The L-type Ca2+ channel of skeletal muscle (CaV1.1) is part of a multi-protein complex involved in excitation–contraction (EC) coupling. Some of the proteins in this structure are essential for the plasma membrane control of internal Ca2+ release, others play a modulatory role. The auxiliary subunit γ1 is highly specific for this channel even though it is not required for voltage-activated Ca2+ release. A recent study by El Ghaleb et al. (2022) in the Journal of General Physiology presents new evidence for a functional interaction of γ1 with the channel molecule that is influenced by alternative splicing.
EC coupling in skeletal muscle
In skeletal muscle fibers, a single action potential triggers Ca2+ release from the sarcoplasmic reticulum (SR) that raises the free myoplasmic Ca2+ concentration from <0.1 to >10 μM within ∼2 ms (Hollingworth and Baylor, 2013). Ca2+ binding to troponin C initiates contraction by unblocking the actin binding sites for myosin cross bridges. The rapid mobilization of an exceptionally large amount of stored Ca2+ is made possible by (1) a steep chemical gradient for Ca2+ across the SR membrane, established by active ATP-driven Ca2+ pumping and efficient SR-luminal buffering, (2) a large increase in SR Ca2+ permeability mediated by ryanodine-sensitive channels (ryanodine receptor RYR1) and (3) a sophisticated protein machinery coupling the RYR1 gating to a voltage sensor in the membrane of the transverse tubules (TTs), i.e., narrow cannels which conduct the electrical excitation from the surface of a muscle cell towards its center. CaV1.1, serves as the voltage sensor in this process (Bannister and Beam, 2013; Hernández-Ochoa and Schneider, 2018). Its original role, i.e., delivering Ca2+ from the external space to the cytoplasm, got suppressed during vertebrate evolution in exchange for functional adjustments to serve as a voltage-dependent controller of the efflux of Ca2+ from the SR (Mackrill and Shiels, 2020). In some vertebrate muscles (all higher teleost fishes), this protein has even become completely non-conductive for Ca2+, caused by point mutations in the selectivity filter region (Schredelseker et al., 2010). Therefore, a trigger Ca2+ influx eliciting SR Ca2+ release, as found in vertebrate heart muscle (Ríos, 2018), is not required in the skeletal muscle of these species. That this is also true for vertebrates possessing Ca2+-conductive CaV1.1 was demonstrated by eliminating extracellular Ca2+ (Armstrong et al., 1972; Spiecker et al., 1979) and most recently by studying homozygous mutant mice presenting one of the Ca2+ permeation-blocking “fish mutations” (Dayal et al., 2017).
The exact mechanism of functionally coupling the TT membrane to the SR membrane across the ∼12 nm junctional gap is still elusive. Very likely, it is a chain of conformational changes involving CaV1.1–RYR1 physical interaction and the CaV1.1 II–III loop (connecting homologous domains II and III) as a major determinant. Other proteins contribute to the molecular machinery for Ca2+ release control (Avila et al., 2019; Shishmarev, 2020). The essential components have recently been identified by reconstituting functional voltage-controlled Ca2+ release from the endoplasmic reticulum in a non-muscle cell line (Perni et al., 2017). The characteristic sigmoidal voltage-dependence of Ca2+ release could be established in tsA201 cells, although the signals remained far from the robust Ca2+ transients found in skeletal muscle cells. The set of co-expressed proteins that did the job consisted of RYR1, STAC3 (SH3 and cysteine-rich domain-containing protein 3), JP2 (junctophilin 2), and the L-type Ca2+ channel subunits CaV1.1 (α1s) and β1a (Fig. 1 A).
The enigmatic γ subunit
In skeletal muscle cells, CaV1.1 is associated with two further auxiliary subunits, α2δ and γ. The γ subunit, a polypeptide exhibiting four transmembrane α helices is highly specific for skeletal muscle (Biel et al., 1991; Jay et al., 1990). Single-particle cryo-EM revealed associations between transmembrane segment 2 (TM2) of this protein and domain IV of α1s (Wu et al. 2015, 2016). Known as γ1, this subunit was the first discovered representative of a protein family whose most other members modulate glutamate receptor function in neurons by serving as transmembrane AMPA receptor regulatory proteins (TARPs; Jackson and Nicoll, 2011). They are structurally related to the claudin family of tight junction proteins.
