TOPOLOGY OF THE P-SEGMENTS IN THE SODIUM-CHANNEL PORE REVEALED BY CYSTEINE MUTAGENESIS

The P segments of the voltage-dependent Na+ channel line the outer mou th and selectivity filter of the pore. The residues that form the cyto plasmic mouth of the pore of the channel have not been identified. To study the structure of the inner pore mouth, the presumed selectivity filter residues (D400, E755, K1237, and A1529), and three amino acids just amino-terminal to each of these residues in the rat skeletal musc le Na+ channel, were mutated to cysteine and expressed in tsA 201 cell s. These amino acids are predicted (by analogy to K+ channels) to be o n the cytoplasmic side of the putative selectivity filter residues. In ward and outward Na+ currents were measured with the whole-cell config uration of the patch-clamp technique. Cysteinyl side-chain accessibili ty was gauged by sensitivity to Cd2+ block and by reactivity with meth anethiosulfonate (MTS) reagents applied to both the inside and the out side of the cell. Outward currents through the wild-type and all of th e mutant channels were unaffected by internal Cd2+ (100 mu M). Similar ly, 1 mM methanethiosulfonate ethylammonium (MTSEA) applied to the ins ide of the membrane did not affect wild-type or mutant outward current s. However, two mutants amino-terminal to the selectivity position in domain III (F1236C and T1235C) and one in domain IV (S1528C) were bloc ked with high affinity by external Cd2+. The Na+ current through F1236 C and S1528C channels was inhibited by MTSEA applied to the outside of the cell. The accessibility of these mutants to externally applied cy steinyl ligands indicates that the side chains of the mutated residues face outward rather than inward. The K+ channel model of the P segmen ts as protein loops that span the selectivity region is not applicable to the Na+ channel.