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Duplex signaling by CaM and Stac3 enhances CaV1.1 function and provides insights into congenital myopathy

CaV1.1 is essential for initiating skeletal muscle contraction. Niu et al. demonstrate that both CaM and stac3 enhance trafficking and gating of CaV1.1. Stac3 mutations associated with congenital myopathy, weaken its binding of CaV1.1, and thus reduce trafficking.


Introduction
Central to excitation-contraction (EC) coupling in skeletal muscle, Ca V 1.1 is an L-type voltage-gated calcium (Ca 2+ ) channel that senses transmembrane depolarization to initiate Ca 2+ release from the SR RYR1 (Schneider and Chandler, 1973;Bannister and Beam, 2013). Although its cardiac counterpart Ca V 1.2 communicates with RYR2 via freely diffusing Ca 2+ ions, Ca V 1.1 is conformationally coupled to RYR1, obviating the intermediary second messenger (Armstrong et al., 1972;Tanabe et al., 1990a;Ríos et al., 1992). This intimate physical linkage warrants a precise geometric arrangement of the two partners in the skeletal myotube: four Ca V 1.1s, termed tetrads, are disposed in ordered arrays that parallel RYR1 arrays at the surface-membrane/SR (peripheral-couplings) or tubular-membrane/SR (triad) interfaces (Franzini- Armstrong and Jorgensen, 1994;Lamb, 2000).
Fitting with this physiology, a cohort of auxiliary subunits such as β 1A (Schredelseker et al., 2009), α 2 δ , γ 1 (Freise et al., 2000), and various SR proteins including RYR1 (Nakai et al., 1996;Avila and Dirksen, 2000;Bannister et al., 2016), JP45 (Anderson et al., 2006), and junctophilin (Golini et al., 2011) tune Ca V 1.1 function. To identify essential signaling partners, a top-down approach using primary cultures of skeletal myotubes obtained from gene knockout (KO) models (Obermair et al., 2008) and cell lines derived from dysgenic and normal myotubes have been insightful (Powell et al., 1996). However, such analyses have often revealed overlapping functions whereby loss of a single protein dramatically alters Ca V 1.1 localization and/ or gating to ultimately disrupt EC coupling. These effects may be either direct or indirect depending on other proteins present in the complex. Thus, quantifying the role of a given modulator on Ca V 1.1 and the underlying regulatory mechanism is challenging. Intriguingly, recent studies have revealed that both calmodulin (CaM; Ohrtman et al., 2008;Stroffekova, 2008) and stac3 regulate Ca V 1.1, although underlying mechanisms remain to be fully elucidated (Horstick et al., 2013;Polster et al., 2015;Linsley et al., 2017a).
The Ca 2+ -binding protein CaM has emerged as a dynamic regulator of neuronal and cardiac Ca 2+ channels (Ca V 1.2/3/4 and Ca V 2.1/2/3; Halling et al., 2006;Minor and Findeisen, 2010;Ben-Johny et al., 2015). The binding of Ca 2+ -free CaM (apoCaM) up-regulates the baseline open probability (P O ), whereas Ca 2+ -CaM interaction relieves this initial enhancement manifesting as Ca 2+ -dependent inactivation (CDI; Adams et al., 2014). For Ca V 1.1, however, CaM regulation has evaded consensus. Exogenously expressed CaM localizes to the skeletal muscle triad (Rodney and Schneider, 2003). CaM interaction with Ca V 1.1 has been controversial in biochemical studies, however, with some reporting weak to no binding (Ohrtman et al., 2008), whereas in vitro surface plasmon resonance measurements and crystallographic analysis suggest a high-affinity interaction with the channel carboxy tail (CT) in the presence of Ca 2+ (Sencer et al., 2001;Black et al., 2005;Halling et al., 2009). Similarly, functional analysis of Ca V 1.1 in skeletal myotubes has revealed the ultra-slow and variable extent of CDI, casting doubt as to whether CaM is relevant for Ca V 1.1 function (Tanabe et al., 1990b;Ohrtman et al., 2008;Stroffekova, 2008). Interestingly, mutations of the CaM binding interface in the Ca V 1.1 CT strongly reduce EC coupling (Stroffekova, 2011).
Likewise, stac3 was recently identified as a component of the EC coupling machinery in association with debilitating congenital human myopathies (Stamm et al., 2008;Horstick et al., 2013;Nelson et al., 2013;Grzybowski et al., 2017). This autosomal-recessive disease was identified in a culturally isolated population of Native Americans (Stamm et al., 2008) but has since been observed in Middle Eastern, African, and South American individuals (Grzybowski et al., 2017;Telegrafi et al., 2017). Patients present with symptoms of muscle weakness, including short stature, kyphoscoliosis, talipes deformities, and drooping facial features, and increased susceptibility to malignant hyperthermia (Stamm et al., 2008;Grzybowski et al., 2017;Telegrafi et al., 2017). Functionally, homozygous KO of stac3 in mouse and zebrafish models led to markedly diminished Ca V 1.1 surface-membrane trafficking, reduced tetrad formation, loss of retrograde signaling, and a near-complete loss of EC coupling (Horstick et al., 2013;Nelson et al., 2013;Polster et al., 2015Polster et al., , 2016Linsley et al., 2017a,b). However, overexpression of a myopathy-associated mutant stac3 partially rescued channel trafficking, although EC coupling remained reduced (Polster et al., 2016;Linsley et al., 2017a). Moreover, the structural determinants of Ca V 1.1 that mediate stac binding also remain unknown . Thus, stac3 may elicit multiple regulatory functions of Ca V 1.1 through direct interactions with the channel or mediated by other triadic proteins (Polster et al., 2016;Linsley et al., 2017a).
To resolve these complex channel-regulatory mechanisms, a bottom-up approach whereby the effects of individual signaling molecules on Ca V 1.1 gating and trafficking are probed in a simplified system without an elaborate SR or t-tubules would be greatly beneficial (Dascal et al., 1992;Polster et al., 2015;Perni et al., 2017). However, functional analysis of Ca V 1.1 and its modulation by various signaling molecules in nonmuscle cell systems remains challenging (Perez-Reyes et al., 1989;Dascal et al., 1992;Johnson et al., 1997;Polster et al., 2015). Although homologous Ca V 1.2, Ca V 1.3, and Ca V 1.4 all exhibit reliable surface-membrane trafficking in heterologous systems in the presence of α 2 δ and β auxiliary subunits (Mikami et al., 1989;Catterall, 2000;Xu and Lipscombe, 2001;McRory et al., 2004), Ca V 1.1 is thought to be retained in internal organelles (Polster et al., 2015;Linsley et al., 2017b). Countering this classical purview, however, a recent functional study demonstrated that the cytosolic adapter protein stac3 with the α 2 δ/β subunits enabled Ca V 1.1 expression in human-derived tsA201 cells (Polster et al., 2015). Further analysis suggested that additional factors including the transmembrane γ 1 subunit may also permit Ca V 1.1 expression in tsA201 cells (Polster et al., 2016). The contrasting molecular requirements that permit Ca V 1.1 expression in heterologous systems obfuscate general principles that underlie channel trafficking and preclude systematic analysis of channel gating.
