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) b lock cardiac Na+ channels when applied from either side of the membrane, bu t 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 (mu 1) and rat heart (rH1) Na+ chan nels were studied by two-electrode voltage clamp in Xenopus oocytes, Mutati ng the mu 1 domain I P-loop Y401, which is the critical residue for isoform differences in tetrodotoxin block, to the heart sequence (Y401C) allowed o utside QX222 block, but its mutation to brain type (Y401F) showed little bl ock. mu 1-Y401C accelerated recovery from block by internal QX222. Block by external QX222 in mu 1-Y401C was diminished by chemical modification with methanethiosulfonate ethylammonium (MTSEA) to the outer vestibule or by a d ouble mutant (mu 1-Y401C/F1579A), which altered the putative local anesthet ic binding site. The reverse mutation in heart rH1-C374Y reduced outside QX 314 block and slowed dissociation of internal QX222, Mutation of mu 1-C1572 in IVS6 to Thr, the cardiac isoform residue (C1572T), allowed external QX2 22 block, and accelerated recovery from internal QX222 block, as reported. Blocking efficacy of outside QX222 in mu 1-Y401C was more than that in mu 1 -C1572T, and the double mutant (mu 1-Y401C/C1572T) accelerated internal QX recovery more than mu 1-Y401C or mu 1-C1572T alone. We conclude that the is oform-specific residue (Tyr/Phe/Cys) in the P-loop of domain I plays an imp ortant role in drug access as well as in tetrodotoxin binding, Isoform-spec ific residues in the IP-loop and IVS6 determine outside drug access to an i nternal binding site.