Accessibility of mid-segment domain IVS6 residues of the voltage-gated Na+ channel to methanethiosulfonate reagents

The inner pore of the voltage-gated Na+ channel is predicted by the structure of bacterial potassium channels to be lined with the four S6 alpha-helical segments. Our previously published model of the closed pore based on the KcsA structure, and our new model of the open pore based on the MthK structure predict which residues in the mid-portion of S6 face the pore. We produced cysteine mutants of the mid-portion of domain IV-S6 (Ile-1575-Leu-1591) in Na(V)1.4 and tested their accessibility to intracellularly and extracellularly placed positively charged methanethiosulfonate (MTS) reagents. We found that only two mutants, F1579C and V1583C, were accessible to both outside and inside 2-(aminoethyl)-methanethiosulfonate hydrobromide (MTSEA) Further study of those mutants showed that efficient closure of the fast inactivation gate prevented block by inside [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) at slow stimulation rates. When fast inactivation was inhibited by exposure to anthropleurin B (ApB), increasing channel open time, both mutants were blocked by inside MTSET at a rate that depended on the amount of time the channel was open. Consistent with the fast inactivation gate limiting access to the pore, in the absence of ApB, inside MTSET produced block when the cells were stimulated at 5 or 20 Hz. We therefore suggest that the middle of IV-S6 is an alpha-helix, and we propose a model of the open channel, based on MthK, in which Phe-1579 and Val-1583 face the pore.