In this study, using a combination of whole-cell electrophysiology, FRET two-hybrid binding assay, and external-epitope labeling with flow cytometry , we demonstrate that Ca V 1.1, in fact, traffics to the plasma membrane of recombinant cell systems in the presence of auxiliary α 2 δ and β subunits alone. However, this baseline expression is lower than that for homologous L-type channels. Moreover, electrophysiological analysis reveals tiny ionic currents, suggesting that Ca V 1.1 has a low baseline P O . Both CaM and stac3 enhance both surface-membrane trafficking and baseline P O of Ca V 1.1. Moreover, we demonstrate that stac3 binds to the CT of Ca V 1.1, and stac3 mutations associated with congenital myopathy weaken this interaction, resulting in reduced channel surface-membrane trafficking. Delivery of CaM to the channel complex can partially reverse this trafficking defect. Interestingly, long-term application of small-molecule Ca V modulators diltiazem and verapamil yields a partial rescue of channel trafficking. These results highlight the utility of reconstituted Ca V 1.1 in HEK293 cells as a simplified platform to distinguish regulatory effects of individual triadic signaling molecules. Additionally, the flow cytometric analysis of plasmalemmal expression may be an attractive venue for high-throughput screens of small molecules that modulate Ca V trafficking. In all, our findings illustrate parallel signaling mechanisms that tune Ca V 1.1 trafficking and gating and shed light on pathophysiological mechanisms for stac3-associated congenital myopathies.

Molecular biology
Ca V 1.3 S was unmodified from previously published rat Ca V 1.3 Δ (GenBank Accession No. AF370009.1; Liu et al., 2010). GFP-Ca V 1.1 was a gift from Kurt Beam (University of Colorado at Denver, Denver, CO). Stac3 human isoform 2 was purchased from Origene. RYR1 P2 domain was synthesized by Genscript (sequence in Table  S1). Ca V 1.1 CT chimeras were generated by first using PCR amplification with primers P01 and P02 (primers listed in Table S1) and restriction enzyme cutting sites XhoI and KpnI to generate a silent mutation to create a unique XbaI site ∼1-2 aa upstream of the EF hand and add an MssI restriction enzyme cutting site. Ca V 1.3 CI region was added to this construct by PCR amplification (P03 and P04) and inserted via XbaI and MssI restriction enzyme cutting sites. The Ca V 1.1 variant was generated by PCR amplification (P05 and P06) and cutting sites BglII-KpnI to insert an XbaI cutting site in place of the stop codon. Then, glycine-(12)-CaM WT was PCR amplified (P07 and P08) and inserted into stopless Ca V 1.1 with XbaI and KpnI. Ca V 1.1 ΔCT was ordered from Genscript with the CT truncated after residue 1,397 (i.e., SIL GPH*) and inserted with XhoI and KpnI. Ca V 1.1 (BBS) was generated by overlap PCR (P09-P12) and restriction enzyme sites SalI-XhoI to insert BBS. β 2A -glycine-(8)-CaM WT was unchanged from previously published rat β 2A modifications (Yang et al., 2014). Using PCR amplification, we cloned CaM 1234 (P08 and P13) into NotI-BsrGI to generate β 2A -glycine-(8)-CaM 1234 . β 2A -glycine-(12)-RYR1 P2 was generated from PCR amplification (P14 and P15) and inserted into β 2A -glycine-(32)-CaM WT from a previously published construct  with BsrGI and compatible ends NheI-XbaI. C1 of stac3 was PCR amplified (P16 and P17) and cloned into pcDNA3 with NheI and BsrGI. Native American myopathy mutation was generated by QuikChange mutagenesis (P18 and P19). Venus-and Cerulean-tagged constructs were generated by PCR amplification (P20-P23) and inserted via NotI and XbaI restriction enzyme cutting sites into previously published constructs . All constructs were verified with DNA sequencing.
For FRET two-hybrid experiments, HEK293 cells were cultured on glass-bottom dishes and transfected using a standard polyethylenimine protocol (Lambert et al., 1996). Epifluorescence was collected 1-2 d after transfection.
For the drug study, drugs were purchased from Sigma-Aldrich. Nifedipine and diltiazem were diluted to 1 mM in DMSO, and verapamil, ranolazine, and mexiletine were diluted to 10 mM in DMSO before being added to cell culture media. Cells were incubated in the respective concentration of drugs for 24 h before bungarotoxin labeling.
Peak current density-voltage curves were fitted with the following equation: where J peak is the peak current density at test potential V, G max is maximal channel conductance, V rev is the reversal potential, V 1/2 is the half-activation voltage, and k G is the slope factor (Table S2). Normalized gating charge-voltage curves were fitted with the following equation: where Q norm is the gating charge movement at voltage V normalized to value at +80 mV. Gating charge movement is composed of a double Boltzmann relation, with Q max as saturating normalized gating charge; V 1/2.a and V 1/2.b are half-activating potentials for the two components; and SF a and SF b are slope factors for the two components.
FRET two-hybrid assay Three-cube FRET fluorescence of transfected HEK293 cells was measured on an inverted fluorescence microscope in 2 mM Ca 2+ Tyrode's under resting Ca 2+ intracellular concentrations and 10 mM Ca 2+ Tyrode's incubated with 4 µM ionomycin (Sigma-Aldrich) under Ca 2+ /CaM conditions. Different concentrations and ratios of DNA were transfected to achieve a range of donor molecule (D free ) concentrations. FRET efficiency (E A ) for each individual cell was calculated (Erickson et al., 2001), and effective dissociation constants (K d,EFF ) were computed by fitting the binding curve E A = [D free ]/(K d,EFF + [D free ]) · E A,max iteratively. For stac3 Native American myopathy constructs where plateaus of FRET binding curves were not clearly defined by data, we assumed that stac3 adopts the same conformation and possesses the same E A,max (Ben Johny et al., 2013).

Bungarotoxin labeling assay
First, we washed transfected cells twice with DPBS (with Mg 2+ and Ca 2+ ; MediaTech). Then, we blocked nonspecific binding sites with 3% BSA/DMEM for 30 min at room temperature. We incubated cells with 1 µM α-bungarotoxin-biotin (Invitrogen) in 3% BSA/DMEM for 1 h at room temperature in the dark. On ice and in the dark, cells were washed twice with DPBS, incubated three times for 5 min with DPBS, and incubated for 1 h with 10 nM Qdot655 for flow cytometry or Qdot605 for confocal imaging (Invitrogen) in 3% BSA/DMEM. Finally, cells were washed with DPBS and imaged on the confocal microscope in 2 mM Ca 2+ Tyrode's or harvested with trypsin, washed with PBS (without Mg 2+ and Ca 2+ ), and resuspended for flow cytometry. The total GFP fluorescence is proportional to the number of channels in a cell, where α G corresponds with the brightness of single GFP given the imaging setup, and I 0 is the intensity of the excitation lamp. Similarly, the number of channels at the plasma membrane is given by where α R corresponds with the brightness of a single quantum dot (QD) molecule when assessed through our imaging setup, I 0 is the intensity of the excitation lamp, and 4 corresponds with the stoichiometry for biotin-streptavidin interaction. The factor ε is the efficiency of QD labeling. The ratio of the two equations yields Eq. 5 and is proportional to the fraction of surfacemembrane channels.

Confocal optical imaging
We captured exemplar images of bungarotoxin-labeled HEK cells with the Olympus FluoView FV300 confocal laser scanning microscope and FluoView software (Olympus). Using the Olympus Plan Apochromat 403 or 603 oil objective (NA 1.40, PLA PO60XO3; Olympus), GFP was excited with an argon laser (488 nm), and Qdot-605 streptavidin conjugate (Invitrogen) was excited with a helium neon (HeNe) green laser. Olympus optical filters used include 442/515-nm excitation splitter (FV-FCV), 570-nm emission splitter (FV-570CH), BA510 immunofluorescence and BA530RIF for GFP emission channel, and 605BP filter for Qdot channel. Images were converted and merged in ImageJ (National Institutes of Health).

