From α₁s splicing to γ₁ function: A new twist in subunit modulation of the skeletal muscle L-type Ca²⁺ channel

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. Ca_V1.1, 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 Ca_V1.1 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 Ca_V1.1–RYR1 physical interaction and the Ca_V1.1 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 Ca_V1.1 (α_1s) and β_1a (Fig. 1 A).

In skeletal muscle cells, Ca_V1.1 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 α_1s (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 Ca_V1.1-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 Na_V channels, the likely evolutionary precursors of Ca_V 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 Ca_V1.1. 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 Ca_V1.2 and skeletal muscle Ca_V1.1 (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 Ca_V1.1 (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 α_1c subunit of the cardiac L-type channel Ca_V1.2 (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 α_1s subunit caused by alternative splicing.

In previous work from the same laboratory, a remarkable change in Ca²⁺ current properties had been discovered when studying (in a Ca_V1.1-null myotube-expression system) a splice variant of Ca_V1.1 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 (Ca_V1.1e), 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 Ca_V1.1a. 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 Ca_V1.1 conductance would be to prevent the corresponding electrical current from interfering with the Na⁺-based action potentials. Continued expression of the Ca_V1.1e 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 Ca_V1.1 and to bind to the II–III loop of α_1s (Polster et al., 2018). These cells were then transfected with plasmids encoding Ca_V1.1a and Ca_V1.1e, respectively. Surprisingly, in this setting, the adult splice variant Ca_V1.1a did not show the expected much-lower current density that was observed when Ca_V1.1-null myotubes were used for expression (Tuluc et al., 2009), whereas it did exhibit the higher-voltage threshold of activation compared to Ca_V1.1e. Some additional determinant for suppressing the current was apparently missing. Because of its structural position adjacent to domain IV of α_1s (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 Ca_V1.1a containing cells but not in those expressing Ca_V1.1e, 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 Ca_V1.1 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 α_1s. 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 α_1s. The present results indicate that the change in conductance caused by γ₁ is possible only in combination with the adult splice variant Ca_V1.1a. 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 Ca_V1.1 (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 β_1a 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 Ca_V1.1 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.