| Several aspects of the molecular mechanisms of calcium- and voltage-activated potassium channels, or BK channels, were investigated in this thesis. The molecular mechanism of this ion channel was probed by several different methods. First of all, the role of different permeant ions in the opening and closing of the channel was assessed. Currents were recorded on a single-channel and macroscopic levels with potassium replaced by an alternate permeant ion, thallium. Thallium was found to inhibit the opening of the channel, causing the channel to require higher potentials and internal calcium concentrations to open. Also, thallium decreased the opening and closing kinetics. Lastly, at the single channel level, thallium decreased the mean open time and increased the incidence of a short-lived flicker state. Current allosteric gaitng models that can describe the gating behavior of BK channels were applied (Horrigan and Aldrich, JGP, 2002), and thallium was found to alter only the energy between the closed and open states. The effect of thallium on the gating of BK channel can be completely accounted for by the addition of a new flicker state in the model. This flicker state may correspond to the collapsed selectivity filter seen in crystal structures of KcsA (Zhou and MacKinnon, JMB, 2003). Additionally, a severe mutant of BK channels was studied. This mutant had the entire intracellular C-terminus removed. Surprisingly, the calcium and voltage activation of this truncated channel was unaltered. These results indicate that the intracellular C-terminus is not required for the gating of this channel, but may play a role in the trafficking or assembly of the channels. |
