Cav1.2 splice variant with exon 9* is critical for regulation of cerebral artery diameter

Abstract

L-type voltage-dependent Ca2+channels (VDCCs) are essential for numerous processes in the cardiovascular and nervous systems. Alternative splicing modulates proteomic composition of Cav1.2 to generate functional variation between channel isoforms. Here, we describe expression and function of Cav1.2 channels containing alternatively spliced exon 9* in cerebral artery myocytes. RT-PCR showed expression of Cav1.2 splice variants both containing (α1C9/9*/10) and lacking (α1C9/10) exon 9* in intact rabbit and human cerebral arteries. With the use of laser capture microdissection and RT-PCR, expression of mRNA for both α1C9/9*/10and α1C9/10was demonstrated in isolated cerebral artery myocytes. Quantitative real-time PCR revealed significantly greater α1C9/9*/10expression relative to α1C9/10in intact rabbit cerebral arteries compared with cardiac tissue and cerebral cortex. To demonstrate a functional role for α1C9/9*/10, smooth muscle of intact cerebral arteries was treated with antisense oligonucleotides targeting α1C9/9*/10(α1C9/9*/10-AS) or exon 9 (α1C-AS), expressed in all Cav1.2 splice variants, by reversible permeabilization and organ cultured for 1–4 days. Treatment with α1C9/9*/10-AS reduced maximal constriction induced by elevated extracellular K+([K+]o) by ∼75% compared with α1C9/9*/10-sense-treated arteries. Maximal constriction in response to the Ca2+ionophore ionomycin and [K+]oEC50values were not altered by antisense treatment. Decreases in maximal [K+]o-induced constriction were similar between α1C9/9*/10-AS and α1C-AS groups (22.7 ± 9% and 25.6 ± 4% constriction, respectively). We conclude that although cerebral artery myocytes express both α1C9/9*/10and α1C9/10VDCC splice variants, α1C9/9*/10is functionally dominant in the control of cerebral artery diameter.