Ca2+- and Voltage-Dependent Gating of Ca2+- and ATP-Sensitive Cationic Channels in Brain Capillary Endothelium

AbstractBiophysical properties of the Ca2+-activated nonselective cation channel expressed in brain capillaries were studied in inside-out patches from primary cultures of rat brain microvascular endothelial cells. At −40mV membrane potential, open probability (Po) was activated by cytosolic [Ca2+]>1μM and was half-maximal at ∼20μM. Increasing [Ca2+] stimulated opening rate with little effect on closing rate. At constant [Ca2+], Po was voltage-dependent, and effective gating charge corresponded to 0.6±0.1 unitary charges. Depolarization accelerated opening and slowed closing, thereby increasing apparent affinity for Ca2+. Within ∼1min of excision, Po declined to a lower steady state with decreased sensitivity toward activating Ca2+ when studied at a fixed voltage, and toward activating voltage when studied at a fixed [Ca2+]. Deactivated channels opened ∼5-fold slower and closed ∼10-fold faster. The sulfhydryl-reducing agent dithiotreitol (1mM) completely reversed acceleration of closing rate but failed to recover opening rate. Single-channel gating was complex; distributions of open and closed dwell times contained at least four and five exponential components, respectively. The longest component of the closed-time distribution was markedly sensitive to both [Ca2+] and voltage. We conclude that the biophysical properties of gating of this channel are remarkably similar to those of large-conductance Ca2+-activated K+ channels