The Shaker voltage-gated potassium channel consists of a central ion conducting pore domain (S5-P-S6) surrounded by a voltage-sensing domain (S1-S4). During depolarisation the voltage-sensing S4 transmembrane segment undergoes a series of motions that lead to opening of the activation gates situated in the pore domain. Here we report the identification of S4 residues that contribute to the stabilisation of the activated state of the channel.

We have previously reported that Cd²⁺ causes a leftward shift in the G-V curve and a slowing of deactivation kinetics of Shaker channels bearing R365C and R368C mutations (Bannister et al. 2000). Here we investigate the effects of Cd²⁺ on cysteine mutants of residues at 356 to 364 of the S4. For this, we expressed mutant channels in Xenopus oocytes and measured currents using two-electrode voltage clamp. 100 mM Cd²⁺ caused leftward shifts in the G-V curves of the L358C and L361C mutant channels, similar to R365C and R368C. The effect at 100 mM concentration indicates Cd²⁺ co-ordination between the S4 cysteines and another co-ordinating residue. Other mutants were either unaffected or showed rightward shifts.

Further studies on L361C showed that extracellular application of Cd²⁺ caused a slowing of activation kinetics, a leftward shift in the G-V curve (-36.2 ± 0.7 mV), and a slowing of the deactivation kinetics (t_d before and after Cd²⁺ were 0.28 ± 0.02 and 14.86 ± 2.82 s, respectively, means ± S.E.M., n = 4). Voltage dependence studies showed that Cd²⁺ may bind to the L361C channels in the closed state but not in the open state.

Effect of Cd²⁺ on a double mutant L361C-R365C was examined. This mutant displayed a leftward shift in its G-V curve (-71.7 ± 13.4 mV, n = 5) similar to the corresponding single mutants. However, the maximal effects were observed at a Cd²⁺ concentration that is 100-fold lower than that required for the single mutants, indicating a high-affinity binding that may involve both the S4 cysteines and an additional, yet to be identified, liganding residue in the channel.

The leftward shifts and slowing of deactivation kinetics reflect stabilisation of the open state of the channel. Since positions 358 and 361 lie on the same face of S4 as 365 and 368, the present data together with the previous work (Bannister et al. 2000) suggest that the face of S4 comprising positions 358, 361, 365 and 368 may contribute to the stabilisation of the activated state of the channel. The question that remains to be addressed is the identification of the residues in the channel with which these residue(s) interact.

The work was supported by The Wellcome Trust.

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