Flow cytometry
Fluorescence of harvested cells was measured with an Attune acoustic focusing flow cytometer (Life Technologies) in high-sensitivity mode with a flow rate of 100 µl/min. We used the blue (488 nm) laser to excite GFP and Qdot to collect green and red fluorescence, respectively. Green fluorescence was measured through the 574/26 optical filter. Likewise, red fluorescence was measured through the 640LP optical filter. Flow cytometer was calibrated and maintained as previously published (Lee et al., 2016). Control experiments included untransfected cells, GFPonly cells, cells transfected with Ca V 1.1 and stac3 as a negative control, and cells with CaV1.1 BBS and stac3 as a positive control. Data were exported as FCS files and analyzed with custom MAT LAB software.
Data processing and statistical analysis Raw data were gated by forward-and side-scatter signals to filter for single and healthy cells, and green signals >1.5 × 10 5 units were excluded because of nonlinearities in flow cytometer measurements (Lee et al., 2016). Red signals >2 × 10 5 units were excluded because of PMT saturation and accounted for <1% of total collected points. To correct for the true green (S G ) and red (S R ) signals, we averaged red signal (Ŝ R,blank ) and green signal (Ŝ G,blank ) of blank cells. We also calculated the slope for GFP bleedthrough into the red channel (f RED,GFP ) to be ∼2.65% because of the broadness of the GFP emission spectrum, yielding two equations: S G = Ŝ G − Ŝ G,blank and Ŝ R = S R − Ŝ R,blank − f RED,GFP · S G , where Ŝ G is the raw green signal and S RED is the red signal. Welch's t test was used to statistically compare two trafficking conditions, and P values report the probability for the null hypothesis that the respective ϕ max for conditions compared are equal. To ensure robustness, we also used a rank-sum test. In all cases, the P value for rank-sum test was similar to that with Welch's t test. The number of independent trials and total number of cells analyzed are listed in Table S3.

Online supplemental material
The supplemental text explicitly derives the Langmuir relationship between plasmalemmal trafficking and affinity of binding for stac3 pertaining to Fig. 7 l. Fig. S1 shows individual peak current densities from individual cells pertaining to Fig. 1. Fig.  S2 shows additional trafficking data and electrophysiological parameters for Ca V 1.1 channels in the presence of β 2A subunit alone Fig. 6 a. Fig. S3 shows additional justification for distinct interfaces for CaM versus stac3 within Ca V 1.1. Fig. S4 shows extended data with the extent of enhancement in trafficking of Ca V 1.1 in the presence of pharmacological chaperones. Table  S1 lists primers used. Table S2 summarizes electrophysiological parameters pertaining to Fig. 1. Table S3 provides supplemental information for trafficking experiments including sample size.

Functional determinants for expression of Ca V 1.1 in heterologous systems
In comparison with other L-type Ca channels, Ca V 1.1 expresses poorly in heterologous cell systems (Perez-Reyes et al., 1989;Polster et al., 2015). Fig. 1 a shows an exemplar inward Ca 2+ current elicited in response to a voltage-step depolarization from a HEK293 cell transiently expressing Ca V 1.3 pore-forming α 1 subunit with auxiliary β 2A and α 2 δ subunits. Population data of mean peak current densities elicited in response to a family of step depolarizations further illustrate robust expression of Ca V 1.3 in HEK293 cells ( Fig. 1 a). In contrast, when Ca V 1.1 α 1 subunit is coexpressed with both β 2A and α 2 δ auxiliary subunits, we observe minimal ionic currents (Figs. 1 b and S1). Given the functional difference between Ca V 1.1 and Ca V 1.3 despite their overall structural similarity, we sought to identify requirements for functional expression of Ca V 1.1 in heterologous systems. First, we reasoned that the Ca V channel cytoplasmic domains may contain critical motifs that differentially enhance channel function (Fang and Colecraft, 2011). In this regard, for related Na V 1.9 sodium channels that also fail to express in nonexcitable cells, a chimeric approach that replaced the CT of Na V 1.9 with that from Na V 1.4 yielded robust currents (Goral et al., 2015). Paralleling this approach, we exchanged the CT of Ca V 1.1 α 1 subunit with that of Ca V 1.3. Electrophysiological analysis revealed robust currents for the chimeric channels ( Fig. 1 c), suggesting that the CT is a key determinant for functional expression.
Second, key Ca V 1.1-interacting proteins may modulate channel function by either serving as chaperones to promote plasmalemmal trafficking or enhancing channel activity. Indeed, recent research shows that Ca V 1.1 currents can be reestablished in HEK293 cell systems by coexpression of stac3, an adapter protein essential for skeletal muscle function (Polster et al., 2015). Exemplar current trace and population data of Ca V 1.1 after coexpression of stac3 in HEK293 cells further confirm these findings ( Fig. 1 d). As CaM is a canonical interacting partner for the CT of various Ca V s, we reasoned that CaM might also permit functional expression of Ca V 1.1. Indeed, overexpression of CaM alone ( Fig. 1 e) or localization of CaM to the Ca V 1.1 complex via fusion to β 2A subunit (Fig. 1 f) reveals a marked enhancement in Ca 2+ currents. Thus, multiple seemingly disparate manipulations permit Ca V 1.1 expression in nonexcitable cells.
Ca V 1.1 exhibits reduced baseline plasmalemmal trafficking Thus informed, we sought to dissect molecular mechanisms that enable Ca V 1.1 function in HEK293 cells. The functional expression of ion channels may be enhanced from changes in three vital parameters: (1) the number of channels at the surface membrane dictated by protein trafficking, (2) ion permeation, and (3) channel gating.
To quantify the relative fraction of channels at the cell surface membrane, we used a dual-labeling approach  whereby the α 1 subunit is tagged with both a GFP on the cytoplasmic amino terminus and an external epitope composed of a 13-aa α-bungarotoxin-binding site (BBS) inserted into the extracellular loop between transmembrane segments 5 and 6 (S5 and S6) of domain II (Fig. 2 a, Ca V 1.1 BBS ). To label surface membrane channels, we incubated cells with cell-impermeable biotin-conjugated α-bungarotoxin and visualized using streptavidin covalently attached to a red QD; the total expression of Ca V 1.1 in a cell is determined by monitoring the GFP fluorescence (Sekine- Aizawa and Huganir, 2004). The high affinity and specificity of bungarotoxin for the BBS site facilitates reliable detection of surface-membrane Ca V 1.1 with minimal background fluorescence (Sekine- Aizawa and Huganir, 2004). We first verified the functionality of Ca V 1.1 BBS by cotransfecting β 2a -CaM WT into HEK cells. The resultant Ca 2+ currents exhibited properties comparable with those of unmodified Ca V 1.1 (Fig. 2 a). We probed baseline plasmalemmal expression for Ca V 1.1 BBS in the presence of β 2A and α 2 δ subunits using confocal imaging (Fig. 2 b). The left subpanel shows the transmitted light image of an exemplar cell, and the middle subpanels show green (S G ) and red (S R ) fluorescence images indicating GFP from total channels and QD emissions from extracellular channels, respectively. The far-right merged image showcases the difference in intracellular and extracellular labeling of Ca V 1.1 BBS . Although strong GFP fluorescence is evident, external QD labeling is sparse, indicating poor surface-membrane expression of Ca V 1.1 (Fig. 2 b). That said, we did observe some surface-membrane labeling in a few cells, suggesting that Ca V 1.1 with just α 2 δ and β subunits might be sufficient for surface-membrane trafficking, albeit with a lower efficacy. Analysis of external epitope labeling from a multitude of individual cells would help resolve such ambiguities.
