Charged residues between the selectivity filter and S6 segments contributeto the permeation phenotype of the sodium channel

The deep regions of the Na+ channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent link ers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers ma y play a role in permeation. To characterize the structural topology and fu nction of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of SG) individually by cysteine substitution. Several cysteine mutan ts displayed enhanced sensitivities to Cd2+ block relative to wild-type and /or were modifiable by external sulfhydryl-specific methanethiosulfonate re agents when expressed in TSX-201 cells, indicating that these amino acids r eside in the permeation pathway. while neutralization of positive charges d id not alter single-channel conductance, negative charge neutralizations ge nerally reduced conductance, suggesting that such charges facilitate ion pe rmeation. The electrical distances Tor Cd2+ binding to these residues revea l a secondary "dip" into the membrane field of the linkers in domains II an d IV. Our findings demonstrate significant functional roles and sur-pr-isin g structural features of these previously unexplored external charged resid ues.