The Shaker potassium channel is a member of the six transmembrane family of voltage-gated potassium channels. Consisting of a voltage-sensing domain (S1-S4) and a pore domain (S5-P-S6), these channels form functional tetrameric channels that activate in response to changes in membrane potential. The positively charged S4 segment is believed to move out of the transmembrane field upon depolarisation, but the exact nature of this movement or the mechanism by which it is translated into opening of the pore activation gate is unknown. There is recent evidence that the N-terminal end of S4 moves close to the pore domain during activation (Elinder et al. 2001).

Using the Cd²⁺ metal bridge approach, we recently demonstrated (Elliott et al. 2002) that four N-terminal residues in S4 (L358, A359, I360 and L361) approach residue E418, just outside S5. This interaction occurs during membrane depolarisation. It has also been demonstrated that S4 residues following L361 do not appear to move close to E418. Here we have examined the two preceding positions (S357 and M356) at the extracellular end of S4 to investigate whether these two residues are also capable of interacting with E418.

Single S4C and double S4C-E418C mutations were introduced into the N-type inactivation-removed Shaker channel. The mutant channels were expressed in Xenopus oocytes and potassium currents were recorded using the two-electrode voltage-clamp technique. These currents were compared in the absence and presence of 100 mM extracellular Cd²⁺, which will form metal bridges between two or more residue side-chains (C, H, M, E or D) if they are approximately 4 ü apart.

The application of Cd²⁺ to the S357C-E418C double mutant but not the corresponding single mutant channels (S357C or E418C) resulted in fast, almost complete loss of conductance (n = 3). However, Cd²⁺ slowed the activation kinetics of the S357C single mutant, and the conductance-voltage curve was also shifted by +22 mV. Cd²⁺ had no effect on the M356C-E418C mutant. The Cd²⁺ effect on S357C-E418C was complete even after holding the oocytes at -120 mV during application (n = 3).

Since residues S357-L361 form a complete helical turn, the data are consistent with the evidence that S4 rotates during depolarisation. Taken together with our previous data, we propose that residue S357 in the extracellular end of S4 is close to E418 in the extracellular end of S5, in the closed state of the channel, but that during depolarisation the subsequent residues move towards E418.

This work was supported by The Wellcome Trust.

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