Accordingly, we used flow cytometric analysis to quantify surface-membrane trafficking at the population level by determining the total GFP (S G ) and QD (S R ) fluorescence from individual cells. For a given cell, the ratio of red to green fluorescence (ϕ = S R / S G ) is proportional to the fraction of surface-membrane channels (f mem ) and serves as a quantifiable metric for trafficking efficacy: The factors α R and α G are brightness of single QD and GFP fluorophores, respectively, and ε is the efficiency of labeling. Given this framework, we plotted S R versus S G obtained from individual cells expressing Ca V 1.1 BBS with β 2A and α 2 δ subunits after 1 d of transfection (Fig. 2 c). Consistent with confocal imaging data, flow-cytometric analysis showed a mixed population of cells: one fraction exhibited minimal surface-membrane labeling (S R = 0), and the other demonstrated reliable QD labeling (S R > 0). Binned data reveal a saturating relationship for S R as S G increases, with a maximal value of ∼1,923 ± 51 fluorescence units. We estimated the saturating surface-membrane trafficking limit (ϕ max ) as the mean ratio ϕ for individual cells exhibiting high GFP fluorescence (i.e., 5.4 × 10 4 ≤ S G ≤ 1.4 × 10 5 ) to be 0.0225 ± 0.0006 ( Fig. 2 f). We excluded values >1.4 × 10 5 GFP fluorescence units because of previously identified nonlinearities in fluorescence measurements from our flow cytometer above this value (Lee et al., 2016). In comparison, ϕ max for Ca V 1.2 BBS is ∼0.0276 ± 0.0004 (Fig. 2 f). Similarly, coexpression of Ca V 1.1 BBS with β 1A subunit that is endogenous to skeletal myotubes also resulted in weak but detectible QD labeling based on confocal imaging (Fig. 2 d) and population analysis (Fig. 2, e and f ). In contrast, Ca V 1.1 BBS exhibited minimal plasmalemmal expression in the absence of β subunits (Fig. 2 f). These findings further demonstrate that β and α 2 δ subunits are sufficient for plasmalemmal trafficking of Ca V 1.1 in nonexcitable cells; however, this baseline trafficking efficacy is diminished in comparison with related Ca V channels.
CaM and stac enhance Ca V 1.1 surface membrane trafficking With baseline plasmalemmal expression levels established, we probed the effect of CaM and stac3 on overall Ca V 1.1 plasmalemmal trafficking. When CaM is delivered locally to Ca V 1.1 BBS via β 2A -CaM, QD labeling is markedly enhanced, suggesting improved plasmalemmal localization (Fig. 3 a). Flow cytometric analysis of Ca V 1.1 coexpressed with β 2A -CaM revealed an overall enhancement in the QD labeling (Fig. 3, b and e) in comparison with levels with the β 2A subunit alone (P < 10 −5 ; Figs. 2 c and 3 e) or with β 2A fused to a sham payload, the P2 domain of RYR1 (P < 10 −5 ; Fig. 3 e). Likewise, the surface membrane expression of Ca V 1.1 bound to the skeletal muscle β 1A subunit was also enhanced significantly upon coexpression of CaM as a separate molecule (P < 10 −5 ; Fig. 3 f). We next explored whether stac3, like CaM, enhances plasmalemmal trafficking of Ca V 1.1. Confocal imaging (Fig. 3 c) and flow cytometric analysis (Fig. 3 d) revealed substantial enhancement in QD labeling for Ca V 1.1 BBS with stac3 in the presence of β 2A (P < 10 −5 ), consistent with improved channel trafficking. Similarly, stac3 enhanced Ca V 1.1 trafficking in the presence of β 1A subunit (P < 10 −5 ; Fig. 3 f). In contrast, coexpression of mutant stac3 containing only the C1 domain only partially enhanced surfacemembrane trafficking (P < 10 −5 ; Fig. 3 f). Together, these results demonstrate that both CaM and stac3 enhance plasmalemmal trafficking of Ca V 1.1.
Molecular determinants for CaM and stac3-mediated enhancement of Ca V 1.1 trafficking We next sought to identify key channel elements that mediate CaM-and stac3-dependent enhancement in Ca V 1.1 trafficking. As the carboxy terminus is critical for Ca V 1.1 functional expression, we tested the binding of CaM and stac3 to this channel domain (Fig. 4 a). Indeed, for nearly all Ca V 1/2 channels, CaM is a well-established partner for the CT known to modulate channel function (Peterson et al., 1999;Qin et al., 1999;Zühlke et al., 1999;Lee et al., 2000;Pitt et al., 2001;Liang et al., 2003;Singh et al., 2006;Yang et al., 2006). Consequently, we used a FRET two-hybrid binding assay (Erickson et al., 2001) in live cells to quantify CaM binding. We coexpressed cerulean-tagged CaM (Cer-CaM WT ) with Figure 2. Quantifying Ca V 1.1 surface-membrane trafficking using a dual-labeling approach. (a) Top: To label surface-membrane Ca V 1.1 channels, we inserted BBS into the domain II S5-S6 linker and a GFP to the amino terminus yielding Ca V 1.1 BBS . Middle: Ca V 1.1 BBS yields robust Ca 2+ currents when coexpressed with β 2A -CaM. Exemplar trace shows Ca 2+ current elicited with a +50-mV voltage-step. Right: J peak (mean ± SEM) computed from indicated number of cells (n). Blue relation shows J peak relationship for WT Ca V 1.1. (b) Ca V 1.1 in the presence of α 2 δ and β 2A auxiliary subunits traffics poorly and exhibits weak extracellular labeling. Left: Schematic shows external-epitope labeling of GFP-tagged Ca V 1.1 BBS in the presence of α 2 δ and β 2A auxiliary subunits. In this figure, the external epitope is α-bungarotoxin conjugated to QD. Right: Transmitted-light, intracellular GFP (S G ), extracellular QD (S R ), and merged images for transfected and labeled cells were collected by confocal microscopy. Bars, 5 μm. (c) Flow-cytometric analysis confirms weak surface-membrane expression for Ca V 1.1 BBS with α 2 δ and β 2A auxiliary subunits cotransfected. QD fluorescence, S R , is plotted as a function of GFP fluorescence, S G . Each dot represents one cell. Black dashed line indicates no extracellular labeling (S R = 0). Black circles and fit denote binned data for QD and GFP fluorescence fitted to a single-exponential function. (d and e) Ca V 1.1 BBS coexpressed with auxiliary β 1A and α 2 δ subunits traffic to the plasma membrane. Format as in b and c. (f) Bar graph summarizes saturating surface-membrane trafficking limit ϕ max , proportional to the maximal fraction of Ca V 1.1 trafficked to the plasma membrane under various conditions. Ca V 1.1 fails to exhibit plasmalemmal trafficking in the absence of β subunits. In the presence of either β 1A or β 2A , Ca V 1.1 exhibits detectible baseline trafficking, albeit reduced in comparison with Ca V 1.2.
