Measurement of the movement of the S4 segment during the activation of a voltage-gated potassium channel.
Voltage-gated ion channels contain a positively charged transmembrane segment termed S4. Recent evidence suggests that depolarisation of the membrane potential causes this segment to undergo conformational changes that, in turn, lead to the opening of the channel pore. In order to define these conformational changes in structural terms, we have introduced single cysteine substitutions into the S4 segment of the prototypical Shaker K+ channel at various positions and expressed the mutants in Xenopus oocytes. The cells were depolarised to induce K+ currents and the effect of application of 100 microM parachloromercuribenzenesulphonate (PCMBS) on these currents was examined by the two-electrode voltage-clamp technique. PCMBS inhibited K+ currents elicited by mutants L358C, L361C, V363C and L366C, but not those by V367C and S376C. Since PCMBS is a membrane-impermeable cysteine-modifying reagent, the data suggest that depolarisation must have caused the S4 segment to move out of the lipid bilayer into the extracellular phase rendering the residues at positions 358, 361, 363 and 366 susceptible to PCMBS attack. The lack of effect of PCMBS on V367C suggests that the exposure of S4 terminates at L366. Detailed analysis of L361C mutant revealed that the S4 movement can occur even below the resting potential of the cell, at which potential voltage-gated K+ channels are normally in a non-conducting closed state.
Yusaf
S P
SP
Department of Pharmacology, University of Leeds, Leeds, LS2 9JT, UK.
Wray
D
D
Sivaprasadarao
A
A
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D004594:Electrophysiology; D005260:Female; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*
D015221:Potassium Channels; D002731:4-Chloromercuribenzenesulfonate