Tonic and phasic tetrodotoxin block of sodium channels with point mutations in the outer pore region

Tonic and use-dependent block by tetrodotoxin (TTX) has been studied in cRNA-injected Xenopus oocytes expressing mutants W386Y, E945Q, D1426K, and D1717Q, of the outer-pore region of the rat brain IIA alpha-subunit of sodium channels. The various phenotypes are tonically half-blocked at TTX concentrations, IC50(t), that span a range of more than three orders of magnitude, from 4 nM in mutant D1426K to 11 mu M in mutant D1717Q. When stimulated with repetitive depolarizing pulses at saturating frequencies, all channels showed a monoexponential increase in their TTX-binding affinity with time constants that span an equally wide range of values ([TTX] approximate to IC50(t), from similar to 60 s for D1426K to similar to 30 ms for D1717Q) and are in most phenotypes roughly inversely proportional to IC50(t). In contrast, all phenotypes show the same approximately threefold increase in their TTX affinity under stimulation. The invariance of the free-energy difference between tonic and phasic configurations of the toxin-receptor complex, together with the extreme variability of phasic block kinetics, is fully consistent with the trapped-ion mechanism of use dependence suggested by Salgado et al. (1986) and developed by Conti et al. (1996). Using this model, we estimated for each phenotype both the second-order association rate constant, k(on), and the first-order dissociation rate constant, k(off), for TTX binding. Except for mutant E945Q, all phenotypes have roughly the same value of k(on) approximate to 2 mu M-1 s(-1) and owe their large differences in IC50(t) to different k(off) values. However, a 60-fold reduction in k(on) is the main determinant of the low TTX sensitivity of mutant E945Q. This suggests that the carboxyl group of E945 occupies a much more external position in the pore vestibule than that of the homologous residue D1717.