Hidden Markov model analysis of intermediate gating steps associated with the pore gate of Shaker potassium channels

Cooperativity among the four subunits helps give rise to the remarkable vol tage sensitivity of Shake potassium channels, whose open probability change s tenfold for a 5-mV change in membrane potential. The cooperativity in the se channels is thought to arise from a concerted structural transition as t he final step in opening the channel. Recordings of single-channel ionic cu rrents from certain other channel types, as well as our previous recordings from T442S mutant Shaker channels, however, display intermediate conductan ce levels in addition to the fully open arid closed states. These sublevels might represent stepwise, rather than concerted, transitions in the final steps of channel activation. here, we report a similar fine structure in th e closing transitions of Shaker channels lacking the mutation. Describing t he deactivation time course oath hidden Markov models, we find that two sub conductance levels are rapidly traversed during most closing transitions of chimeric, high conductance Shaker channels. The lifetimes of these levels are voltage-dependent, with maximal values of 52 and 22 its at -100 mV, and the voltage dependences of transitions among these states suggest that the y anise from equivalent conformational changes occurring in individual subu nits. At least one subconductance level is found to be traversed in normal conductance Shaker channels. We speculate that voltage-dependent conformati onal changes in the subunits give rise to changes in a "pore gate" associat ed with the selectivity filter region of the channel, producing the subcond uctance states. As a control for the hidden Markov analysis, we applied the same procedures to recordings of the recovery from N-type inactivation in Shaker channels. These transitions are found to be instantaneous in compari son.