Charged residues between the selectivity filter and S6 segments contribute to 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 linkers 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 may play a role in permeation. To characterize the structural topology and function 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 mutants displayed enhanced sensitivities to Cd2+ block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSX-201 cells, indicating that these amino acids reside in the permeation pathway. while neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances Tor Cd2+ binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and sur-pr-ising structural features of these previously unexplored external charged residues.