A CaV1.1 Ca2+ Channel Splice Variant with High Conductance and Voltage-Sensitivity Alters EC Coupling in Developing Skeletal Muscle

Abstract

The Ca(2+) channel alpha(1S) subunit (Ca(V)1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca(2+) release from internal stores and conducts a slowly activating Ca(2+) current. However, this Ca(2+) current is not essential for skeletal muscle EC coupling. Here, we identified a Ca(V)1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged alpha(1S) subunit and expressed it in dysgenic (alpha(1S)-null) myotubes. GFP-alpha(1S)Delta 29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca(2+) currents through GFP-alpha(1S)Delta 29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca(2+) influx contributed substantially to the depolarization-induced Ca(2+) transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary alpha(2)delta-1 subunit. Thus, characterizing the Ca(V)1.1 Delta 29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca(2+) channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.

Non–Ca 2+ -conducting Ca 2+ channels in fish skeletal muscle excitation-contraction coupling

“…However, Ca 2+ influx in mammalian skeletal muscle seems to be variably regulated, e.g., by the accessory Ca V 1.1 subunits α 2 δ-1 (33) and γ (34) or by retrograde current amplification, induced by RyR1-Ca V 1.1 interaction (35). Ca 2+ current density is also massively up-regulated in developing skeletal muscle by expression of an alternatively spliced α 1S subunit (36). A possible role of this regulated Ca 2+ influx might be in Ca 2+ homeostasis by, e.g., SR store filling.…”

Role of putative voltage-sensor countercharge D4 in regulating gating properties of CaV1.2 and CaV1.3 calcium channels

“…Whereas in Ca V 1.1 charge neutralization of D4 caused a  ̴20 mV right-shift of V½ and reduction of current density to 15% of control [8,9], in Ca V 1.2 and Ca V 1.3 current density was reduced to about half of control levels and the right-shift of V½ was only about 5 mV, and in Ca V 1.2 did not reach statistical significance. Thus, it appears that this mechanism, although present, is of lesser importance for voltage sensing of VSD IV in Ca V 1.2 and Ca V 1.3.…”

“…Whereas the interaction between D4 and R1/R2 in VSD IV appears to contribute to voltage-sensing of VSD IV in all three examined Ca V 1 channels it is not the general mechanism for the regulation of gating properties in all L-type calcium channels. On a broader scale, the S3-S4 linker of the fourth VSD appears to be a hotspot for modulation of gating properties by alternative splicing and toxin binding in many voltage-gated cation channels [9,16–18,27]. This highlights the overall importance of this VSD for channel gating.…”

“…When D4 or either one of the putative interaction partners (R1, R2) were mutated in Ca V 1.1e, the voltage-dependence of activation was shifted by  ̴20 mV towards more positive potentials and the current amplitude was dramatically reduced. Interestingly, this mirrored the effect of insertion of exon 29 into the extracellular loop connecting helices IVS3 and IVS4 (IVS3-S4 linker) that causes a similar 30 mV shift in voltage-dependence of activation and reduction of current density [9,10]. Therefore, this molecular interaction within VSD IV of Ca V 1.1 facilitates channel gating and provides a means for regulation of the gating properties by alternative splicing.…”

“…Ca V 1.2 – the predominant L-type calcium channel in the cardio-vascular system and in the brain – has an intermediate voltage-dependence of activation [1]. Interestingly, the negatively charged amino acid D4 in IVS3 is highly conserved throughout the Ca V family and all L-, PQ-, and N-type channels comprise functionally important alternatively spliced exons in the IVS3-S4 linker (Figure 1(A)) [9,15–18]. Therefore we hypothesized that the D4-R1/R2 interaction mechanism, previously identified in Ca V 1.1, may be involved in adjusting the specific voltage-dependences of activation and its regulation by alternative splicing also in other Ca V 1 channels.…”