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Article

From the Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101-6430

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 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.

Key Words: barium block • skeletal muscle • microscopic reversibility • asymmetric gating • preopening

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