We propose to determine the three-dimensional architecture of the outer vestibule/pore region of the rat skeletal muscle subtype 1 (rSkM1) voltage-sensitive sodium channel. To accomplish this we will use mu- conotoxin GIIIA, the three-dimensional structure of which is known, as a molecular caliper to map out the position of channel residues on the extracellular surface of rSkM1. We will achieve this goal by several types of experiments. first, we will determine the residues on the channel surface that interact with the toxin. this will be accomplished by constructing and analyzing chimeric and site-specifically mutated channels and measuring the effects of these changes on wild-type (WT) toxin affinity. Second, we will mutate the toxin and measure the affinities of mutant toxins on the wild-type and mutant channels. From these thermodynamic measurements, mutant cycles can be constructed that reflect the interaction between a site on the channel and one in the toxin. If there is not interaction between two mutated sites, one in toxin and the other in rSkM1, the degree of interaction or coupling energy is zero (coupling coefficient of unity). Non-unity coupling coefficients enable the assignment of pairwise interactions. Using the toxin as a molecular caliper and electrostatic compliance measurements, estimates will be obtained for the distance between the two sites. Third, we will use di-cysteine substituted channels to form cross-links by oxidation (-SS-) or Me 2+ chelation and assess the effects on channel function including toxin binding. Positive results indicate proximal side chains and place distance constraints between these residues. Triangulation using a set of distance measurements will define the spatial arrangement of channel amino acids. Computer models of the rSkM1 external vestibule will be constructed and tested by further mutant cycle analysis. In the long-term, from the surface architecture, synthetic molecules will be designed as new candidate drugs that modify channel function.
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