The resulting structure predicts a putative interaction of residues D1223 and D1225 of the IVS3-S4 linker of Ca V 1.1a with residue R160 in the second extracellular loop of the γ 1 subunit (Figs. 7 A and 6 B). To test whether the observed inhibition of the Ca V 1.1a current amplitude by γ 1 is dependent on this ionic interaction, we performed site-directed mutagenesis to substitute the involved residues with alanines, which deletes all interactions made by side-chain atoms beyond the β carbon (Wells, 1991). However, mutation of residue R160 of the γ 1 subunit to an alanine did not diminish its ability to inhibit the current amplitude of Ca V 1.1a (Fig. 7, A-D; and Table 3). Also, simultaneously mutating both D1223 and D1225 of Ca V 1.1a did not alter the ability of γ 1 to reduce the current amplitude of Ca V 1.1a (Fig. 7, E-H; and Table 3). Together, these results indicate Figure 5. γ 1 Increases the surface density of both Ca V 1.1a and Ca V 1.1e isoforms. (A) Scheme displaying the strategy to detect Ca V 1.1 channels expressed on the plasma membrane of HEK cells (stably expressing β 3 and α 2 δ-1).
The introduction of the 13 amino acid BBS in the extracellular domain of GFP-Ca V 1.1a or GFP-Ca V 1.1e allowed the selective labeling of channels in the membrane by sequentially incubating the unpermeabilized cells with biotinylated BTX and streptavidin-conjugated quantum dots (QD 655 ). (B) From top to bottom, representative images of HEK cells expressing the adult GFP-Ca V 1.1a isoform alone, with STAC3, with γ 1 , and with both STAC3 and γ 1 . (C) The same for HEK cells expressing the embryonic GFP-Ca V 1.1e isoform. Scale bar, 2 µm. (D and E) Representative raw data from flow cytometry experiments showing the GFP and the QD 655 signal for cells expressing GFP-Ca V 1.1a (D) or GFP-Ca V 1.1e (E) alone, with STAC3, with γ 1 , and with both STAC3 and γ 1 . The vertical and horizontal lines represent threshold values determined using untransfected cells, untreated cells, and cells exposed only to QD 655 . Single cells are depicted as dots, which have been colored in gray (untransfected), green (transfected, lacking surface expression), or red (transfected with appreciable surface expression). (F and G) Normalized mean QD 655 fluorescence signals across separate flow cytometry experiments (n = 4). Data were normalized to the QD 655 signals of cells expressing only GFP-Ca V 1.1. In F, the conditions with STAC3 (***, P = 0.0003), γ 1 (*, P = 0.0143), and STAC3 + γ 1 (***, P = 0.0002) are significantly different from the control GFP-Ca V 1.1a using oneway ANOVA and Tukey post-hoc mean comparison. In G, the conditions with STAC3 (****, P < 0.0001), γ 1 (**, P = 0.0019), and STAC3 + γ 1 (***, P = 0.0002) are significantly different from the control GFP-Ca V 1.1e using one-way ANOVA and Tukey post hoc mean comparison. that this putative interaction between the IVS3-S4 linker of Ca V 1.1a and the γ 1 subunit is dispensable for current amplitude inhibition by γ 1 . Previously, it has been suggested that the N-terminal half of the γ 1 subunit, including the first two transmembrane domains, mediates its interaction with the calcium channel and is responsible for suppressing the current amplitude of Ca V 1.1 (Arikkath et al., 2003). Because the analyzed R160A mutation is located outside of this region in the C-terminal half of the γ 1 subunit protein, we further modeled the structure of the extensive extracellular loop located in the first half of the γ 1 subunit and searched it for possible interaction sites with the IVS3-S4 linker of Ca V 1.1a. We identified putative ionic interactions of residues D1225 and R1229 in the IVS3-S4 linker of Ca V 1.1a with K102 and E103 positioned in the first extracellular domain of the γ 1 subunit (Fig. 7 I, Table 3, and Fig. 6). However, mutation of K102 and E103 to alanines did not alter the ability of γ 1 to inhibit the calcium channel current amplitude (Fig. 7, J-L; and Table 3). Finally, to exclude the possibility that the interaction between the IVS3-S4 linker of Ca V 1.1a with either one of the two extracellular loops of γ 1 was sufficient to suppress the calcium channel current amplitude, we combined the R160A and K102A/E103A mutations (Fig. 7 M). However, this triple-mutant γ 1 was also capable of inhibiting the current amplitude of Ca V 1.1a to levels similar to the wild type γ 1 (Fig. 7, N-P; and Table 3). Together, these mutagenesis experiments suggest that the current-inhibiting effect of γ 1 is not mediated by the direct ionic interactions between γ 1 and the IVS3-S4 loop of Ca V 1.1a, at least not those predicted by our structure modeling.

