A critical residue for isoform difference in tetrodotoxin affinity is a molecular determinant of the external access path for local anesthetics in the cardiac sodium channel.

Membrane-impermeant quaternary derivatives of lidocaine (QX222 and QX314) block cardiac Na(+) channels when applied from either side of the membrane, but they block neuronal and skeletal muscle channels poorly from the outside. To find the molecular determinants of the cardiac external QX access path, mutations of adult rat skeletal muscle (micro1) and rat heart (rH1) Na(+) channels were studied by two-electrode voltage clamp in Xenopus oocytes. Mutating the micro1 domain I P-loop Y401, which is the critical residue for isoform differences in tetrodotoxin block, to the heart sequence (Y401C) allowed outside QX222 block, but its mutation to brain type (Y401F) showed little block. mu1-Y401C accelerated recovery from block by internal QX222. Block by external QX222 in mu1-Y401C was diminished by chemical modification with methanethiosulfonate ethylammonium (MTSEA) to the outer vestibule or by a double mutant (mu1-Y401C/F1579A), which altered the putative local anesthetic binding site. The reverse mutation in heart rH1-C374Y reduced outside QX314 block and slowed dissociation of internal QX222. Mutation of mu1-C1572 in IVS6 to Thr, the cardiac isoform residue (C1572T), allowed external QX222 block, and accelerated recovery from internal QX222 block, as reported. Blocking efficacy of outside QX222 in mu1-Y401C was more than that in mu1-C1572T, and the double mutant (mu1-Y401C/C1572T) accelerated internal QX recovery more than mu1-Y401C or mu1-C1572T alone. We conclude that the isoform-specific residue (Tyr/Phe/Cys) in the P-loop of domain I plays an important role in drug access as well as in tetrodotoxin binding. Isoform-specific residues in the IP-loop and IVS6 determine outside drug access to an internal binding site.