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Voltage-gated sodium ion channels are required for the generation and propagation of action potentials in vertebrate nerve and muscle cells. The neurotoxin tetrodotoxin (TTX) binds in the channel pore and blocks sodium channel function. Some populations of garter snakes (Thamnophis sirtalis) have evolved resistance to TTX in part due to amino acid substitutions in the pore of the voltage-gated sodium channel expressed in skeletal muscle fibers (tsNaV1.4). We have previously demonstrated that the four amino acid substitutions in tsNaV1.4 from snake populations in California reduce TTX binding. These four amino acid substitutions are at or near an amino acid position that binds TTX. In this study, we investigated if these four amino acid substitutions alter TTX binding by measuring the TTX binding affinity of a homology model of tsNaV1.4. We created a homology model of tsNaV1.4 using Swiss-Model with the full amino acid sequence from a TTX sensitive snake. We altered amino acids in the pore of the homology model to match the substitutions identified in TTX resistant snakes using the program PyMol. Then we modeled TTX binding in its pore binding site and compared the interaction energy of tsNaV1.4 and TTX for the TTX sensitive and TTX resistant channel sequence using AutoDock Vina. Surprisingly, we measured the same binding energy values for both the TTX sensitive and the TTX resistant channel. These results do not support the hypothesis that amino acid substitutions in the TTX binding site alter the binding affinity of this site for TTX. Rather, further evaluation of the homology model suggests that one of the amino acid substitutions may affect TTX entry into the pore binding site instead of TTX binding at the site. Our future plans include modeling how amino acid substitutions in tsNaV1.4 alter the interaction energy between the channel and toxin for the length of the outer pore using the Poisson-Boltzmann equation and directly measuring the on-rate for TTX binding of heterologously expressed tsNaV1.4 channels.
Utah State University
Mitchell, Kenyon, "Altered Toxin Binding or Access to the Binding Site, What Changes in the Tetrodotoxin Resistant Sodium Channels of Garter Snakes?" (2020). Fall Student Research Symposium 2020. 52.