Time-irreversible subconductance gating associated with Ba²⁺ block of large conductance Ca²⁺-activated K⁺ channels

Ba²⁺ block of large conductance Ca²⁺-activated K⁺ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba²⁺ ion would dissociate to the external solution (150 mM N-methyl-D- glucamine⁺(o), 500 mM K⁺(i), 10 μM Ba²⁺(i), +30 mV, and 100 μM Ca²⁺(i) to fully activate the channel), Ba²⁺ blocks with a mean duration of ~2 s occurred, on average, once every ~100 ms of channel open time. Of these Ba²⁺ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ~0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ~10,000 Ba²⁺ blocks. Thus, the preopenings represent Ba²⁺-induced time- irreversible subconductance gating. The fraction of Ba²⁺ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba²⁺](i) or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during the normal gating in the absence of Ba²⁺ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba²⁺(o) and no added Ba²⁺(i). Adding K⁺, Rb⁺, or Na⁺ to the external solution decreased the fraction of Ba²⁺ blocks with preopenings, with K⁺ and Rb⁺ being more effective than Na⁺. These results are consistent with models in which the blocking Ba²⁺ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba²⁺ blocking site and acts directly as the preopening gate.