Venus-tagged Ca V 1.3 CT, including the dual vestigial EF hands and the pre-IQ and IQ domains (Ven-Ca V 1.3 Ca 2+ inactivation [CI]), and measured FRET efficiency (E A ) between the donor-acceptor pairs (Fig. 4 b). Strong binding of Cer-CaM WT to Ven-Ca V 1.3 CI was observed under both basal and elevated Ca 2+ conditions (Fig. 4 b), consistent with prior research (Ben Johny et al., 2013). In contrast, FRET two-hybrid analysis of Venus-tagged Ca V 1.1 CI (Ven-Ca V 1.1 CI) and Cer-CaM WT showed weak binding under both basal and elevated Ca 2+ conditions (Fig. 4 c). This weak affinity is consistent with a significant fraction of Ca V 1.1 lacking prebound CaM in endogenous conditions in HEK293 cells. In like manner, FRET two-hybrid analysis of Cer-tagged stac3 with Ven-Ca V 1.1 CI also revealed strong binding (Fig. 4 d), with K d,EFF ∼12,000 D free units ∼400 nM. Fig. 4 e compares relative binding affinities for both CaM and stac3 with Ca V CT obtained from FRET two-hybrid experiments. Altogether, these findings raise the possibility that the binding of CaM or stac3 to the Ca V 1.1 CT may be critical for its function.
Accordingly, we reasoned that the deletion of the CT would abrogate stac3-and CaM-mediated enhancement in Ca V 1.1 trafficking, a prediction that could be assessed readily using the flow cytometric assay. With β 1A and α 2 δ coexpressed, Ca V 1.1(ΔCT) BBS with a truncated CT showed significant enhancement in trafficking in comparison with the WT channels (P < 10 −5 ; Fig. 4 f). However, coexpression of either β 2A -CaM (P = 0.4) or stac3 (P = 0.11) did not further enhance surface-membrane trafficking of Ca V 1.1 (Fig. 4 f). These results suggest that CaM and stac3 binding to the CT is functionally critical to enhance plasmalemmal trafficking. Moreover, FRET two-hybrid experiments in Fig. 4  . In all, these results suggest that the occupancy of Ca V 1.1 CT is closely linked to proper channel function, and an emerging repertoire of CT-binding proteins may modify Ca V function via parallel mechanisms (Park et al., 2010;Wang et al., 2010;Marshall et al., 2011;Flynn and Altier, 2013;Hall et al., 2013).
Distinct binding sites on the CT allow CaM and stac3 to act independently Given that both CaM and stac3 bind to the channel CT to enhance surface-membrane expression, we examined whether these agents act independently or through a shared endpoint. Consequently, to further delineate the CT binding interface for stac3 and CaM, we parsed the CT into three distinct segments: dual vestigial EF hands and pre-IQ and IQ domains. Using FRET two-hybrid assay, we probed binding between Venus-tagged channel segments and cerulean-tagged CaM or stac3. Ca 2+ /CaM exhibits a markedly higher affinity to the pre-IQ and IQ domains in comparison with the dual vestigial EF hand segments (Fig. 5, a  and b). In contrast, stac3 preferentially binds to the dual vestigial EF hand segments in comparison with the pre-IQ and IQ domains (Fig. 5, c and d). Importantly, these findings are in contrast with a recent study that suggested direct IQ binding based on reduced colocalization of Ca V 1.1 and stac3 after mutations in the IQ domain (Campiglio et al., 2018). In light of our present findings, it is possible that mutations in the IQ may indirectly alter stac3 interaction with upstream elements. In all, these findings demonstrate that stac3 and CaM prefer distinct CT interfaces.
Thus, we probed surface-membrane labeling of Ca V 1.1 BBS in the presence of both β 2A -CaM and stac3. If the two agents act through a shared endpoint, then their combination will not further increase trafficking. However, flow cytometric analysis revealed that the two agents combinatorially enhance the trafficking of Ca V 1.1 BBS nearly sixfold, suggesting that they act Confocal imaging and flow cytometry show that cotransfection of β 2a -CaM augments Ca V 1.1 surface-membrane labeling. α 2 δ was cotransfected. Format as in Fig. 2 (b and c). Blue circles and fit in d correspond with binned data in the presence of β 2a -CaM, and black fit is the relation with β 2A reproduced from Fig. 2 c to facilitate comparison with baseline Ca V 1.1 trafficking. (c and d) Confocal imaging and flow cytometry of Ca V 1.1 BBS cotransfected with stac3, β 2A , and α 2 δ subunits confirms enhanced surface-membrane trafficking of Ca V 1.1. Format as in Fig. 2 (b and c). Bars, 5 µm. (e) Bar graph summary shows marked enhancement in saturating surface-membrane trafficking limit ϕ max , with coexpression of β 2A -CaM but not β 2A tethered to RYR1 P2 domain (β 2A -RYR1[P2]) in comparison with β 2A alone. Similarly, stac3 coexpression also enhances ϕ max . (f) Bar graph summary illustrates enhancement in surface-membrane trafficking of Ca V 1.1 bound to β 1A subunit after coexpression of CaM or stac3 alone. *, P < 10 −5 with Welch's t test. Error bars show mean ± SEM.
independently through distinct sites on the CT (P < 10 −5 ; Fig. 5, e-g). In contrast, coexpression of freely diffusing CaM with Ca V 1.1 BBS and β 2A -CaM did not further enhance trafficking (P = 0.13; Fig. 5 g), suggesting that the additive effect here did not result from incomplete saturation of channel CT by CaM. Together, these findings suggest that Ca V 1.1 plasmalemmal trafficking is enriched by a duplex signaling mechanism.
CaM and stac3 enhance the open probability of Ca V 1.1 With the role of CaM and stac3 on Ca V 1.1 trafficking established, we probed their effects on channel gating. However, as the activation of Ca V 1.1 is right-shifted to near its reversal potential (Table S2), detecting single-channel openings reliably in an on-cell configuration is challenging as the unitary currents at these voltages are small. Thus, to estimate changes in the maximal open probability, we analyzed macroscopic I tail and overall gating charge movement. More specifically, the peak I tail is linearly proportional to both the steady-state P O of the channel at the activating prepulse potential and the number of surface-membrane channels. However, the total gating charge moved at the reversal potential (q rev ) is proportional to the number of surface-membrane channels. Gating charges can be isolated by blocking ion currents with heavy metals Cd 2+ / La 3+ . Thus, the ratio I tail /q rev is linearly proportional to P O and serves as a convenient proxy to estimate changes in P O,max under various conditions. Although our initial functional research failed to detect appreciable Ca V 1.1 currents with auxiliary β 2A and α 2 δ subunits coexpressed ( Fig. 1 b), these experiments were conducted 1 d after transient transfection. Our trafficking research instead showed that Ca V 1.1 surface-membrane expression with the same subunits is substantially enhanced (P < 10 −5 ) several days after transient transfection (Fig. S2 a). As such, we conducted wholecell patch-clamp experiments of Ca V 1.1 with auxiliary β 2A and α 2 δ subunits 2-4 d after transfection (Fig. S2 b). Scrutiny of current recordings revealed substantial gating currents in response to a 100-ms activating pulse to +80 mV, indicating the presence of surface membrane channels (Fig. 6 a, labeled Q). The duration of the activating pulse was chosen to accommodate the ultra-slow activation of Ca V 1.1, but the tail currents (I tail ) elicited at 0 mV after this activation pulse were comparatively small. Moreover, blockade of ionic currents revealed both ON gating current, in response to a depolarizing pulse, and OFF gating current during repolarization (Fig. 6 b). Computing I tail /q rev demonstrated low saturating values consistent with a diminished baseline P O,max of Ca V 1.1 channels (Fig. 6 c). Moreover, normalized ON and OFF gating charges plotted as a function of voltage overlays on each other demonstrated that Q ON and Q OFF were similar in magnitude and voltage dependence (Figs. 6 d and S2 c). In contrast, with CaM or β 2A -CaM coexpressed, Ca V 1.1 produce markedly enhanced I tail (Fig. 6, e and i) with similar gating currents (Fig. 6, f and j). Further analysis shows that the saturating value of I tail /q rev is approximately fivefold enhanced in the presence of CaM (Fig. 6 g, P = 0.006) or β 2A -CaM (Fig. 6 k, P = 0.004), suggesting that CaM up-regulates P O,max . Reassuringly, normalized Q ON and Q OFF were similar in magnitude in the presence of CaM and β 2A -CaM (Fig. 6, h and l). In like manner, overexpression of stac3 also resulted in enhanced I tail /q rev (P = 0.006) for Ca V 1.1 (Fig. 6, m-p). These results indicate that both CaM and stac up-regulate the maximal P O of Ca V 1.1 (Fig. 6 q). Reassuringly, further quantification of gating charge density at +80 mV (Q density [+80]) showed a significant increase for β 2A -CaM (P = 0.039) and stac3 (P = 0.045), confirming that modulatory agents also enhance trafficking of Ca V 1.1 to the plasma membrane (Fig. 6 r). Together, these data suggest that both modulators not only boost surface-membrane expression but also up-regulate the activity of Ca V 1.1. The CaM-dependent change in maximal P O is reminiscent of findings with related Ca V 1.3 channels (Adams et al., 2014). Myopathy-associated stac3 mutants diminish Ca V 1.1 surfacemembrane trafficking Recent genetic screens have identified multiple mutations within stac3 that are associated with severe congenital myopathies as illustrated in Fig. 7 a. The first autosomal recessive mutation observed in patients of the Lumbee Native American tribe were homozygous autosomal recessive (W[284]S) in the first SH3 domain of stac3 (Stamm et al., 2008). Subsequently, compound heterozygous variants (K[288]* and L[111]Δ) were identified in a patient of Turkish heritage (Grzybowski et al., 2017). Given that stac3 binds to the Ca V 1.1 CT, we considered whether myopathy-associated mutants may disrupt this interaction and diminish surface-membrane trafficking.
