An investigation of the properties of large-conductance Ca^2+-activated K^+ channels of rat arterial smooth muscle and their modulation by vasoconstrictors

Large-conductance Ca^2+ -activated K^+ (BKCa) channels play an important role in the regulation of vascular tone. They are activated by membrane depolarization and increases in local Ca^2+ concentration ([Ca^2+]). Their location in the plasma membrane allows them to be activated by transient releases of Ca^2+ from ryanodine receptors (RyR) in the sarcoplasmic reticulum, termed Ca^2+ sparks, leading to the efflux of K^+ known as a spontaneous transient outward current (STOC). Activation of BKCa channels in this manner provides a negative feedback mechanism to regulate vasoconstriction by hyperpolarizing the cell membrane and so reducing Ca^2+ influx through L-type voltage dependent Ca2+ channels. In this thesis I have investigated the relationship between [Ca^2+]i and membrane potential using inside-out patches excised from smooth muscle cells isolated from rat mesenteric artery. Whole-cell BKCa currents in these cells were also investigated both in the form of STOCs and by using voltage pulses to activate BKCa channels. The effects of the vasoconstrictors endothelin-1 (ET- 1) and angiotensin II (Ang II) on both pulse-induced BKCa currents and STOC amplitude and frequency were investigated. Single BKCa channels with a slope conductance of 189 pA were recorded and their activation was shown to be dependent on [Ca^2+]i and membrane potential. Membrane depolarization also increased BKCa whole-cell current and the frequency and amplitude of STOCs. ET-1 and Ang II were found to inhibit pulse-induced BKCa currents and this effect of ET-1 could be inhibited using a peptide PKC inhibitor. ET-1 and Ang II also caused a decrease in both STOC amplitude and frequency, although the decrease in frequency may be the result of the reduction in amplitude. Finally, 1, 2-dioctanoyl-sn-glycerol (DOG), an analogue of the endogenous PKC activator diacylglycerol (DAG), was seen to inhibit both BKCa whole-cell and single channel currents, possibly due to direct inhibition of BKCa channels.