Discussion
Whereas the role of the auxiliary α 2 δ and β subunits in subcellular targeting and gating modulation have been extensively studied for high voltage activated calcium channels in heterologous cells, this has not been the case for the γ 1 subunit. γ 1 is a specific subunit of the skeletal muscle Ca V 1.1 isoform and, until recently, Ca V 1.1 had resisted efficient functional expression in heterologous expression systems. Only since the discovery of STAC3 as an essential component of the Ca V 1.1 channel complex, permitting the reliable heterologous expression of Ca V 1.1, have such analyses been possible (Horstick et al., 2013;Nelson et al., 2013;Polster et al., 2015). Here, we developed and validated two HEK cell lines stably expressing STAC3 (plus α 2 δ-1 and β 3 ), which proved to be a convenient and efficient heterologous expression system for Ca V 1.1. By coexpression of Ca V 1.1 and γ 1 in these cells, we found three effects of the γ 1 subunit: facilitated membrane expression of Ca V 1.1, a reduction of the current density, and a shift of steady-state inactivation to hyperpolarizing potentials. The effects of the γ 1 subunit on the two splice variants of Ca V 1.1 expressed in our new STAC3-HEK cell lines revealed a novel, isoform-dependent mechanism of channel modulation by this subunit. Although γ 1 equally supports membrane expression of Ca V 1.1a and Ca V 1.1e, it functions only as a negative regulator of the adult Ca V 1.1a splice variant. This differential regulation of current density is mediated by the inclusion of the alternatively spliced exon 29 in the extracellular loop connecting helices S3 and S4 in repeat IV, but it does not require the direct ionic interactions between this loop and the γ 1 subunit. Another novel finding is that in both the adult and embryonic Ca V 1.1 splice variants, γ 1 reduces steadystate inward current at more negative voltages by shifting the voltage dependence of steady-state inactivation, but not of activation, to more negative voltages and by promoting the time course of current inactivation.
The γ 1 subunit supports membrane expression of Ca V 1.1 The substantially increased surface expression induced by coexpression of γ 1 observed with extracellular BTX labeling and flow cytometry did not translate into increased current densities. This is consistent with the observation that in γ 1 -null mouse muscle, in which STAC3 is endogenously expressed, the expression levels of Ca V 1.1 are similar to those of wild type mice (Arikkath et al., 2003). In our experiments, this is explained by Table 3. Current activation properties of Ca V 1.1a-D1223A-D1225A, γ 1 -R160A, γ 1 -K102A-E103A, and γ 1 -R160A-K102A-E103A (RKE AAA) mutants the observation that the effects of γ 1 and STAC3 on membrane expression are not additive, and therefore γ 1 does not significantly increase Ca V 1.1 beyond the level already achieved by STAC3. Apparently, an independent component must be limiting for membrane targeting. The effect of γ 1 on membrane targeting in heterologous cells is consistent with a previous immunocytochemistry and charge movement analysis showing that in the absence of STAC3, the γ 1 subunit supports robust membrane expression of Ca V 1.1 in tsA201 cells, while sustaining only very small currents (Polster et al., 2016). In contrast, an earlier Western blot analysis of tsA201 cells lysates reported that coexpression of γ 1 reduces the levels of Ca V 1.1 protein expression (Sandoval et al., 2007). In sum, our results corroborate the findings that the γ 1 subunit supports membrane expression of Ca V 1.1 in heterologous cell systems in a splice variantindependent manner, possibly by masking retention motifs on the C-terminus (Niu et al., 2018), but without adding to the calcium influx.
