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 ra t brain IIA sodium channel a subunit. The mutant sodium channels were expressed in Xenopus oocytes and analyzed by two-microelectrode voltag e clamping with respect to voltage-dependent activation, inactivation, ion selectivity, and sensitivity to the pore-blocking neurotoxins tet rodotoxin (TTX) and saxitoxin (STX). None of the mutations had signifi cant 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. Neutralizatio n of negative charges at positions 942 and 945 greatly reduced the blo ck by TTX and STX, suggesting that these two residues interact directl y with the toxins. Substitution of a nearby negative charge at positio n 949 resulted in a smaller decrease in TTX and STX block, although an alysis of TTX block of this mutant at low ionic strength suggests that the interaction is not simply by an electrostatic through-space mecha nism. None of the other five mutations had any effects on block by eit her TTX or STX. The two acidic residues that had dramatic effects on t oxin binding had significantly smaller effects at a depolarized membra ne potential. The sodium channel interacts with TTX and STX with highe r affinity at depolarized potentials, so these two residues must make a greater contribution to toxin binding in the low affinity state. The se results define a small segment of the sodium channel alpha subunit domain II S5-S6 region that interacts with TTX and STX and therefore m ust lie near the mouth of the channel pore.