Toxin-Resistant Sodium Channels: Parallel Adaptive Evolution across a Complete Gene Family

Abstract

Approximately 75% of vertebrate proteins belong to protein families encoded by multiple evolutionarily related genes, a pattern that emerged as a result of gene and genome duplications over the course of vertebrate evolution. In families of genes with similar or related functions, adaptation to a strong selective agent should involve multiple adaptive changes across the entire gene family. However, we know of no evolutionary studies that have explicitly addressed this point. Here, we show how 4 taxonomically diverse species of pufferfishes (Tetraodontidae) each evolved resistance to the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX) via parallel amino acid replacements across all 8 sodium channels present in teleost fish genomes. This resulted in diverse suites of coexisting sodium channel types that all confer varying degrees of toxin resistance, yet show remarkable convergence among genes and phylogenetically diverse species. Using site-directed mutagenesis and expression of a vertebrate sodium channel, we also demonstrate that resistance to TTX/STX is enhanced up to 15-fold by single, frequently observed replacements at 2 sites that have not previously been implicated in toxin binding but show similar or identical replacements in pufferfishes and in distantly related vertebrate and nonvertebrate animals. This study presents an example of natural selection acting upon a complete gene family, repeatedly arriving at a diverse but limited number of adaptive changes within the same genome. To be maximally informative, we suggest that future studies of molecular adaptation should consider all functionally similar paralogs of the affected gene family.

Constraint shapes convergence in tetrodotoxin-resistant sodium channels of snakes

“…S1) clearly demonstrates that high sensitivity to TTX is the ancestral condition and that TTX resistance has evolved independently at least six times in snakes. Functionally similar (and sometimes identical) P-loop replacements occur in Na v 1.4 of TTX-bearing pufferfish (39)(40)(41) (the only other TTX-resistant vertebrate whose sodium channels have been characterized) in all but two of the sites modified in TTX-resistant snakes (Fig. 1).…”

“…couchii (positions follow Na v 1.4 CDS from Th. sirtalis AY851746) produces a 15-fold decrease in TTX binding to the channel (40); the I1561V substitution in Th. sirtalis halves TTX-binding affinity (32); and the shared D1568N replacement in Th.…”

The evolutionary origins of beneficial alleles during the repeated adaptation of garter snakes to deadly prey

“…TTX-bearing pufferfish (44, 45) (teleost fish possess functional duplicates of most Na v genes), and when this replacement was constructed in rat Na v 1.4 and functionally expressed, the amount of TTX required to block Na ϩ current (IC 50 ) increased 15-fold (45). T. atratus possess 3 amino acid changes in the P loops of Na v 1.4: 2 in DIII (D1277E and A1281P) and 1 in DIV (D1568N).…”

“…The 2 DIII replacements, D1277E and A1281P, have not been functionally expressed; however, other replacements at D1277 do lead to minor changes in TTX-binding affinity (39,42). P-loop replacements with only small effects on TTX ligation by themselves appear to have nearly ordinal effects on TTX sensitivity when combined with other resistant replacements (28,47), so it is not surprising that both the D3E and A3P DIII replacements are also found in some pufferfish (44,45).…”

“…On the contrary, diverse patterns of phenotypic variation in snake TTX resistance (23) and surveys of the sodium channels from pufferfish (45) suggest that variation in whole-animal performance is likely to involve multiple, perhaps convergent, adaptive changes across the entire gene family (45).…”