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Selective modulation of ion channel subunit expression to probe regional differences in vascular smooth muscle BKCa function

M.A. Hill,1 Y. Yang,1 Y. Sohma,2 Z. Nourian,1 M.J. Davis1 and A.P. Braun,3 1Dalton Cardiovascular Research Center and Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri 65211, USA, 2Dept of Pharmacology, Keio University, Tokyo 160-8582, Japan and 3Dept of Physiology and Pharmacology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

β1-subunits enhance the gating properties of BKCa channels formed by α-subunits. In arterial vascular smooth muscle cells (VSMC) β1-subunits are vital in coupling SR–generated Ca2+ sparks to BKCa activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMC show heterogeneity of BKCa activity due to apparent differences in the β1:α subunit ratio. To define these differences studies were conducted at the single channel level while siRNA was used to manipulate specific subunit expression.

Methods and Results: β1 modulation of the α-subunit Ca2+ sensitivity was studied using patch clamp techniques. Significant leftward shifts in BKCa channel open probability (NP0) versus membrane potential (Vm) curves (at [Ca2+]i from 0.5 to 100μM), were observed in cerebral versus cremaster VSMC. As [Ca2+]i increased from 0.5 to 100μM, the V1/2 values of channels decreased from 72.0 ± 6.1 to −89 ± 9mV in cerebral compared to 101 ± 10 to −63 ± 7mV in cremaster VSMC. Ca2+ set points (Ca0) were 12.1 and 5.0μM in cremaster and cerebral VSMC, respectively. Thus, at Vm of −30mV, a mean [Ca2+]i of 39μM was required to open half of the channels in cremaster versus 16μM [Ca2+]i in cerebral VSMC. Further, shortened mean open and longer mean closed times were evident in BKCa events from cremaster VSMC at either −30 or 30mV and any given [Ca2+]. Uptake of siRNA into VSMCs was verified by studies of both a fluorescently labeled unrelated siRNA and β-subunit directed siRNA. Further, Western blotting confirmed a decrease in protein subunit expression. siRNA directed at the α subunit caused a decrease in BKCa function in both cell types. β-subunit directed siRNA decreased the Ca2+ sensitivity of BKCa in cerebral VSMCs and the appearance of STOCs such that the cells more closely resembled the activity seen in cremaster VSMC.

Conclusion: The data are consistent with a higher ratio of β1:α subunit of BKCa channels in cerebral compared to cremaster VSMC. Functionally, this leads to both higher Ca2+ sensitivity and NPo for BKCa in the cerebral vasculature relative to that of skeletal muscle.