Fig. 3. Independent, adaptive molecular evolution in the skeletal muscle sodium channel (Nav1.4). Amino acid replacements at sites important in TTX ligation in the P loops of Nav1.4 are found only in TTX-resistant garter snakes and appear uniquely derived. (A) Structure of the-subunit of Nav1.4 showing the 4 domains (DI–DIV), their 6 transmembrane segments (S1–S6), and the linkers that connect segments. The 4 polypeptide chains that link S5 to S6 (bold) form the outer pore of the channel that allows selective permeation of Na ions; however, a number of residues that form the outer pore bind strongly to TTX, which occludes the pore and halts Na movement (see text for further discussion and citations). Approximate location of amino acid substitutions in DIII and DIV P loops (color-coded to species) discussed in text. (B) Phylogeny of Nav1.4 alleles from Thamnophis and relatives based on 2.9 kb of Nav1.4 sequence data (4.3-kb alignment), including the coding regions of all 4 P loops (1.0 kb) and portions of 3 linked introns (1.9 kb). The gene tree closely resembles independent estimates of garter snake phylogeny based on mtDNA (Fig. 2), and shows that elevated TTX resistance has evolved multiple times in garter snakes. Topology and nodal support values were estimated via Bayesian tree searches; some outgroups were pruned for simplicity; locality information is in Table S1. (C) Measures of whole-animal resistance to TTX (50% MAMU) alongside amino acid sequences of the DIII and DIV P loops (Table S1). Amino acid substitutions (arrows and replacements color-coded to species) occur at critical residues that change the structure and electrostatic environment of the pore and alter TTX binding affinity. Human sequence is given for comparison (M81758), but amino acid positions follow Nav1.4 CDS from T. sirtalis AY851746; structures of the pore labeled below human sequence (*, selectivity filter; , -helix; , -strand).
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