The γ 1 subunit promotes steady-state inactivation in Ca V 1.1a and Ca V 1.1e Functionally, the two negative actions of γ 1 on Ca V 1.1 currents dominate. The observed decrease in current amplitude and leftshift of steady-state inactivation are in general agreement with previous studies in muscle cells (Ahern et al., 2001;Freise et al., 2000) as well as in tsA201 cells expressing Ca V 1.1a (Polster et al., 2016). Limiting calcium influx through Ca V 1.1 during muscle activity is tolerable because of the principal role of Ca V 1.1 as a voltage sensor in skeletal muscle EC coupling (Schneider and Chandler, 1973;Rios and Brum, 1987). At the same time, it is important to limit interference of calcium influx with other calcium signaling events, such as those regulating fiber type specification, and to avoid adverse effects of calcium overload on mitochondrial integrity (Sultana et al., 2016). Previously, we pointed out how intrinsic mechanisms in the Ca V 1.1 α 1S subunit and the actions of auxiliary subunits cooperate in limiting the calcium currents in skeletal muscle (Tuluc et al., 2009;Flucher et al., 2005). Whereas the α 2 δ-1 subunit slows down the activation, the γ 1 subunit promotes voltage-dependent inactivation at more negative voltages. This effect was equally observed in the adult and, as shown here for the first time, the embryonic splice variant. Together with the observed increase in membrane targeting, this is the first experimental evidence demonstrating that the γ 1 subunit functionally interacts with the embryonic splice variant Ca V 1.1e. Therefore, this modulatory effect is independent of the length of the extracellular loop connecting helices IVS3 and IVS4.
The γ 1 subunit reduces the current amplitude specifically in Ca V 1.1a The most interesting finding of this study is the differential down-regulation of calcium currents in Ca V 1.1a versus Ca V 1.1e. The small current size is one of the hallmarks of skeletal muscle calcium currents. Our results demonstrate that the γ 1 subunit is a major determinant of this reduced current density. Whereas in skeletal muscle the adult and embryonic Ca V 1.1 splice variants differ substantially in voltage dependence of current activation and in current size, the currents recorded in the HEK cells (stably expressing α 2 δ-1, β 3 , and STAC3) reproduced the difference in V 1/2 of activation, but not in current density. Apparently, this difference is due to the lack of one or more musclespecific factors in the heterologous expression system. As coexpression of γ 1 restored the reduced current density in Ca V 1.1a compared with Ca V 1.1e, the γ 1 subunit is such a factor. Quantitatively, the difference in current density between the two splice variants was still smaller than that observed when the same constructs were expressed in dysgenic myotubes Tuluc et al., 2009). Therefore, it is likely that other modulatory mechanisms present in the native environment of the channel in the skeletal muscle triads contribute to the full expression of this splice variant-specific difference. The γ 1 subunit is one of two proteins shown to differentially modulate the current properties of the two Ca V 1.1 splice variants, along with RYR1 (Benedetti et al., 2015), demonstrating the importance of the native cellular environment for the accurate expression of physiological current properties. Notably, γ 1 does not reduce the current density of Ca V 1.1a by decreasing its plasma membrane expression. As previously shown, Ca V 1.1e has a higher open probability than Ca V 1.1a in skeletal myotubes (Tuluc et al., 2009). Therefore, the most likely explanation is that γ 1 decreases the channel's maximal open probability in a splice variant-specific manner. The sole difference in the primary structure between the embryonic and adult splice variants is the inclusion of 19 amino acids coded in exon 29 in the IVS3-S4 loop of Ca V 1.1a. Apparently, this difference determines the action of the γ 1 subunit on current size. There are two possible mechanisms how inclusion of exon 29 can enable this functional interaction with γ 1 : direct interactions between the IVS3-S4 loop and γ 1 or the stabilization of a conformation of the channel complex by inclusion of exon 29 that renders Ca V 1.1a susceptible to this particular γ 1 modulation. As the first possibility is amenable to experimental testing, we examined it by identifying and mutating putative interaction sites on both channel subunits. However, none of these ion pairs seemed to be essential for the current-reducing action of γ 1 . This result is in agreement with previous findings showing that Ca V 1.1 current reduction is mediated by the first two transmembrane domains of γ 1 , and that the extracellular loop is dispensable for this interaction (Arikkath et al., 2003). Therefore, it is unlikely that this effect is mediated by the direct interaction of the γ 1 subunit with the IVS3-S4 loop, although our experiments do not entirely rule out this possibility.
