Histidine scanning mutagenesis of basic residues of the S4 segment of the shaker k+ channel.

The voltage sensor of the Shaker potassium channel is comprised mostly of positively charged residues in the putative fourth transmembrane segment, S4 (Aggarwal, S.K., and R. MacKinnon. 1996. Neuron. 16:1169-1177; Seoh, S.-A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Neuron. 16:1159-1167). Movement of the voltage sensor in response to a change in the membrane potential was examined indirectly by measuring how the accessibilities of residues in and around the sensor change with voltage. Each basic residue in the S4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the voltage sensor will include the histidine charge. Accessibility of the histidine to the bulk solution was therefore monitored as pH-dependent changes in the gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since histidine accessibility is detected by labeling with solution protons, very confined local environments can be resolved and labeling introduces minimal interference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible. Histidine replacement of residues R365, R368, and R371, on the other hand, showed that each of these residues traverses entirely from internal exposure at hyperpolarized potentials to external exposure at depolarized potentials. This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient. In the case of 371H, depolarization drives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduction are presented. Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.