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

Nystoriak MA, Murakami K, Penar PL, Wellman GC. Ca(v)1.2 splice variant with exon 9* is critical for regulation of cerebral artery diameter. Am J Physiol Heart Circ Physiol 297: H1820-H1828, 2009. First published August 28, 2009; doi:10.1152/ajpheart.00326.2009.-L- type voltage-dependent Ca2+ channels (VDCCs) are essential for numerous processes in the cardiovascular and nervous systems. Alternative splicing modulates proteomic composition of Ca(v)1.2 to generate functional variation between channel isoforms. Here, we describe expression and function of Ca(v)1.2 channels containing alternatively spliced exon 9* in cerebral artery myocytes. RT-PCR showed expression of Ca(v)1.2 splice variants both containing (alpha C-1(9/9*/10)) and lacking (alpha C-1(9/10)) exon 9* in intact rabbit and human cerebral arteries. With the use of laser capture microdissection and RT-PCR, expression of mRNA for both alpha C-1(9/9*/10) and alpha C-1(9/10) was demonstrated in isolated cerebral artery myocytes. Quantitative real-time PCR revealed significantly greater alpha C-1(9/9*/10) expression relative to alpha C-1(9/10) in intact rabbit cerebral arteries compared with cardiac tissue and cerebral cortex. To demonstrate a functional role for alpha C-1(9/9*/10), smooth muscle of intact cerebral arteries was treated with antisense oligonucleotides targeting alpha C-1(9/9*/10) (alpha C-1(9/9*/10)-AS) or exon 9 (alpha C-1-AS), expressed in all Ca(v)1.2 splice variants, by reversible permeabilization and organ cultured for 1-4 days. Treatment with alpha C-1(9/9*/10)-AS reduced maximal constriction induced by elevated extracellular K+ ([K+](o)) by similar to 75% compared with alpha C-1(9/9*/10)-sense-treated arteries. Maximal constriction in response to the Ca2+ ionophore ionomycin and [K+](o) EC50 values were not altered by antisense treatment. Decreases in maximal [K+](o)-induced constriction were similar between alpha C-1(9/9*/10)-AS and alpha C-1-AS groups (22.7 +/- 9% and 25.6 +/- 4% constriction, respectively). We conclude that although cerebral artery myocytes express both alpha C-1(9/9*/10) and alpha C-1(9/10) VDCC splice variants, alpha C-1(9/9*/10) is functionally dominant in the control of cerebral artery diameter.