Determining the molecular structure and function of sodium channel

The structure and functions of Na+ channel were studied using mutagenesis in combination with biophysical probes such as Cd 2+, sulfhydryl-specific modifiers, various toxins and local anesthetics (LA). Since high-affinity blockade of cardiac Na+ channels by Cd2+ is mediated by a cysteine residue in the pore, we first developed a strategy of studying the Na+ channel pore structure by systematically replacing single residues in the putative pore region of Cd2+ insensitive rat skeletal muscle (rSkM1) Na+ channels with cysteine. Further 3-dimensional structural information was obtained by simultaneously substituting pairs of residues in distinct P-loops. Dual-replacements created channels with either enhanced or reduced Cd2+ sensitivity relative to the corresponding single mutants, suggesting coordinated Cd 2+ binding and cross-linking by the inserted paired sulfhydryls. Since both processes require stringent geometric constraints (<3.5Å) and multiple consecutive adjacent residues in one P-loop could very often approach a single residue in another P-loop, our results indicate that the Na + channel pore is highly flexible and most of that flexibility resides in Domain IV (DIV). We next looked for correlations between pore flexibility and channel functions by examining the role of pore-lining residues in Na+ channel ion-selectivity. Specifically, we measured permeability and ionic current ratios of single mutant channels in the presence of different monovalent and divalent cations. Mutations of members of the proposed selectivity filter of Na+ channels had little effects except K1237 in DIII. However, we identified three other residues, all in DIV, which are critical determinants of ionic selectivity. These data not only redefine the Na+ channel selectivity filter but further suggest a possible role of pore flexibility in selectivity. Pharmacologically, we examined the effects of pore mutations on <math> <f> <g>m</g></f> </math>-conotoxin (<math> <f> <g>m</g></f> </math>-CTX) and LA binding. We identified several pore residues which are critical for interacting with the peptide toxin via either electrostatic or hydrophobic interactions. Pore mutations also affected LA binding. In particular, mutations of W1531 in domain IV completely abolish lidocaine binding to Na + channels without affecting drug access. We conclude that this trytophan forms part of the local anesthetic binding site (LABS). Taken together with its role in ionic selectivity and toxin binding, our results suggest that W1531 is critical for normal channel functioning. To map the spatial relationship between the pore and the LABS, we employed the novel strategy A_nchor-L_inker-D_rug (ALD). Specifically, the LA benzocaine was tethered to the sulfhydryl specific chemical group methanethiosulfonate (MTS) via a hydrocarbon chain. Benzocaine-based methanethiosulfonate derivatives produced typical but irreversible local anesthetic actions in cardiac channels and the mutant rSkM1 channels containing a cysteine at the equivalent location as the cardiac channels. Such effects were readily reversible in rSkM1 upon drug washout indicating the specificity of these drugs to the pore cysteine. By varying the linker length, ALD also allows determinations of the spatial relationship between the pore cysteine and the LABS. We find that the two functional domains are structurally adjacent to each other. Our strategy also provides a potential method for developing a heart-specific antiarrhythmic using the novel strategy. (Abstract shortened by UMI.)