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 p ositively 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). Mo vement of the voltage sensor in response to a change in the membrane potent ial was examined indirectly by measuring how the accessibilities of residue s in and around the sensor change with voltage. Each basic residue in the S 4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the volt age sensor will include the histidine charge. Accessibility of the histidin e to the bulk solution was therefore monitored as pH-dependent changes in t he gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since his tidine accessibility is detected by labeling with solution protons, very co nfined local environments can be resolved and labeling introduces minimal i nterference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was not 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. Histidin e replacement of residues R365, R368, and R371, on the other hand, showed t hat each of these residues traverses, entirely from internal exposure at hy perpolarized potentials to external exposure at depolarized potentials. Thi s translocation enables the histidine to transport protons across the membr ane in the presence of a pH gradient. In the case of 371H, depolarization d rives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduc tion are presented. Finally, the results presented here are incorporated in to existing information to propose a model of voltage sensor movement and s tructure.