Using a FRET two-hybrid assay, we assessed the binding of Ven-tagged Ca V 1.1 CI and Cer-tagged stac3 variants (Fig. 7 b). In comparison with WT, all three disease-associated stac3 variants exhibited a spectrum of weakened binding affinities (Fig. 7 c; black, WT; red, mutant). Stac3 variants L[111]Δ and W[284]S showed a nearly 10-fold weakened affinity, whereas the mutation K[288]* resulted in a twofold reduced binding of Ca V 1.1 carboxy terminus (Fig. 7 j). To discern analogous functional changes, we compared the surface-membrane trafficking of Ca V 1.1 BBS with WT or mutant stac3 in the presence of both β 1A and α 2 δ subunits. Upon coexpression of WT stac3, Ca V 1.1 BBS showed strong QD labeling, confirmed by confocal imaging (Fig. 7 d) and flow cytometric analysis (Fig. 7, e and k), suggesting robust surfacemembrane expression. In contrast, coexpression of stac3 variant W[284]S with Ca V 1.1 BBS resulted in sharply diminished QD labeling visualized via confocal imaging (Fig. 7 f). Population analysis using flow cytometric analysis further confirmed this result (P < 10 −5 ; Fig. 7, g and k). Likewise, analysis of two additional disease-associated stac3 variants, L[111]Δ (P < 10 −5 ) and K[288]* (P < 10 −5 ), revealed variably diminished channel surface-membrane trafficking as evident from reduced ϕ max (Fig. 7 k). Quantitatively, if the binding of stac3 to Ca V 1.1 BBS genuinely underlies the enhancement in channel surface-membrane trafficking, then this functional increase will follow a Langmuir function with the binding affinity of the stac-channel interaction as follows: where ϕ stac3 and Λ are constants and ϕ max stac3 and ϕ max baseline represent the saturating surface-membrane trafficking limit in the presence and absence of stac3, respectively (see supplemental text). For stac3 variants, we assume that their relative binding affinity for the Ca V 1.1 CI deduced from FRET two-hybrid binding assays (K a,EFF ) is proportional to that for the holochannel interface (K a ). This theoretical framework for channel trafficking mirrors individually transformed Langmuir analysis previously developed to deduce binding interfaces critical for channel gating (Ben Johny et al., 2013). Plotting the experimentally determined saturating surface-membrane trafficking ratio ϕ max versus the relative Ca V 1.1 CI binding affinities (K d,EFF ) for the stac3 variants reveals the predicted Langmuir relationship (Fig. 7 l). These results (e and f) Coexpression of CaM and stac3 results in distinct extracellular labeling and supralinear increase of channels on the membrane. Format as in Fig. 2 (b and c). Bar, 5 µm. (g) Bar graph summarizing ϕ max for CaM and stac3 separately as well as together. Expressing CaM and β 2A -CaM does not appreciably change ϕ max . In comparison, coexpression of β 2A -CaM with stac3 results in a sixfold increase in ϕ max . *, P < 10 −5 . Error bars show mean ± SEM. demonstrate that stac3 binding to Ca V 1.1 promotes plasmalemmal trafficking and that myopathy-associated stac variants exhibit weakened trafficking resulting from disrupted binding to the Ca V 1.1 CT.
Given that both CaM and stac3 independently enhance surface-membrane trafficking of Ca V 1.1, we next investigated whether CaM might rescue the defects in trafficking associated with myopathy-associated stac3. Consequently, we assessed surface-membrane trafficking of Ca V 1.1 BBS in the presence of both stac3 W[284]S and CaM WT . Confocal imaging showed an increase in QD labeling (Fig. 7 h), and flow cytometry confirmed a modest rescue at the population level (P < 10 −5 ; Fig. 7 i). Similar analysis with other myopathy-associated stac3 variants (L[111]Δ and K[288]*) further confirmed the partial rescue of Ca V 1.1 BBS trafficking when CaM WT is coexpressed (P < 10 −5 for both variants; Fig. 7 k). Intriguingly, the net magnitude of CaM-dependent enhancement in Ca V 1.1 surface-membrane trafficking is similar in the presence of all stac3 variants irrespective of their binding affinities (Fig. S3). These results suggest that the CaM effect on channel trafficking is independent of stac, consistent with the two regulatory proteins using distinct binding interfaces (Fig.  S3), and raise the possibility that CaM delivery to Ca V 1.1 furnishes an orthogonal strategy for partially reversing functional defects resulting from myopathy-associated mutations in stac3. Moreover, Ca V 1.1 CT represents a prime interface for screening small molecules that promote Ca V 1.1 trafficking and function.
