SPECIFICITY FOR BLOCK BY SAXITOXIN AND DIVALENT-CATIONS AT A RESIDUE WHICH DETERMINES SENSITIVITY OF SODIUM-CHANNEL SUBTYPES TO GUANIDINIUMTOXINS

Tyrosine 401 of the skeletal muscle isoform (mu 1) of the rat muscle N a channel is an important determinant of high affinity block by tetrod otoxin (TTX) and saxitoxin (STX) in Na-channel isoforms. In mammalian heart Na channels, this residue is substituted by cysteine, which resu lts in low affinity for TTX/STX and enhanced sensitivity to block by Z n2+ and Cd2+. In this study, we investigated the molecular basis for h igh affinity block of Na channels by STX and divalent cations by measu ring inhibition of macroscopic Na+ current for a series of point mutat ions at residue Tyr401 of the rat mu 1 Na channel expressed in Xenopus oocytes. Substitution of Tyr401 by Gly, Ala, Ser, Cys, Asp, His, Trp, and Phe produced functional Na+ currents without major perturbation o f gating or ionic selectivity. High affinity block by STX and neosaxit oxin (NEO) with K-i values in the range of 2.6-18 nM required Tyr, Phe , or Trp, suggestive of an interaction between an aromatic ring and a guanidinium group of the toxin. The Cys mutation resulted in a 7- and 23-fold enhancement of the dissociation rate of STX and NEO, respectiv ely, corresponding to rapid toxin dissociation rates of cardiac Na cha nnels. High affinity block by Zn2+ (k(i) = 8-23 mu M) required Cys, Hi s, or Asp, three residues commonly found to coordinate directly with Z n2+ in metalloproteins. For the Cys mutant of yl and also for the card iac isoform Na channel (rh1) expressed in the L6 rat muscle cell line, inhibition of macroscopic Na+ conductance by Zn2+ reached a plateau a t 85-90% inhibition, suggesting the presence of a substate current. Th e Asp mutant also displayed enhanced affinity for inhibition of conduc tance by Ca2+ (K-i = 0.3 mM vs similar to 40 mM in wild type), but blo ck by Ca2+ was incomplete, saturating at similar to 69% inhibition. In contrast, Cd2+ completely blocked macroscopic current in the Cys muta nt and the L6 cell line. These results imply that the magnitude of sub state current depends on the particular residue at position 401 and th e species of divalent cation. The His mutant also exhibited enhanced s ensitivity to block by H+ with a pK(a) of similar to 7.5 for the His i midazole group. Our findings provide further evidence that residue 401 of mu 1 is located within the outer vestibule of the Na channel but e xternal to the single-filing region for permeant ions.