γ1 knockout mice showed neither movement abnormalities nor changes in electrically evoked contraction in fast and slow twitch muscle (Ursu et al., 2001; Ahern et al., 2001). Voltage-dependent Ca2+ current and Ca2+-release activation measured in single adult muscle fibers of the γ1-null mice were indistinguishable from wild type (Ursu et al., 2004). However, voltage-dependent inactivation (VDI) of both Ca2+ current and Ca2+ release was found to be altered such that the voltage of half-maximal availability was displaced by 16 and 14 mV, respectively, to more depolarized potentials, i.e., a stronger prolonged depolarization is needed to obtain the same degree of inactivation in γ1-null muscle. Probably resulting from this reluctance to inactivate, muscle fiber bundles of the γ1-null mouse showed significantly larger contractures during application of high-K+ solutions, causing long-lasting depolarization to about −17 mV (Ursu et al., 2001; Melzer et al., 2006).
The very slow VDI (taking seconds for completion) and the even slower recovery (requiring minutes for full restauration) are characteristics of CaV1.1-mediated Ca2+ current next to its remarkably slow activation kinetics. Ca2+ release, even though activated much more rapidly by depolarization than the L-type Ca2+ current, shares the slow kinetics of VDI. Structural studies on bacterial NaV channels, the likely evolutionary precursors of CaV channels, indicate that VDI results from a collapse of the pore caused by movements of the S6 segments of the four homologous domains (Catterall et al., 2017). This mechanism may also apply to CaV1.1. Certain Ca2+-antagonistic drugs affect Ca2+ release in skeletal muscle by enhancing VDI (Ríos and Pizarro, 1991; Melzer et al., 1995; Zhao et al., 2019). We could show that such antagonists (a phenylalkylamine and a benzothiazepine drug) and γ1 influenced each other with regard to their effects on VDI and dihydropyridine binding, respectively, qualifying γ1 as a muscle-intrinsic Ca2+ antagonist (Andronache et al., 2007). Consistent with this notion, binding sites for those groups of antagonists have been identified on S6 segments, notably in domains III and IV of cardiac CaV1.2 and skeletal muscle CaV1.1 (Catterall and Swanson, 2015; Catterall et al., 2020; Zhao et al., 2019) and for γ1 in nearby regions, i.e., the III–IV linker and S4 of domain IV of CaV1.1 (Wu et al., 2016).
The change in VDI was a consistent effect of the γ1 subunit, even when it was experimentally co-expressed with the α1c subunit of the cardiac L-type channel CaV1.2 (Sipos et al., 2000) and when studying mature (fibers) and immature skeletal muscle cells (myotubes; Ursu et al., 2004; Ahern et al., 2001; Freise et al., 2000). In myotubes derived from mice younger than 4 wk, a second effect, a lower Ca2+ current amplitude as compared to wild type, has been reported (Freise et al., 2000; Ahern et al., 2001; Held et al., 2002). Both changes could be reversed by transient expression of γ1 in the knockout myotubes. The difference in amplitude but not in the shifted voltage dependence of inactivation got lost when myotubes were cultured from older animals indicating independence of these two functional modifications (Held et al., 2002). The paper by El Ghaleb et al. (2022) likewise describes a dissociation of γ1 effects on Ca2+ current amplitude and fractional VDI and relates the impact on current size to a structural change in the α1s subunit caused by alternative splicing.
CaV1.1 splicing changes the functional impact of γ1
In previous work from the same laboratory, a remarkable change in Ca2+ current properties had been discovered when studying (in a CaV1.1-null myotube-expression system) a splice variant of CaV1.1 that lacks exon 29 encoding 19 amino acids in the loop linking segments S3 and S4 of homologous domain IV (Tuluc et al., 2009; Benedetti et al., 2015). The characteristics of this variant (CaV1.1e), which predominates in embryonic muscle cells, are (1) a lower-voltage threshold of activation, (2) a larger maximal conductance, and (3) a more rapid turn-on during step depolarization compared to the adult splice variant CaV1.1a. Thus, the presence of the 19 amino acid stretch in the IV S3–S4 linker helps to suppress Ca2+ influx in adult muscle. One advantage of reducing CaV1.1 conductance would be to prevent the corresponding electrical current from interfering with the Na+-based action potentials. Continued expression of the CaV1.1e variant in adult muscle is of clinical relevance, as it is correlated with weakness in myotonic dystrophy (Tang et al., 2012).