Small-molecule modulators reverse myopathy-associated Ca V 1.1 trafficking defects Recently, pharmacological chaperones have emerged as a promising strategy to rescue surface-membrane trafficking deficits observed in a variety of genetic disorders involving both G protein-coupled receptors (Beerepoot et al., 2017) and ion channels such as cystic fibrosis transmembrane conductance regulator associated with cystic fibrosis (Hanrahan et al., 2013), K ATP channels associated with congenital hyperinsulinism of infancy (Martin et al., 2013), and Na V 1.5 channels associated with Brugada syndrome (Valdivia et al., 2004;Moreau et al., 2012). In many of these cases, small-molecule modulators that alter channel gating may offer a dual purpose as chaperones Figure 6. P O of Ca V 1.1 is increased with CaM and stac3. (a) Ca V 1.1 with only its auxiliary β 2A and α 2 δ subunits elicits small tail current (I tail ) despite large gating charge movement, indicating a low P O for these channels at baseline. Top: Cartoon depicts Ca V 1.1 α 1 subunit bound to auxiliary β 2A and α 2 δ subunits in HEK293 cells. Bottom: Exemplar currents elicited in response to voltage step depolarization from −80 mV to +80 mV show large gating-charge movement at the reversal potential (q rev ) and I tail evoked upon repolarization to 0 mV. (b) Exemplar current trace for gating charge movement after pore block with Cd 2+ /La 3+ with voltage step depolarization from −80 mV to +80 mV and back to −80 mV. Trace shows equivalent gating charge movement into the open conformation (Q ON ) and gating charge movement into the closed conformation (Q OFF ). (c) Population data reveal low P O . In this figure, I tail evoked in response to a voltage-step family with varying prepulse potentials is normalized by the gating charge movement at the reversal potential, q rev . This ratio (I tail /q rev ) is proportional to P O of the channel. (d) Population data of normalized Q OFF to Q ON confirms equivalent charge movement and also the reliability for approximating P O .  (j) Bar graph summarizes binding affinities of stac variants to Ca V 1.1 CI, mean ± SEM. (k) Bar graph summarizes the saturating surface-membrane trafficking limit ϕ max of Ca V 1.1 in the presence of myopathy-associated mutant stac3 and corresponding rescue with CaM. Stac3 mutations that weaken binding to Ca V 1.1 CT also reduce surface-membrane trafficking (gray bar) and may be partially rescued with CaM coexpression (green bar). Control (white) bar is Ca V 1.1 expressed with basic auxiliary subunits α 2 δ and β 1A for comparison. (l) For all stac3 variants, plotting ϕ max versus the association constant (K a,EFF = 1/K d,EFF ; d) for the binding of mutant stac3 to Ca V 1.1 CI module reveals a Langmuir relationship, suggesting that the binding of stac3 is a key determinant for Cav1.1 surface-membrane trafficking. Dashed gray lines are the ϕ max for with (top) and without (bottom) stac3 WT . by stabilizing key channel conformations. Moreover, as Ca 2+ influx through Ca V 1.1 channels is not necessary to trigger muscle contraction (Armstrong et al., 1972;Dayal et al., 2017), we reasoned that clinically relevant small-molecule Ca V 1 antagonists that traditionally block Ca 2+ influx may be repurposed to reverse trafficking defects of Ca V 1.1 observed in the presence of myopathy-associated mutant stac3 (Fig. 8 a). To evaluate this possibility, bungarotoxin-labeling assays and flow-cytometric analysis were used to quantify drug-induced changes in Ca V 1.1 trafficking coexpressed with mutant stac3 W[284]S, the most prevalent myopathy-associated stac variant, and α 2 δ and β 1A auxiliary subunits. We tested three L-type Ca 2+ -channel modulators, nifedipine, diltiazem, and verapamil ( Fig. 8 b, cyan bars), as well as two Na channel modulators, mexiletine and ranolazine (Fig. 8 b, blue bars), clinically approved for various cardiovascular conditions, at two concentrations reflecting typical low and high therapeutic plasma concentrations. Remarkably, among Ca V channel modulators, incubation with verapamil resulted in ∼40% recovery of Ca V 1.1 trafficking (Fig. 8 b) at low (>60%) drug concentration and ∼67% recovery at high (>80%) drug concentration (P < 10 −5 ). Diltiazem also increased channel trafficking by ∼34% at high (>80%) drug concentration (P < 10 −5 ). In contrast, incubation with nifedipine, mexiletine, and ranolazine resulted in minimal change (<10%) in the saturating fraction of surface-membrane channels (ϕ max ; Fig. 8 b). Of note, in all five conditions, the total GFP fluorescence remained the same, suggesting that the increase in the fraction of surface-membrane channels (ϕ max ) observed in the presence of verapamil and diltiazem reflects genuine potentiation of channel plasmalemmal trafficking. Verapamil application increased Ca V 1.1 trafficking in the absence of stac3 by approximately twofold (Fig. 8 b) but increased channel trafficking in the presence of CaM and stac3 by only 24% and 33%, respectively (Fig. S4 a). Importantly, as stac3 is also thought to directly mediate EC coupling, the partial rescue of trafficking observed in this study may not suffice to rescue deficits in muscle contraction. Nonetheless, these results highlight the utility of the bungarotoxin-labeling assay for small-molecule screens of pharmacological chaperones.

Discussion
Ca V 1.1 has often appeared atypical among L-type channels with seemingly poor conservation of regulatory mechanisms and idiosyncratic requirements for membrane expression manifesting as a loss of function in heterologous systems. Our results indicate that reduced function stems from two deficits. First, quantitative flow-cytometric analyses of surface-membrane expression show that Ca V 1.1 with β and α 2 δ subunits traffics to the plasma membrane, albeit at reduced levels in comparison with related L-type channels. Second, electrophysiological analyses reveal that Ca V 1.1 exhibits a low P O . Both deficits in function depend on the channel CT harboring distinct binding interfaces for CaM and stac3, and coexpression of these proteins markedly enhances channel function. In addition, multiple myopathy-associated mutations weaken stac3 binding to Ca V 1.1 CT and fail to promote channel trafficking. Further analysis of trafficking demonstrated that clinically used Ca V 1 antagonists verapamil and diltiazem reverse trafficking defects of Ca V 1.1. In all, these findings highlight parallel mechanisms that buttress Ca V 1.1 function in heterologous expression systems, lend insight into pathophysiological deficits of Ca V 1.1 associated with congenital myopathy, and posit pharmacological strategies for rescue of channel function. Molecular determinants for Ca V 1.1 trafficking in heterologous systems The requirements for Ca V 1.1 surface-membrane trafficking in heterologous systems have long evaded consensus. Although related Ca V 1/2 channels exhibit robust plasmalemmal trafficking with β and α 2 δ subunits, additional components such as cytosolic stac3 and the transmembrane γ 1 subunit are thought to be obligatory for Ca V 1.1 currents in heterologous systems (Tuluc et al., 2009;Bannister and Beam, 2013;Polster et al., 2015Polster et al., , 2016. How do these modifications at disparate channel interfaces influence trafficking? Our results point to a unified trafficking scheme (Fig. 9), with the requirements for Ca V 1.1 trafficking paralleling those for related Ca V channels (Fang and Colecraft, 2011). Specifically, the β subunit is a dominant effector necessary for Ca V 1.1 plasmalemmal trafficking (Fig. 9 a). This requirement of β subunits for Ca V 1.1 trafficking fits well with the reduced channel expression and diminished tetrad formation observed in β 1A -KO mice (Schredelseker et al., 2005). Upon binding the β subunit, however, Ca V 1.1 exhibits only low baseline trafficking (Fig. 9 b). The binding of either CaM or stac3 alone leads to only a partial enhancement in membrane trafficking (Fig. 9, c and d). Finally, the binding of both CaM and stac3 to the Ca V 1.1 CT yields a supralinear increase in membrane trafficking (Fig. 9 e). Interestingly, complete removal of the CT results in a basal increase in channel trafficking, suggesting that there may be retention motifs encoded within the CT that are masked upon the interaction of either stac3 or CaM (Fig. 4 f). This simplified scheme captures the experimentally observed effects of stac3 and CaM on Ca V 1.1 and provides a platform for other indirect mechanisms to be assessed.
