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Paper Review:

Evolutionary diversification of TTX-resistant sodium channels in a predator-prey interaction

Shana L. Geffeney, Esther Fujimoto, Edmund D. Brodie Ill, Edmund D. Brodie Jr & Peter C. Ruben. 2005. Nature 434, 759-763.

This paper examines changes in the molecular structure of tsNav1.4--a sodium channel expressed in snake skeletal muscle. Those changes are responsible for differences in TTX (tetrodotoxin) resistance in garter snake populations that are coevolving with toxic newts. The authors argue that their results show that functional changes in a gene that is otherwise highly conserved have have caused the evolution of a physiological trait.

One idea is that phenotypic diversity may be explained by regulation of genes of major effect. This is the result of the emerging thought that adaptive radiation may be more predictable at the genetic level. There are many examples where changes in the regulation of single genes in developing tissues have produced ecologically important morphological differences between lineages. However, in other cases, adaptive differences have been explained by functional changes in protein-coding regions of structural genes.

Coevolution has resulted in geographic variation in TTX resistance in the predators. TTX binds to the outer pore of voltage-gated sodium channels and blocks nerve and muscle fiber activity. This results in paralysis and/or death. TTX was used by the newts as a defense against their snake predators. High levels of tetrodotoxin are found in their skin. But, elevated resistance to TTX has evolved at least twice within the radiation of the snakes. The expression of TTX-resistant sodium channels in the garter snakes is at least partially responsible for this adaptation.

TTX-sensitive animals have an aromatic amino acid in the outer pore of domain 1 that causes the high affinity for TTX binding. TTX-resistant animals, on the other hand, have a substitution at this critical position. The authors propose that TTX resistance in garter snakes may be caused by a change in sodium channel expression or mutations in TTX-sensitive members of the sodium channel gene family.

To test their hypotheses, they first made a cDNA library from skeletal muscle of a snake with elevated TTX resistance to identify the TTX resistant sodium channel gene expressed in skeletal muscle tissue. They probed the library to find clones with skeletal and cardiac muscle sodium channel sequences. These clones were then sequenced and an open reading frame of 5,625 nucleotides was found. tsNa v1.4 has a tyrosine residue in the critical domain 1 position. Two novel amino acid substitutions were found in other regions important for TTX binding and pore structure—the pore helix and β-strand of the domain IV outer pore.

Figure 1 shows the amino acid sequences for four different snake populations and their relatedness in a phylogenetic tree. The Willow Creek population is the most closely related to Illinois (the root population with no resistance) and has the most resistance to TTX. The next closest to Illinois is Bear Lake (non-resistant population) followed by the two most closely related populations Warrenton and Benton. Compared to Bear Lake (a non-resistant population), Warrenton has one difference in sequence in the pore helix, and Benton and Willow Creek have two and four differences, respectively, in the pore helix and the β-strand. This figure also shows that, although each population has a different evolutionary history, they all have a substitution of valine for isoleucine at position 1,561.

The researchers next tested whether these differences affected TTX sensitivity of Na v1.4 by expressing the channels in Xenopus oocytes. They constructed a human-snake chimaera channel for each population. Each chimaera consisted of a human Na v1.4 sequence with each snake population's tsNa v1.4 sequence substituted for the outer pore sequence in this domain. They also tested each snake population’s tsNa v1.4 channel and measured the effects of these changes on TTX sensitivity.

Figure 2 consists of four graphs showing the effect of different TTX concentrations on tsNa v1.4 and the snake-human chimaera channels. Part a shows that entirely snake sequences are not any more resistant to TTX block than chimaeric channels. This graph serves as a control and a basis for comparison for parts b and c. Part b shows chimaeric channels where domain IV sequences are blocked by different concentrations of TTX. It shows that chimaeras with different domain IV pore sequences have different TTX-binding affinities. Part c shows if the channel is blocked when a valine is changed to an isoleucine in the position 1,561 of the pore helix. In the Willow Creek population, this change halves the K d value. Part d is a current recording from chimaeric channels expressed in the oocytes before and after the addition 100nM TTX to the external solution. It shows that Bear Lake channels are blocked, but Willow Creek channels are not. From figure 2, table 1 was constructed. The table shows the TTX concentration that is needed to block 50% of the sodium channel. This shows the TTX sensitivity of each chimaera.

Figure 3 compares the K d of the chimaeric channels to the K d of the skeletal muscle. The K d for the skeletal muscle was calculated by looking at the rates of rise in the action potential of the skeletal muscle fibers over varying TTX concentrations. The p value < 0.0001, so there is a significant relationship between the K d value of the chimaeric channel and the K d value of the skeletal muscle. The TTX resistance in the channel, therefore, is a good indicator of TTX resistance in the whole animal.

This paper is a very good beginning and raises many important questions linking molecular and evolutionary biology. There are, however, a few limitations to their experiments. All of their work was done on cDNA so they cannot necessarily be generalized to the genomic DNA level. It could be that a change in the non-coding region of the DNA the sodium ion channel causes TTX resistance. Also, as admitted by the authors, the possibility that differences reflect post-transcriptional modifications cannot be eliminated by the data presented in this paper.

The figures and methods are fairly clear. I would like to see a table explaining the different populations, and I find the placement of figure 3 odd. Though it is mentioned in conjunction with figure 1, it is presented at the end of the paper. Obviously, this figure presents their conclusion, but maybe they could have another intermediate figure that puts figure 1 and figure 3 together and makes comparison easier. The controls in each figure are good and fairly obvious.

Overall, I found these data convincing due mostly to the fact that they come from functional tests with fairly undisputable mathematical results.

This paper is really a springboard. The possibilities for future research are huge. First, as mentioned by the authors, an analysis of parallel genotypic evolution would add a lot to these results. This could be carried out through the use of microarrays that show which genes for each population are repressed or expressed when a snake is exposed to varying amounts of TTX.

To test the molecular mechanism of the TTX resistance, someone could mutate specific amino acids and perform functional tests on the protein and look at how these mutations change the binding mechanism. This could also be explored through crystallization of TTX and domain IV. This would really help explore the structure/function relationship.

Another way to look at the binding mechanism would be to use the yeast-two-hybrid method. You use TTX as the bait and different garter snake proteins as possible targets. This would show if the TTX does bind with different sodium channels and could also show which proteins interact with TTX.

Work Cited:

Geffeney, Shana, et al. 2005. Evolutionary diversification of TTX-resistant sodium channels in a predator-prey interaction. Nature 434, 759-763.