K+ channels open and close, a process described as """"""""gating"""""""", in response to signals as varied as membrane potential or ligand binding. This tightly controlled activity is critical to the biological role the channels play, for example, in action potential generation and propagation. The molecular details of how a channel opens and closes remain unknown. To date, the only K+ channel studied by structural techniques is the bacterial KcsA channel from Streptomyces lividans. However, the dearth of functional data from KcsA limits the inferences about function merely to analogy with the eukaryotic channels. The overall objective of this proposal is to characterize the gating properties of purified reconstituted KcsA K+ channel using electrophysiological techniques. Our first major aim is to determine the molecular basis of pH sensitivity. KcsA is activated by protons, but the mechanism underlying this activation and the identity of the pH sensor are unknown. We will detail the pH dependence of KcsA, and examine how proton activation is coupled to the presence of permeant ions within the pore. Using both chemical modification and site-specific mutagenesis, we will identify residues that form the pH sensor. Our second major aim is to examine the origin of voltage-dependence in KcsA. Although KcsA does not contain an S4 sequence, it is sensitive to voltage. We will quantify this voltage sensitivity, and then examine the ionic contribution to this voltage-dependence. Our third major aim is to examine the functional consequences of structural rearrangements of the inner vestibule during gating. First we will gauge the dimensions of the inner vestibule of the open channel using a series of pore blockers. Then we will examine how accessibility to the blocking site changes with gating.
| Silverman, William R; Heginbotham, Lise (2007) The MlotiK1 channel transports ions along the canonical conduction pore. FEBS Lett 581:5024-8 |
