Opening and closing of the central ion-conducting pore in voltage-dependent ion channels is gated by changes in membrane potential. Although a gate residue in the eukaryotic voltage-gated sodium channel has been identified, the minimal molecular determinants of this gate region remain unknown. Here, by measuring the closed- and open-state reactivity of MTSET to substituted cysteines in all the pore-lining helices, we show that the state-dependent accessibility is delineated by four hydrophobic residues at homologous positions in each domain. Introduced cysteines above these sites do not react with intracellular MTSET while the channels are closed and yet are rapidly modified while the channels are open. These findings, in conjunction with state-dependent metal cross-bridging, support the notion that the gate residues in each of the four S6 segments of the eukaryotic sodium channel form an occlusion for ions in the closed state and are splayed open on activation.
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