Abstract
Ba
2+
block of large conductance Ca
2+
-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 Ba
2+
ion would dissociate to the external solution (150 mM
N
-methyl-
d
-glucamine
+
o
, 500 mM K
+
i
, 10 μM Ba
2+
i
, +30 mV, and 100 μM Ca
2+
i
to fully activate the channel), Ba
2+
blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba
2+
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 Ba
2+
blocks. Thus, the preopenings represent Ba
2+
-induced time-irreversible subconductance gating. The fraction of Ba
2+
blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba
2+
]
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 Ba
2+
was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba
2+
o
and no added Ba
2+
i
. Adding K
+
, Rb
+
, or Na
+
to the external solution decreased the fraction of Ba
2+
blocks with preopenings, with K
+
and Rb
+
being more effective than Na
+
. These results are consistent with models in which the blocking Ba
2+
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 Ba
2+
blocking site and acts directly as the preopening gate.