In the present study (El Ghaleb et al., 2022), a non-muscle system was employed to investigate both variants further. HEK293 cells already constitutively expressing muscle α2δ-1 and a β subunit (non-muscle β3) were used to generate two cell lines hosting STAC3 in addition. STAC3 is known to significantly enhance the expression of CaV1.1 and to bind to the II–III loop of α1s (Polster et al., 2018). These cells were then transfected with plasmids encoding CaV1.1a and CaV1.1e, respectively. Surprisingly, in this setting, the adult splice variant CaV1.1a did not show the expected much-lower current density that was observed when CaV1.1-null myotubes were used for expression (Tuluc et al., 2009), whereas it did exhibit the higher-voltage threshold of activation compared to CaV1.1e. Some additional determinant for suppressing the current was apparently missing. Because of its structural position adjacent to domain IV of α1s (Wu et al., 2016), γ1 was considered as a candidate for the missing factor. Indeed, co-expressing γ1 (Fig. 1 B) reduced the current maximum in the CaV1.1a containing cells but not in those expressing CaV1.1e, therefore re-establishing a similar situation as found in the myotube expression system (Fig. 1 C). Using an elegant fluorescence-labeling approach, the increase in surface expression caused by γ1 was found to be comparable for both CaV1.1 variants. Consequently, a difference in channel density incorporated in the plasma membrane was ruled out by the authors as a possible cause for the difference in current density.
The team went on to look for possible determinants enabling direct ionic interactions between γ1 and α1s. Based on structure modelling, they applied side-directed alanine mutations to remove charged residues on both the S3–S4 linker and the γ1 subunit. Because these changes lacked the expected result, it is concluded that γ1 affects the channel conformation by a different allosteric mechanism involving the S3–S4 linker of domain IV that leads to reduced conductance. Obviously, the effect of γ1 on VDI is independent of this mechanism.
In summary, this study adds further pieces to the EC coupling puzzle. It is in line with previous results obtained using myocytes from young γ1 knock-out mice (Ahern et al., 2001; Freise et al., 2000; Held et al., 2002) showing that the γ subunit can exert two independent inhibitory effects on the L-type channel, (1) enhancing voltage-dependent inactivation and (2) reducing maximal Ca2+ conductance; and it highlights the importance of alternative splicing of α1s. The present results indicate that the change in conductance caused by γ1 is possible only in combination with the adult splice variant CaV1.1a. Yet, in mature muscle fibers and in myotubes of adult γ1-null mice Ca2+ current was not significantly affected whereas the absence of γ1 led to an increase at an earlier developmental stage (e.g., myotubes cultured from neonatal γ1-null mice; Ursu et al. 2001, 2004; Freise et al., 2000; Held et al., 2002). The reason for this apparent discrepancy requires further investigation. The presence of the ryanodine receptor may be an important factor because of its reciprocal interactions with CaV1.1 (Huang et al., 2011; Benedetti et al., 2015).
The approach of assembling proteins of the EC coupling machinery in a non-muscle cellular environment is a powerful supplement to targeting these components in muscle cells. Obviously, it would be of interest to see if the present results are invariant to adding further elements of the EC coupling system, primarily RYR1 (and the muscle-specific β1a in replacement of β3). One also wonders whether there are any consequences of these findings for the Ca2+ release control by voltage. Further efforts are required to identify the molecular interactions leading to the differential γ1 effects on conductance and inactivation. Generating chimeras between γ1 and one of its non-muscle relatives, as has been done by Arikkath et al. (2003) may be promising. Interesting in this context is also the observation by Held et al. (2002) of a comparable differential response to cAMP analogs pointing to different levels of protein kinase-A–dependent phosphorylation as a cause of the conductance differences seen in their experiments (see above). Finally, the surface expression of CaV1.1 in the HEK293 cell expression system may permit to determine, by patch clamping, which alterations in single channel properties underlie the observed changes in current density. In any case, using this general experimental approach will hopefully continue to uncover important structure–function relations on the way to a full understanding of the link between muscle electricity and force development.
Eduardo Ríos served as editor.
The author declares no competing financial interests.
This work is part of a special issue on excitation–contraction coupling.