DEPTH ASYMMETRIES OF THE PORE-LINING SEGMENTS OF THE NA+ CHANNEL REVEALED BY CYSTEINE MUTAGENESIS

We used serial cysteine mutagenesis to study the structure of the oute r vestibule and selectivity region of the voltage-gated Na+ channel. T he voltage dependence of Cd2+ block enabled us to determine the locati ons within the electrical field of cysteine-substituted mutants in the P segments of all four domains. The fractional electrical distances o f the substituted cysteines were compared with the differential sensit ivity to modification by sulfhydryl-specific modifying reagents. These experiments indicate that the P segment of domain II is external, whi le the domain IV P segment is displaced internally, compared with the first and third domain P segments. Sulfhydryls with a steep voltage de pendence for Cd2+ block produced changes in monovalent cation selectiv ity; these included substitutions at the presumed selectivity filter, as well as residues in the domain IV P segment not previously recogniz ed as determinants of selectivity. A new structural model is presented in which each of the P segments contribute unique loops that penetrat e the membrane to varying depths to form the channel pore.