Given that the Ca V 1.1 structure identified the II and III transmembrane domains of γ 1 as the ones involved in the interaction with the IVS3-S4 of Ca V 1.1 (Wu et al., 2016), and that the first two transmembrane domains of γ 1 were sufficient for reconstituting the current reduction (Arikkath et al., 2003), we can deduce that an interaction between the second transmembrane domain of γ 1 and the IVS4 of Ca V 1.1 is the most likely scenario for mediating the current-reducing effect. We therefore conclude that insertion of exon 29 into the IVS3-S4 loop alters the conformation of the channel in a way that enables it to respond to the inclusion of γ 1 with a reduced current density (Fig. 8 A). Notably, the left-shifted activation in Ca V 1.1e compared with Ca V 1.1a is observed with or without γ 1 , and the left-shifted inactivation is observed with or without exon 29, whereas the decreased current amplitude requires their cooperation. Evidently, the interdependence of the analyzed gating properties on the IVS3-S4 loop and the γ 1 subunit is highly specific. Each of the partners independently exerts its specific action on the voltage dependence of activation and inactivation (Fig. 8 B).
Uncoupling of the effects of the γ 1 subunit on current size and inactivation The finding that the current amplitude of the embryonic variant Ca V 1.1e is not modulated by the γ 1 subunit, unlike the adult isoform, is surprising. In fact, in a previous study in γ 1 knockout mice, it was reported that the difference in current density between wild type and knockout mice is age dependent, as it was detected only in mice <4 wk of age, but not in older animals (Held et al., 2002). However, this observation cannot be explained by the differential current regulation of Ca V 1.1a and Ca V 1.1e reported here. There is no evidence that primary cultures derived from muscles at different times after birth express different ratios of Ca V 1.1e and Ca V 1.1a. Moreover, if there were such differences, muscles of mice at ≥4 wk would be expected to express predominantly the adult isoform and thus be more susceptible to modulation by γ 1 than younger muscles, not the opposite (Tang et al., 2012;Sultana et al., 2016;Tuluc et al., 2009) More importantly, unlike the age-dependent reduction in current amplitude, in muscles of γ 1 knockout mice, the shift in the steady-state inactivation was found to be age independent (Held et al., 2002), suggesting that these two functional effects of the γ 1 subunit are not coupled with each other. Here, we observed a similar lack of coupling of the two γ 1 effects in Ca V 1.1e, which is subject to the shift in steady-state inactivation but not to the reduction in current density in cells coexpressing γ 1 . Together, these data strongly suggest that the two γ 1 functional effects are independent of each other and possibly mediated by different domains. Figure 8. Model of differential γ 1 modulation on Ca V 1.1a and Ca V 1.1e currents and its consequences for retrograde coupling. (A) In both Ca V 1.1 splice variants, the γ 1 subunit limits calcium currents by shifting the voltage dependence of inactivation to more hyperpolarizing potentials and rendering inactivation more complete. Inclusion of exon 29 in the extracellular IVS3-S4 loop stabilizes a conformation of the Ca V 1.1a channel complex, which enables the γ 1 subunit to reduce the current amplitude. (B) The IVS3-S4 loop including exon 29 and the γ 1 subunit require each other for reducing the current amplitude. In contrast, this cooperation is not required to shift the voltage dependence of activation and inactivation, which occurs in a splice variant-dependent