Immobilizing the moving parts of voltage-gated ion channels

Voltage-gated ion channels have at least two classes of moving parts, volta ge sensors that respond to changes in the transmembrane potential and gates that create or deny permeant ions access to the conduction pathway. To exp lore the coupling between voltage sensors and gates, we have systematically immobilized each using a bifunctional photoactivatable cross-linker, benzo phenone-4-carboxamidocysteine methanethiosulfonate, that call be tethered t o cysteines introduced into the channel protein by mutagenesis. To validate the method, we first tested it on the inactivation gate of the sodium chan nel. The benzophenone-labeled inactivation gate of the sodium channel can b e trapped selectively either in an open or closed state by ultraviolet irra diation at either a hyperpolarized or depolarized voltage, respectively. To verify that ultraviolet light can immobilize S4 segments, we examined its relative effects on ionic and gating currents in Shaker potassium channels, labeled at residue 359 at the extracellular end of the S4 segment. As pred icted by the tetrameric stoichiometry of these potassium channels, ultravio let irradiation reduces ionic current by approximately the fourth power of the gating current reduction, suggesting little cooperativity between the m ovements of individual S4 segments. Photocross-linking occurs preferably at hyperpolarized voltages after labeling residue 359, suggesting that depola rization moves the benzophenone adduct out of a restricted environment. Imm obilization of the S4 segment of the second domain of sodium channels preve nts channels from opening. By contrast, photocross-linking the S4 segment o f the fourth domain of the sodium channel has effects on both activation an d inactivation. Our results indicate that specific voltage sensors of the s odium channel play unique roles in gating, and suggest that movement of one voltage sensor, the S4 segment of domain 4. is at least a two-step process , each step coupled to a different gate.