Alternative splicing of Cav1.2 channel exons in smooth muscle cells of resistance-size arteries generates currents with unique electrophysiological properties

Abstract

Voltage-dependent calcium (Ca(2+), Ca(V)1.2) channels are the primary Ca(2+) entry pathway in smooth muscle cells of resistance-size (myogenic) arteries, but their molecular identity remains unclear. Here we identified and quantified Ca(V)1.2 alpha(1)-subunit splice variation in myocytes of rat resistance-size (100-200 microm diameter) cerebral arteries. Full-length clones containing either exon 1b or the recently identified exon 1c exhibited additional primary splice variation at exons 9*, 21/22, 31/32, and +/- 33. Real-time PCR confirmed the findings from full-length clones and indicated that the major Ca(V)1.2 variant contained exons 1c, 8, 21, and 32+33, with approximately 57% containing 9*. Exon 9* was more prevalent in clones containing 1c (72%) than in those containing 1b (33%), suggesting exon-selective combinatorial splicing. To examine the functional significance of this splicing profile, membrane currents produced by each of the four exon 1b/c/ +/- 9* variants were characterized following transfection in HEK293 cells. Exon 1c and 9* caused similar hyperpolarizing shifts in both current-voltage relationships and voltage-dependent activation of currents. Furthermore, exon 9* induced a hyperpolarizing shift only in the voltage-dependent activation of channels containing exon 1b, but not in those containing exon 1c. In contrast, exon 1b, 1c, or +9* did not alter voltage-dependent inactivation. In summary, we have identified the Ca(V)1.2 alpha(1)-subunit splice variant population that is expressed in myocytes of resistance-size arteries and the unique electrophysiological properties of recombinant channels formed by exon 1 and 9* variation. The predominance of exon 1c and 9* in smooth muscle cell Ca(V)1.2 channels causes a hyperpolarizing shift in the voltage sensitivity of currents toward the physiological arterial voltage range.

Fig. 1.

Major regions that undergo splicing in cerebral artery smooth muscle cell voltage-dependent Ca²⁺ (Ca_v1.2) channel α₁-subunits. The 4 major regions are illustrated as bold lines or hatched cylinders. The presence of only exon 8 is also highlighted. The S4 segments that function as voltage sensors are shaded in light gray.

Fig. 2.

Additional splicing sites identified in cerebral artery Ca_V1.2 α₁-subunits. Insertions are illustrated as black font shaded in light gray, with deletions as white font shaded with dark gray. *Stop codon. Amino acids introduced by insertions are indicated in bold font. Exon/exon junctions are presented as underlined bold font. A: 66-nt insert (155117087-155117024) between exons 4 and 5 in clone C4. B: 108-nt insert (155229797-155229690) between exons 4 and 5 in clone C8. C: 73-nt deletion (154984855-154984783) within exon 15 in clone C17. D: 21- nt insert (154984876-154984856) between exons 14 and 15 in clone C5. E: 71-nt insert (155174200-155174130) between exon 32 and 33 in clone B9. F: 18-nt deletion (154911308-154911291) within exon 41 in clone C16.

Fig. 3.

Real-time quantitative RT-PCR identifies relative expression of major Ca_V1.2 exon variants in isolated cerebral artery smooth muscle cells and cardiac myocytes. A: exon 9* expression is higher in cerebral artery smooth muscle cells (n = 6) than in cardiac myocytes (n = 5). *P < 0.05 when compared with exon 9 and 9* expression in arterial smooth muscle cells. B: in cerebral artery smooth muscle cells, Ca_V1.2 subunits preferentially express exon 21 (n = 4). *P < 0.05 when compared with exon 21 expression. C: exon 32+33 is the major splice variant in cerebral artery smooth muscle cells (n = 5). *P < 0.05 when compared with exon 32 and exon 31+33 expression. Each n is the calculated mean of real-time PCR experiments that were performed in triplicate.

Fig. 4.

Electrophysiological properties of currents produced by exon 1b/c and ± 9* Ca_v1.2 splice variants. A: representative current traces generated by the exon 1c+9+9* channel variant in response to 1-s conditioning pulses to −80, +10, or +30 mV followed by 200-ms test pulses to 0 mV. B: current-voltage relationships of Ca_v1.2 e1b+9 (n = 21), Ca_v1.2 e1c+9 (n = 10), Ca_v1.2 e1b+9+9* (n = 7), Ca_v1.2 e1c+9+9* (n = 21) variants. C: mean steady-state inactivation of all Ca_v1.2 splice variants [see Table 3 for half-inactivation voltage of steady-state inactivation (V_1/2) values and experimental number]. D: mean current inactivation rate for all variants. I/I_max, normalized peak current.

Fig. 5.

Voltage-dependent activation of Ca_v1.2 splice variants. A: representative tail currents generated by the e1c+9+9* variant evoked by repolarization to −80 mV after depolarizing test pulses to −20, 0, +20, and +40 mV. B: mean voltage-dependent activation for all variants (see Table 3 for V_1/2 values and experimental number).

Fig. 6.

Window currents of Ca_v1.2 splice variants. Graphs illustrate the voltage-dependent relative window current (I/I_max) produced by channels containing exon 1b (black lines) or exon 1c (gray lines) and 9 or 9+9* as indicated.