Effect of cysteine substitutions on the topology of the S4 segment of the Shaker potassium channel: implications for molecular models of gating

1. The gating properties of voltage-gated potassium channels are largely de termined by the amino acid sequence of their S4 segments. To investigate th e nature of S4 movement during gating, we introduced single cysteines into the S4 segment of the Shaker potassium channel and expressed the mutants in Xenopus oocytes. We then measured the conductance-voltage (g-V) relationsh ips and the rate and the voltage dependence of movement of the engineered c ysteines, using p-chloromercuribenzene sulphonate (pCMBS) as a probe. 2. Mutation of charged residues at positions 362, 365 and 368, but not the uncharged residues (positions 360, 361, 363, 364 and 366), to cysteines shi fted the g-V relationships to more positive potentials. Mutant channels in which cysteines replaced the charged residues at positions 362 and 365 (R36 2C and R365C) reacted faster with pCMBS than those in which cysteines were introduced in place of uncharged residues at positions 360 and 361 (I360C a nd L361C). Furthermore, the R365C mutant channel reacted with pCMBS even at hyperpolarised (-120 mV) potentials. Currents expressed by the doubly muta ted R365S/V367C and R368S/V367C channels, but not the singly mutated V367C channel, were inhibited by pCMBS. Moreover, the R368C mutant channel was al so affected by pCMBS. 3. Voltage dependence of block by pCMBS (2 min exposure) was steeper for L3 66C than for L361C and V363C mutant channels (effective charge 2.19, 1.41 a nd 1.45, respectively). The voltage dependence of the pCMBS effect was also shifted to more depolarising potentials the deeper in the membrane the pos ition of the residue mutated to cysteine (voltages for half-maximal effect -107, -94 and -73 mV for positions 361, 363 and 366, respectively). 4. Our data show firstly that charge-neutralising mutations in S4 alter the topology of this region such that the membrane-spanning portion of S4 is r educed. Secondly, our data for the other mutant channels suggest that S4 mi ght move in at least two sequential steps, and can mow up to its maximal li mit even at the resting potential of the cell.