Alternative splicing of Cav1.2 channel exons in smooth muscle cells of resistance-size arteries generates currents with unique electrophysiological properties.
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.
Cheng
Xiaoyang
X
Department of Physiology , University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Pachuau
Judith
J
Blaskova
Eva
E
Asuncion-Chin
Maria
M
Liu
Jianxi
J
Dopico
Alejandro M
AM
Jaggar
Jonathan H
JH
D017398:Alternative Splicing / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D002478:Cells, Cultured; D002536:Cerebral Arteries / Q000166:cytology*; D002560:Cerebrovascular Circulation / Q000502:physiology*; D005091:Exons / Q000235:genetics; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D014655:Vascular Resistance / Q000502:physiology
C504728:Cacna1c protein, rat; D020746:Calcium Channels, L-Type