Parallel Evolution of Tetrodotoxin Resistance in Three Voltage-Gated Sodium Channel Genes in the Garter Snake Thamnophis sirtalis

Abstract

Members of a gene family expressed in a single species often experience common selection pressures. Consequently, the molecular basis of complex adaptations may be expected to involve parallel evolutionary changes in multiple paralogs. Here, we use bacterial artificial chromosome library scans to investigate the evolution of the voltage-gated sodium channel (Nav) family in the garter snake Thamnophis sirtalis, a predator of highly toxic Taricha newts. Newts possess tetrodotoxin (TTX), which blocks Nav’s, arresting action potentials in nerves and muscle. Some Thamnophis populations have evolved resistance to extremely high levels of TTX. Previous work has identified amino acid sites in the skeletal muscle sodium channel Nav1.4 that confer resistance to TTX and vary across populations. We identify parallel evolution of TTX resistance in two additional Nav paralogs, Nav1.6 and 1.7, which are known to be expressed in the peripheral nervous system and should thus be exposed to ingested TTX. Each paralog contains at least one TTX-resistant substitution identical to a substitution previously identified in Nav1.4. These sites are fixed across populations, suggesting that the resistant peripheral nerves antedate resistant muscle. In contrast, three sodium channels expressed solely in the central nervous system (Nav1.1–1.3) showed no evidence of TTX resistance, consistent with protection from toxins by the blood–brain barrier. We also report the exon–intron structure of six Nav paralogs, the first such analysis for snake genes. Our results demonstrate that the molecular basis of adaptation may be both repeatable across members of a gene family and predictable based on functional considerations.

Is there more than one way to skin a newt? Convergent toxin resistance in snakes is not due to a common genetic mechanism

“…In contrast, the allopatric H. platirhinos and H. nasicus showed only base levels of resistance, on par with other nonresistant snakes (Table 2) (Feldman et al, 2012). No H. platirhinos possessed derived SCN4A alleles found in the other TTX-resistant taxa or paralogs that have been examined thus far (Figure 3) (Geffeny et al, 2005;Soong and Venkatesh, 2006;Jost et al, 2008;Feldman et al, 2012;McGlothlin et al, 2014;Hanifin and Gilly, 2015). Thus, the resistance-conferring mutations in SCN4A known to reduce the binding affinity of TTX to the channel appear to be entirely lacking in TTX-resistant H. platirhinos.…”

“…Given the highly specific action of TTX, one mechanism of TTX resistance seems obvious: functional changes to the outer pore of sodium channels that reduce the affinity of TTX to the protein. Indeed, all TTX-resistant vertebrates examined thus far have remarkably similar mutations that alter the structure of the outer pore and reduce TTX-binding affinity to the channel (Geffeny et al, 2005;Soong and Venkatesh, 2006;Jost et al, 2008;Feldman et al, 2012;McGlothlin et al, 2014;Hanifin and Gilly, 2015). In fact, this mechanism of TTX resistance appears nearly identical across several independent, coevolutionary arms races involving predatory snakes and their TTX-defended amphibian prey (Feldman et al, 2009(Feldman et al, , 2012, as well as across sodium channel paralogs within species (McGlothlin et al, 2014), suggesting that adaptation to TTX is highly predictable (Jost et al, 2008;Feldman et al, 2012;McGlothlin et al, 2014).…”

3D Structures and Molecular Evolution of Ion Channels

“…Interestingly, such changes are seen in the sodium channel paralogs, which are expressed in the skeletal muscle and peripheral nervous system of snakes and thus should be exposed to the ingested tetrodotoxin. In contrast, sodium channel paralogs, which are expressed solely in the central nervous system, showed no evidence of the TTX resistance, indicating that the blood-brain barrier protects from the toxin [32]. The observed genetic changes represent only a small fraction of the experimentally validated mutations known to increase the sodium channels resistance to TTX.…”

Predictably Convergent Evolution of Sodium Channels in the Arms Race between Predators and Prey

“…Analysis of the coding sequence of these genes from T. sirtalis revealed familiar amino acid replacements in both of these newly explored channels ( fig. 3 ) [McGlothlin et al, 2014]. Na V 1.6, which is expressed in peripheral nerves, had the same Ile-Val substitution in DIV that is common in the Na V 1.4 of resistant populations of the same species.…”

“…Relatively little is known about convergence and parallelism in the genetic mechanisms involved with neurophysiological traits. Even less is known about the predictability of convergence among gene paralogs that code for proteins expressed in different tissues [Jost et al, 2008;McGlothlin et al, 2014].…”