β2 and β3a subunits coassemble in single BK channels. (A) The indicated voltage protocol was used to activate single channels in excised patches (10 μM cytosolic Ca²⁺). Traces are from one patch with a single β2-containing BK channel, with ensemble average of all sweeps from the patch shown on bottom. For these 10 trials, 10 openings result in inactivation, but in 9 of 10 trials, the channel recovers from inactivation without reopening during the 20 ms repolarization to −160 mV. (B) Single-channel traces from a patch from an oocyte injected with a 1:8 β2:β3a injection ratio. 3 of 10 trials exhibit an instantaneous reopening upon repolarization with a reduced current amplitude (β3a-like; red stars) compared with a full BK tail opening. 5 of 10 trials show recovery from inactivation without any reopening during the repolarization (β2-like). (C) For a patch expressing a single β3a-containing BK channel, 9 of 10 traces show instantaneous reopening upon repolarization, with the reduced current amplitude relative to a full BK tail opening, The 5^th and 10^th traces show a brief full BK opening at the end of the reduced current burst. (D) Likelihood of observing β3a reduced conductance tail openings over all trials is plotted for 5 patches from β2-only patches, 16 patches from 1:8 β2:β3a injection, and 6 patches from β3a-only. (E) Red symbols plot likelihood of β3a-tail openings for the channels from the 16 β2:β3a patches, arranged in accordance with 5 potential stoichiometric assemblies: 4:0, 3:1, 2:2, 1:3, and 0:4 (β3a:β2). Lines plot predictions based on different ratios of likelihood (from 1.0 to 4.0) that a β3a N terminus produces inactivation at the time of repolarization relative to a β2 N terminus, assuming that this is defined solely by differences in rate of inactivation onset.