Transcript Scanning Reveals Novel and Extensive Splice Variations in Human L-type Voltage-gated Calcium Channel, Cav1.2 α1 Subunit

Abstract

The L-type (Ca v 1.2) voltage-gated calcium channels play critical roles in membrane excitability, gene expression, and muscle contraction. The generation of splice variants by the alternative splicing of the poreforming Ca v 1.2 ␣ 1 -subunit (␣ 1 1.2) may thereby provide potent means to enrich functional diversity. To date, however, no comprehensive scan of ␣ 1 1.2 splice variation has been performed, particularly in the human context. Here we have undertaken such a screen, exploiting recently developed "transcript scanning" methods to probe the human gene. The degree of variation turns out to be surprisingly large; 19 of the 55 exons comprising the human ␣ 1 1.2 gene were subjected to alternative splicing. Two of these are previously unrecognized exons and two others were not known to be spliced. Comparisons of fetal and adult heart and brain uncovered a large IVS3-S4 variability resulting from combinatorial utilization of exons 31-33. Electrophysiological characterization of such IVS3-S4 variation revealed unmistakable shifts in the voltage dependence of activation, according to an interesting correlation between increased IVS3-S4 linker length and activation at more depolarized potentials. Steady-state inactivation profiles remained unaltered. This systematic portrait of splice variation furnishes a reference library for comprehending combinatorial arrangements of Ca v 1.2 splice exons, especially as they impact development, physiology, and disease.Rapid influx of Ca 2ϩ through the Ca v 1.2 channels initiates physiological responses like gene expression, neurotransmitter release, cardiac or smooth muscle contraction, and regulation of Ca 2ϩ -dependent ion channels (1-4). In these capacities, the functional profile of Ca v 1.2 calcium channels can be customized by combinatorial assembly of the Ca v 1.2 ␣ 1 with several different auxiliary ␤-and ␣ 2 ␦-subunits (5). Even greater flexibility in functional tuning could arise from alternative splicing of the ␣ 1 -subunit genes (6); splicing of the human ␣ 1 1.2 subunit gene is known to generate variants with tissue-specific biases and with distinct pharmacological properties (7). At present, 15 of 53 known exons (Fig. 1) of the human ␣ 1 1.2 gene have been reported to be subjected to alternative splicing (8). However, the full set of possible splice loci and variations and their distributions in heart and brain could far exceed this initial view.Recently, genome-wide analyses suggest that as high as 74% of human genes are alternatively spliced (9). Alternative splicing of pre-mRNA has been implicated in development, physiology, and pathophysiology, and the inclusion or exclusion of exons can be regulated in a tissue-specific or temporal manner (10, 11). Splice variations of human ␣ 1 1.2 subunit confer on the channel isoforms altered properties such as sensitivity to blockade by antagonists, regulation by protein kinase, current density, and activation and inactivation characteristics (12)(13)(14). However, these studies have reported generally on the impact of al...

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

“…Consistent with published reports [15] exclusion of exon 33 caused a 6.3 mV (P = 0.052) shift of V½ to less depolarizing potentials without affecting peak current density (Figure 4(D–G); Table 2). Thus, alternative splicing of the Ca V 1.2 IVS3-S4 linker shares a common effect on voltage-sensitivity with the analogous splicing events in Ca V 1.1 and Ca V 1.3, but unlike these channels it does not modify current density.

10.1080/19336950.2018.1482183-F0004Figure 5.Charge neutralization of E4 in VSD II of Ca V 1.2 affects the voltage sensitivity.

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“…Conversely, the big effects of exon 29 insertion and D4N charge neutralization in Ca V 1.1 clearly demonstrate that in the skeletal muscle channel isoform the contribution of VSD IV to channel gating is substantially greater than in its close relatives Ca V 1.2 and Ca V 1.3. This explanation is corroborated by the different magnitudes of effects of alternative splicing in the IVS3-S4 linker which are also considerably smaller in Ca V 1.2 [15] and Ca V 1.3 [16], compared to Ca V 1.1 [8,10]. …”

“…Alike these two channels also the gating properties of Ca V 1.2 are modulated by alternative splicing in the IVS3-S4 linker [15]. Therefore we next examined the potential involvement of the D4-dependent mechanism in the modulation of voltage-dependence of activation in wildtype and mutant (D1327N) Ca V 1.2 with and without exon 33.…”

“…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.…”

Mutations in voltage-gated L-type calcium channel: implications in cardiac arrhythmia

“…Splawski et al discovered a de novo G-to-A mutation, which caused a p.G406R substitution in translational sequence at position 1216 (c.G1216A) of CACNA1C exon 8A (also known as exon 8 [60]) in all 13 individuals with TS. However, the p.G406R mutation was not identified in 180 ethnically matched controls [61], indicating that p.G406R mutation is associated with TS.…”