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Action of halothane on large-conductance, calcium-activated potassium channels in rat cerebrovascular smooth muscle cells Yan, Hong


Halothane anesthesia is associated with increased cerebral blood flow and dilatation of cerebral vessels. The mechanisms which underlie this drug action are presently unclear. Cerebrovascular tone is probably regulated in part by the opening of large conductance, Ca²⁺-activated potassium channels (BK channels) in cerebral artery smooth muscle cells. The present experiments utilized extra cellular patch clamp techniques to investigate whether clinically relevant concentrations of halothane directly alter the biophysical properties of these channels. Cerebrovascular smooth muscle cells (CVSMCs) were dispersed from the basilar, middle, posterior communicating, and posterior cerebral arteries of adult Wistar rats using collagenase and trypsin and maintained in vitro for 48 hrs prior to use. Recordings were made from isolated inside-out membrane patches at room temperature (21-23°C) using a List EPC-5 patch clamp amplifier. Under control conditions, amplitude distributions of single BK channel currents were well described by a single Gaussian term. This behavior was maintained during exposure to halothane. The mean conductance of single BK channels, which was 194 ± 6.1 pS in symmetrical 140 mM K⁺ solutions, was unchanged by application of halothane at any of the concentrations tested (0.5, 1.6, 2.8 mM). Halothane caused a dose-dependent, reversible decrease in the open probability (Po) of BK channels. Halothane reduced Po by 14 % and 55 % on application of 1.6 mM (n=11) and 2.8 mM (n=11) halothane respectively, while 0.5 mM halothane had no significant effect on the open probability (n=7). Kinetic analysis of BK channel currents showed that halothane altered the gating of these channels. Halothane reduced the mean channel open time by 23 % and 56 %, and increased the mean channel closed time by factors of 2.1 and 9.3 when applied at concentrations of 1.6 mM or of 2.8 mM, respectively. The inhibitory effect of halothane on BK channel function is unlikely to result solely from the fluidization of membrane lipids by the anesthetic, since this would probably increase the channel opening probability. Rather, halothane appears to alter BK channel function by binding to hydrophobic domains within the channel protein, or by interfering with protein-lipid interactions in the membrane. A halothane-induced decrease in the open probability of BK channels in CVSMCs might be expected to reduce outward potassium current, resulting in enhanced contraction of blood vessel walls. Hence, the direct inhibitory effects of halothane on BK channels obtained from cerebral artery cells cannot explain the marked cerebral vasodilation caused by the anesthetic. This vasodilation must therefore result from other drug actions on vascular smooth muscle cells, which include reduction in calcium influx through voltage-dependent calcium channels, decreased accumulation of intracellular free calcium, and lowered sensitivity of contractile proteins to calcium.

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