MOLECULAR ANALYSIS OF POTENTIAL HINGE RESIDUES IN THE INACTIVATION GATE OF BRAIN-TYPE IIA NA+ CHANNELS

During inactivation of Na+ channels, the intracellular loop connecting domains III and IV is thought to fold into the channel protein and oc clude die pore through interaction of the hydrophobic motif isoleucine -phenylalanine-methionine (IFM) with a receptor site. We have searched for amino acid residues flanking the IFM motif which may contribute t o formation of molecular hinges that allow this motion of the inactiva tion gate. Site-directed mutagenesis of proline and glycine residues, which often are components of molecular hinges in proteins, revealed t hat G1484, G1485, P1512, P1514, and P1516 are required for normal fast inactivation. Mutations of these residues slow the time course of mac roscopic inactivation. Single channel analysis of mutations G1484A, G1 485A, and P1512A showed that the slowing of macroscopic inactivation i s produced by increases in open duration and latency to first opening. These mutant channels also show a higher probability of entering a sl ow gating mode in which their inactivation is further impaired. The ef fects on gating transitions in the pathway to open Na+ channels indica te conformational coupling of activation to transitions in the inactiv ation gate. The results are consistent with the hypothesis that these glycine and proline residues contribute to hinge regions which allow m ovement of the inactivation gate during the inactivation process of Na + channels.