Characterization of the high-conductance Ca²⁺-activated K⁺ channel in adult human skeletal muscle

Ca²⁺-activated K⁺ channels of a large conductance (BK_Ca) in human skeletal muscle were studied by patch clamping membrane blebs and by using the three microelectrode voltage-clamp recording technique on resealed fibre segments. Single-channel recordings in bleb-attached and inside-out modes revealed BK_Ca conductances of 230 pS for symmetrical and 130 pS for physiological K⁺ distributions. Open probability increased with membrane depolarization and increasing internal [Ca²⁺]. The Hill coefficient was 2.0, indicating that at least two Ca²⁺ ions are required for full activation. Kinetic analysis revealed at least two open and three closed states. An additional long-lived inactivated state, lasting about 0.5-20 s, was observed following large depolarizations, when extracellular K⁺ was lowered to physiological values. BK_Ca were blocked by three means: (1) externally by tetraethylammonium which reduced single-channel amplitude (IC₅₀ approx. 0.3 mM); (2) internally by polymyxin B which decreased the open probability (IC₅₀ approx. 5 μg/ml); and (3) externally by charybdotoxin which caused long-lasting periods of inactivation (IC₅₀ <10 nM). Measurements on resealed fibre segments at physiological [K⁺] were in accordance with the single-channel data: only when intracellular [Ca²⁺] was elevated did charybdotoxin (50 nM) reduce the macroscopic membrane K⁺ conductance with depolarizing voltage steps.