Mechanistically, the Ca V 1.1 CT is a critical determinant for surface-membrane trafficking by harboring both CaM and stac3, a finding that resonates with early research that identified a vital role for this domain in triad localization (Flucher et al., 2000). As CaM is enriched in the triad via transient association with cytoplasmic loops of RYR1 (Mochca et al., 2001;Sencer et al., 2001;Xiong et al., 2002), its weak binding to Ca V 1.1 may promote colocalization of the channels at the tubular or surface membranes (Rodney and Schneider, 2003). Recurrent large-amplitude Ca 2+ transients in the triadic space may further reinforce this localization. Indeed, the role of Ca 2+ /CaM in mediating activity-dependent trafficking has emerged as a pervasive theme in Ca V channel physiology, yet the precise motifs that orchestrate this phenomenon are yet to be elucidated (Wang et al., 2007;Hall et al., 2013;Tseng et al., 2017). Similarly, our results indicate that stac3 potentiates Ca V 1.1 trafficking also via interaction with the CT. Even so, CaM and stac3 likely act through distinct sites as their combination supraadditively enhanced channel trafficking. In this regard, recent studies have shown that multiple channel segments including the II-III loop could bind stac3 (Wong King Yuen et al., 2017;Polster et al., 2018), although with weak affinity. It is possible that stac3 interaction with multiple Ca V 1.1 segments may concurrently enhance its affinity. Analysis of stac3 −/− zebrafish and mouse skeletal myotubes revealed a partial reduction of Ca V 1.1 at the triad, leading to incomplete tetrads and a loss of EC coupling (Polster et al., 2015;Linsley et al., 2017a,b). The magnitude of reduction varied between the two models, suggesting that other regulators such as CaM may play a role in channel trafficking in the muscle. As various stac isoforms promote trafficking of Ca V 1.2 and Ca V 3 (Rzhepetskyy et al., 2016), stac may be a shared modulator across the Ca V family (Weiss and Zamponi, 2017). Our quantitative framework and flow-cytometric analysis of external-epitope labeling may delineate vital signals for membrane trafficking of Ca V channels in skeletal muscle and other native cell types.
CaM and stac3 modulate channel gating The ability to resolve Ca V 1.1 currents in heterologous systems in the presence of β and α 2 δ subunits alone enables systematic analysis of channel gating modulation by regulatory partners. For nearly all Ca V 1/2 channels, CaM confers a potent feedback mechanism (Halling et al., 2006;Minor and Findeisen, 2010;Ben-Johny et al., 2015). Our analysis shows that local enrichment of CaM to Ca V 1.1 results in a fivefold increase in maximal P O (Fig. 6 k). As potentiation in gating occurs at high voltages where channels convey minimal Ca 2+ influx, this effect likely depends on apoCaM interaction. Excitingly, these results are evocative of recent findings that apoCaM binding augments the baseline P O of Ca V 1.3 variants (Adams et al., 2014), hinting at a conserved mechanism across the Ca V superfamily (Ben-Johny et al., 2015). Of note, effects on channel gating and trafficking were both elicited by CaM fused to β subunit. As β subunits have a 1:1 stoichiometry with α subunits (Dalton et al., 2005;Wu et al., 2016), a single CaM mediates both functional effects. Thus, CaM signaling may be bifurcated, whereby the apo form enhances channel gating and the Ca 2+ -bound form enriches channels at the plasma membrane.
Stac3 coexpression up-regulates the baseline P O of Ca V 1.1 to the same extent as CaM depending on the CI module (Fig. 6 o). Interestingly, in skeletal myotubes, homozygous stac3 KO (Polster et al., 2015;Linsley et al., 2017a) and Ca V 1.1 mutants with weakened CaM binding (Stroffekova, 2011) lead to a dramatic loss of EC coupling despite the presence of gating charge movements. Thus, robust EC coupling may require a permissive Ca V 1.1 CT conformation along with that for the II-III loop (Tanabe et al., 1990a). Synthesizing a general framework of Ca V modulation by CaM and stac is an exciting frontier, and the ability to express Ca V 1.1 in heterologous systems under a wide range of conditions facilitates this pursuit.
Pathophysiology and treatment of myopathyassociated stac mutants Stac3 has been identified as a vital genetic locus for debilitating congenital myopathy that encompasses an expanding list of mutations. Patients exhibit a plethora of myopathy-associated symptoms including facial weakness with ptosis, hypotonia, small stature, scoliosis, cleft palate, and susceptibility to malignant hyperthermia (Stamm et al., 2008;Zaharieva et al., 2014;Grzybowski et al., 2017;Telegrafi et al., 2017). Current treatment strategies focus on early diagnosis and symptom management, particularly anticipatory management of malignant hyperthermia, and novel small-molecule agents that reverse pathogenesis are highly desired.
Our analysis reveals that disease-associated stac3 variants weaken binding to the CT, resulting in variably diminished Ca V 1.1 surface-membrane trafficking, highlighting potential pathogenic mechanisms. Indeed, reconstitution of myopathy-associated mutant stac3 (W[284]S) in stac3 −/− KO zebrafish and mouse models led to diminished trafficking, triadic organization, and activity of Ca V 1.1, resulting in marked loss of EC coupling (Polster et al., 2016;Linsley et al., 2017a,b). As patients are either homozygous or compound heterozygous for stac mutations, it is likely that the weakened affinity of stac3 for Ca V 1.1 CT results in incomplete saturation of Ca V 1.1 by this regulatory protein. Our findings point to three distinct avenues for developing effective pharmacological strategies. First, given that CaM can both partially rescue reduced Ca V 1.1 trafficking and enhance Ca V 1.1 activation gating, local enrichment of CaM may be an effective strategy for reversing the pathophysiology of stac3-associated myopathies. In this regard, a CRI SPR-interference approach was recently developed to selectively manipulate CaM expression for a subset of cardiac arrhythmogenic long-QT syndrome (Limpitikul et al., 2017). Second, as we identify Ca V 1.1 CT as the primary effector interface for stac3, FRET two-hybrid binding assay may be repurposed to devise high-throughput screens for small-molecule modulators that enhance this interaction and tune skeletal muscle function (Janzen, 2014). Third, certain Ca V channel antagonists such as diltiazem and verapamil at low therapeutic plasma concentrations partially rescue these trafficking defects, depending on continual exposure to the drug. Structurally, phenylalkylamines such as verapamil bind pore-lining residues of the domain III-IV S6 segments adjacent to the beginning of the carboxy terminus (Striessnig et al., 1990;Tang et al., 2016). Moreover, mutations within the CaM-binding IQ domain in the Ca V 1 carboxy terminus have been shown to allosterically modify binding of phenylalkylamines and other Ca channel antagonists (Dilmac et al., 2004;Huang et al., 2013). Thus, it is possible that the binding of verapamil may either directly stabilize the CT or allosterically switch its conformation to ultimately promote plasmalemmal trafficking. As EC coupling in skeletal muscle does not depend on freely diffusing Ca 2+ ions, blockade of Ca 2+ influx resulting from Ca V antagonists may not significantly alter the strength of EC coupling (Dayal et al., 2017). Paradoxically, recent research has shown that verapamil can potentiate contractions in mouse skeletal muscle (Dayal et al., 2017). Nonetheless, as stac3 is thought to be directly involved in EC coupling, a simple rescue of plasmalemmal channels may be insufficient to reverse pathogenesis in the case of congenital myopathies. Further functional analysis of EC coupling in stac3 mutant animal models after longterm application of verapamil is necessary to assess therapeutic potential. Nonetheless, the quantitative flow-cytometric assay promises to facilitate discovery of small-molecule trafficking modulators. Indeed, similar pharmacological chaperones have emerged as a potential therapeutic avenue for rescue of trafficking deficits associated with cystic fibrosis (Hanrahan et al., 2013), congenital hyperinsulinism of infancy (Martin et al., 2013), and Brugada syndrome (Valdivia et al., 2004;Moreau et al., 2012).
In all, our results hint at a conserved mechanism by which multiple signaling molecules tune Ca V 1.1 gating and localization, inform on mechanisms of disease pathogenesis for congenital myopathy, and suggest potential avenues for development of therapeutic strategies.