Molecular Characterization of Large Conductance, Ca2+‐activated, K+ Channels (BK) in Arteries from Cerebral and Skeletal Muscle Vasculatures

Abstract

Previous studies have shown functional differences in BK of smooth muscle cells (VSMC) from cerebral and cremaster muscle vasculatures. Specifically, BK from cremaster showed decreased Ca2+ sensitivity resulting in equivalent channel opening at more positive membrane potential compared to cerebral VSMC. Studies were performed to determine if variation in molecular features of BK contributes to differences in activity. The existence of splice variants in the intracellular C terminus domain of the α‐subunit was examined by RT‐PCR. Expression of β1, β2, β3 and β4 subunits was also determined. Total RNA was extracted from isolated arterioles and quantified by absorbance (260/280nm). Equal amounts of RNA were reverse‐transcribed into cDNA and quantitative‐PCR was performed. Two α‐subunit variants, Zero (without splice insert) and STREX‐1 (with a 58 amino acid insert at splice site 2), were identified as identical in cremaster and cerebral arteries by sequencing of RT‐PCR products. Q‐PCR showed the β1 subunit to be the predominant accessory subunit expressed in both vessels. A low level of β4 compared to β1 was observed in both preparations. No evidence was obtained for either β2 or β3 mRNA expression. The results suggest the molecular features of BK are likely similar in the two arteries. Differences in functional properties may relate to subunit stoichiometry of the intact channel or post‐translational mechanisms.