Kinetics of voltage- and Ca2+ activation and Ba2+ blockade of a large-conductance K+ channel from Necturus enterocytes

Abstract

Potassium channels in membranes of isolated Necturus enterocytes were studied using the patch-clamp technique. The most frequent channel observed had a conductance of 170 pS and reversal potential of 0 mV in symmetrical potassium-rich solutions. Channels were highly K- selective. Channel activity was modulated by membrane potential and cytosolic Ca2+ concentration. Channel openings occurred in characteristic bursts separated by long closures. During bursts openings were interrupted by brief closures. Two gating modes controlled channel opening. The primary gate's sensitivity to intracellular Ca2+ concentration and membrane potential crucially determined long duration closures and bursting. In comparison, the second gate determining brief closures was largely insensitive to voltage and intracellular Ca2+ concentration. The channel was reversibly blocked by cytosolic barium exposure in a voltage-sensitive manner. Blockade reduced open-state probability without altering single-channel conductance and could be described, at relatively high Ca2+ concentration, by a three-state model where Ba2+ interacted with the open channel with a dissociation constant of about 10(-4) M at 0 mV.