manner. (C) In skeletal muscle cells, the negative regulation of calcium currents by the γ 1 subunit is a prerequisite of retrograde current amplification by the RYR1 in Ca V 1.1a (red arrow from RYR1 to γ 1 ; Grabner et al., 1999;Nakai et al., 1996). Without exon 29 in embryonic Ca V 1.1e, no γ 1 -dependent reduction of current amplitude and no RYR1dependent relief of this inhibition occurs (Benedetti et al., 2015). The red loop in Ca V 1.1a indicates inclusion of exon 29. The role of the γ 1 subunit in orthograde and retrograde coupling of Ca V 1.1 and RYR1 The γ 1 subunit was previously demonstrated to be dispensable for EC coupling, i.e., the orthograde coupling between Ca V 1.1 and the RYR1. In fact, in γ 1 -null myotubes, neither the amplitude nor the voltage dependence of the calcium transients was affected (Ahern et al., 2001;Freise et al., 2000). Likewise, calcium release was unaffected in γ 1 -null myotubes, and twitch and tetanic force development of adult γ 1 -null mice was very similar in both fast and slow muscles (Ursu et al., 2001). However, long-lasting potassiuminduced contractures were significantly larger, and the shift of the steady-state inactivation in Ca V 1.1 currents was shown to translate into a similar shift in the inactivation curve of calcium release of adult skeletal muscle fibers (Ursu et al., 2004). Our finding that γ 1 equally shifts the voltage dependence of inactivation of Ca V 1.1a and Ca V 1.1e indicates that the corresponding shift in the inactivation curve of calcium release also may be present. In skeletal muscle, not only does Ca V 1.1 activate RYR1, but Ca V 1.1a calcium currents are also augmented by an interaction of its cytoplasmic II-III loop with the RYR1 (Grabner et al., 1999), a phenomenon termed retrograde coupling. Previously we demonstrated that this function is specific for the adult Ca V 1.1a splice variant (Benedetti et al., 2015). The currents of Ca V 1.1e are not reduced when the connection with RYR1 is severed. The dependence of the current augmentation by retrograde coupling on inclusion of exon 29 in the IVS3-S4 loop of Ca V 1.1 mirrors the importance of exon 29 for the current reduction by γ 1 . Based on the results of the earlier study, we had proposed a mechanistic model according to which retrograde coupling partially relieves the inhibition of Ca V 1.1 currents by an unknown, exon 29-dependent factor. Our current study suggests that the γ 1 subunit may be this inhibitory factor. According to this hypothetical model, in the simultaneous presence of exon 29 and the γ 1 subunit, the currents of Ca V 1.1a are reduced, and this effect is partially counteracted by the interaction with RYR1. If either exon 29 or the γ 1 subunit is missing, this inhibition is absent and there is nothing to be relieved by retrograde coupling (Fig. 8 C).

Conclusions
This analysis of the actions of the γ 1 subunit on the two splice variants of Ca V 1.1 in heterologous cells revealed multiple functions of γ 1 in membrane targeting and functional modulation of the skeletal muscle calcium channel. Interestingly, some of the γ 1 effects are general for both splice variants, while another is specific for the adult Ca V 1.1a. Inclusion of exon 29 in Ca V 1.1a appears to allosterically render the channel susceptible to the reduction of its currents by γ 1 , as well as to the simultaneous relief of this block by RYR1. Newly generated mammalian cell systems proved highly valuable for this type of coexpression study of Ca V 1.1, but at the same time highlight the multitude of factors involved in shaping the physiological current properties in the native environment of skeletal muscle.