Site-directed mutagenesis of the putative pore region of the rat IIA sodium channel.

We have used site-directed mutagenesis to examine the functional role of each of the eight acidic amino acid residues in the region between proposed transmembrane segments 5 and 6 (S5-S6) of domain II of the rat brain IIA sodium channel alpha subunit. The mutant sodium channels were expressed in Xenopus oocytes and analyzed by two-microelectrode voltage clamping with respect to voltage-dependent activation, inactivation, ion selectivity, and sensitivity to the pore-blocking neurotoxins tetrodotoxin (TTX) and saxitoxin (STX). None of the mutations had significant effects on voltage-dependent gating, ion selectivity, or block by protons or calcium. Three of the mutations had significant effects on the sensitivity of the channel to block by TTX and STX. Neutralization of negative charges at positions 942 and 945 greatly reduced the block by TTX and STX, suggesting that these two residues interact directly with the toxins. Substitution of a nearby negative charge at position 949 resulted in a smaller decrease in TTX and STX block, although analysis of TTX block of this mutant at low ionic strength suggests that the interaction is not simply by an electrostatic through-space mechanism. None of the other five mutations had any effects on block by either TTX or STX. The two acidic residues that had dramatic effects on toxin binding had significantly smaller effects at a depolarized membrane potential. The sodium channel interacts with TTX and STX with higher affinity at depolarized potentials, so these two residues must make a greater contribution to toxin binding in the low affinity state. These results define a small segment of the sodium channel alpha subunit domain II S5-S6 region that interacts with TTX and STX and therefore must lie near the mouth of the channel pore.