Time-irreversible subconductance gating associated with Ba(2+) block of large conductance Ca(2+)-activated K(+) channels

Ion channels play an integral role in regulating the movement of ions across cell membranes. In addition to the fully open and closed conductance states, most ion channels enter partially open (subconductance) states. Although previous observations in several channels indicate that transitions to and from subconductance states can violate microscopic reversibility, little is known about the mechanism(s) by which this occurs. In this study, I show that Ba2+ can block and induce irreversible subconductance gating in BK channels. Ba2+ enters and blocks the channel for typically 1--15 s, after which Ba2+ unblocks. When NMDG+ is the only cation present in the external solution, Ba2+ preferentially unblocks to the external solution. Inspection of the blocking and unblocking transitions reveals that Ba2+ always blocks the channel in a single step, but only 78% of the time unblocks in a single step. Twenty-two percent of the unblocking transitions contain a subconductance level (preopening) with a mean duration of 0.75 ms and with a conductance which is 26% that of the fully open channel at +30 mV. The fraction of blocks with a preopening, their mean duration, and fractional conductance all appeared to be independent of the internal Ba2+ concentrations over the range of 0.1 muM to 10 mM. The fraction of blocks with preopenings and their mean duration were also independent of the membrane potential over the range of +10 to +90 mV. The fractional conductance of the preopenings increased from 0.23 to 0.39 over this range of membrane potential. Addition of 100 mM K+, Rb+, or Na+ to the external solution, ions which are known to force Ba2+ to unblock to the internal solution, decreased the fraction of blocks with a preopening to 0%, 0%, and 5%, respectively. When 10 mM external Ba 2+ was used to block the channel, preopenings were still observed, but in reduced numbers. These results are consistent with models in which the blocking Ba2+ ion either induces a preopening gate before dissociating to the external solution, or moves to a site located on the external side of the Ba2+ blocking site where it acts directly as the preopening gate.The full text of two additional studies performed for this dissertation can be found in Appendices I and II