Characterization of the large-conductance Ca-activated K channel in myocytes of rat saphenous artery

Abstract

We used the patch-clamp method to characterize the BK channel in freshly isolated myocytes from the saphenous branch of the rat femoral artery. Single-channel recordings revealed that the BK channel had a conductance of 187 pS in symmetrical 150 mM KCl, was blocked by external tetraethylammonium (TEA) with a KD(TEA) of approx. 300 microM at +40 mV, and by submicromolar charybdotoxin (CTX). The sensitivity of the BK channel to Ca was especially high (KD(ca) approx. 0.1 microM at +60 mV) compared to skeletal muscle and neuronal tissues. We also investigated the macroscopic K current, which under certain conditions is essentially sustained by BK channels. This conclusion is based on the findings that the macroscopic current activated upon depolarization follows a single exponential time course and is virtually fully blocked by 100 nM CTX and 5 mM external TEA. We made use of this occurrence to assess the voltage and Ca dependence of the macroscopic BK current. In intact myocytes, the BK channel showed a strong and voltage-dependent reduction of the outward current (62% at +40 mV), most likely due to block by intracellular Ba and polyamines. The results obtained from macroscopic and unitary current indicate that approx. 2.5% of the BK channels are active under physiological conditions, sustaining approx. 20 pA of outward current. Given the high input resistance of these cells, few BK channels are required to open in order to cause a significant membrane hyperpolarization, and thus function to limit the contraction resulting from acute increases in intravascular pressure, or in response to hypertensive pathologies.

Preserved BK Channel Function in Vasospastic Myocytes from a Dog Model of Subarachnoid Hemorrhage

“…The resulting slope factors resemble those reported for cloned dog BK channels expressed in human embryonic kidney cells (15.1 mV, α subunit alone compared to 14–15 mV in this study) [33] and oocytes (11.9–14.1 mV, α + β1 subunits) [34] and do not differ from those observed by other investigators in resistance cerebral arteries from rats (14–16 mV) [35, 36]. Additionally, investigators have found close agreement between the macroscopic methods employed here to determine BK voltage dependency and estimates from single-channel records from the same arterial tissue [11, 34]. …”

“…To minimize this, K + was lowered and replaced with choline. This method has been employed by other investigators when examining BK current voltage dependency [11]. Standard intracellular solution consisted of (in m M ): KCl 20, choline-Cl 120, MgCl 2 1.5, CaCl 2 2.65, EGTA 5, HEPES 5, glucose 5, pH 7.2 with KOH.…”

BK Channels – Focus on Polyamines, Ethanol/Acetaldehyde and Hydrogen Sulfide (H2S)

“…Blocking polyamine synthesis with the ornithine decarboxylase inhibitor DFMO (difluoromethylornithine) released BK channel rectification supporting the notion of a rectifying action imposed by polyamines. Similar data were reported for BK channels in myocytes from the saphenous branch of the rat femoral artery (Catacuzzeno et al, 2000). These discoveries remind to the mechanism of current rectification caused by polyamines at inward rectifier channels (K ir ) (Fakler et al, 1995).…”

Inhibition of endothelium-dependent vasorelaxation by extracellular K+: a novel controlling signal for vascular contractility

“…These results indicate that an increase of [K ϩ ]o in the physiological range (6-12 mM) inhibits [Ca 2ϩ ]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K ϩ efflux, and activating the Na ϩ -K ϩ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone. (5,7,29). This activation, in turn, induces an efflux of intracellular K ϩ .…”