Introduction

In this paper, the researchers are examining the cause of the same phenotype in different groups of related animals. There are two lines of thought in this debate: either there are mutations in genes that change the functional product of a gene, or are there mutations that change the regulation of a gene, either through expression levels or alternative splicing.

TTX-resistant sodium channels make for an interesting model to test this hypothesis because both types of mutations seem to play a role in different organisms. The toxin TTX is produced by T. granulosa as a defense against predators. TTX binds strongly to susceptible sodium channels and inhibits their ability to propagate an action potential through a muscle cell. However, not every sodium channel is susceptible to the toxin. Prior to the publication of this paper, it had been determined that the sodium channels in the newt possess a non-aromatic amino acid substitution that seems to inhibit the binding of the toxin to the channel itself. This would be an example of a mutation changing the function of the protein itself. However, in rats, there are two different kinds of sodium channels: sensitive and resistant. Previous research has found that changing the expression levels of the two types of channels can alter the susceptibility of rat tissue to TTX. In this paper, the researchers examine populations of garter snakes with elevated resistance to TTX in an attempt to further examine the evolutionary causes of this resistance to TTX.

The researchers began by isolating the cDNA sequence responsible for coding the resistant sodium channel. They took a cDNA library of skeletal muscle of snake from Benton County, probed the library using sodium channel sequence from rats (both the resistant and the susceptible kind), and found one open reading frame. After sequencing the cDNA construct, it was found that this cDNA construct possesses much similarity to other sodium channel genes in other organisms. They deduced the sequence of the protein from the cDNA transcript, and then built subsequent libraries from other populations of the same species of snake that also show resistance to TTX, and compared their sequence homologies to each other and to susceptible snakes.

Results

Figure 1a shows the phylogenetic tree of these different snakes sampled based on mitochondrial DNA analysis. Along with the degree of relatedness is the dosage amount (adjusted for mass) of TTX that it takes to incapacitate a snake. When compared by this dosage amount, we see 3 statistically significant populations of snake. I assume the “ Illinois” snake to be a negative control of sorts—a baseline level of toxin resistance, although the authors never mention the snake explicitly in the text of the paper. We see though, that the amount of mass units required to incapacitate a snake ranges from 3.6 units for “ Bear Lake” to 730 units for “Willow Creek.”

Figure 1b shows the sequence homology for the sodium channel cDNA transcript in the three snake populations. We see that there are some differences in sequence between the resistant and susceptible populations, most notably a valine for isoleucine at position 1,561. A few other sequence differences are pointed out as well; refer to the paper for these.

Figure 2 shows the manipulation of these various cDNA constructs expressed in frog embryos. The researchers utilized both snake cDNA and human/snake chimeras. The chimera consisted of human sodium channel (which is susceptible to TTX), and replaced its S5-S6 linker position with the sequence deduced from a particular snake. Table 1 lists the amounts of TTX needed to sufficiently inhibit sodium transport through the channel. These values seem to correlate rather well with the physiological results of the experiment described in Figure 1a; snakes that were highly resistant to TTX toxicity had sodium channels that were resistant to TTX binding and vice versa.

The results are presented graphically in Figure 2. Parts a-c show inhibition curves of the various snakes’ sodium channels. Figure 2a shows the curves for the Bear Lake population and the Benton population, and illustrate that there is no significant difference between the chimeric channel and the wild type channel. As expected from the tree in Figure 1, there is an order of magnitude difference between the two populations in the amount of TTX required to inhibit current flow.

Figure 2b shows the ratios required to inhibit sodium flow for each chimeric transcript. Again, much like the tree would suggest, the amount of TTX required to inhibit ion flow is different for each chimeric transcript, and correlates well with the initial data in Figure 1a. Figure 2c illustrates the difference in a point mutation in the chimeric transcript of the Willow Creek population. The highly conserved valine residue is replaced with the isoleucine found in susceptible populations. The subsequent concentration of TTX required for inhibition of the channel is halved as a result of this mutation.

Figure 2d shows the current differences between a susceptible channel and a resistant channel after exposure to a certain quantity of TTX. The susceptible channel showed nearly total inhibition of function, while the resistant channel showed seemingly no change whatsoever.

Figure 3 better illustrates the relationship between the binding of TTX to chimeric cDNA products and TTX resistance in snake populations. In this figure, the researchers plot the amount of TTX needed to inhibit current flow in sodium channels versus the amount of current flow needed to disrupt the propagation of action potentials in snake muscle. There is a linear correlation between the functionality of the sodium channel (which is determined by amino acid sequence) and the level of resistance to TTX in muscle fibers.

Critique and future experiments

The researchers are quite sensitive to their experimental design, and acknowledge that the experiment was done with cDNA constructs rather than genomic DNA. I think this strongly aids in the paper’s credibility, because while their findings are interesting, I am not entirely sure that the mechanism by which the snakes are resistant to TTX is simply a matter of gene function itself. The researchers rightly point out that by using cDNA, the possibility for post-transcriptional modification still exists, but ignore it by saying it has never been observed before. While that’s probably not a bad assumption, it does not rule out that possibility. Perhaps one way to determine whether or not post-transcriptional modification occurs is to examine the genomic sequence DNA in that region. By designing PCR primers that begin in the regions surrounding the S5-S6 linker area, the sequence of the genomic DNA could be easily found. If it is found that there are no introns or sequences where the RNA could splice itself in this region, then I will feel much surer in the results of the experiment. Another, more nitpicky critique of the paper comes from failure to mention the “Illinois” snake population anywhere in the paper except in the picture. Even a sentence in the Methods section would have been helpful. Also, while the researchers were successful in inhibiting the function of a highly resistant sodium channel in Figure 2c, the data would have been much more convincing if there had been a modification to a susceptible transcript that made it resistant. We know that the valine residue at position is 1,561 is necessary for TTX resistance, but we do not know if it is sufficient in and of itself to produce resistance. Such a demonstration would strongly support the functional hypothesis the researchers talk about in the introduction.

It would be interesting to observe the resistance levels of transgenic snakes expressing the chimeric sodium channels, and see if their muscle fibers are as resistant to the toxin as wild type. While perhaps not economically feasible in the real world, it would certainly support their conclusions. Another interesting possibility would be to see if these resistance levels are additive; perhaps by inserting a non-aromatic amino acid (like the one present in the newt itself) into chimeric cDNA transcripts, we could see increased levels of resistance. The researchers acknowledge that there are many differences in resistance levels within a given population that cannot be explained by this model they have just described. This makes me suspect the gene regulation hypothesis discussed in the introduction. One future project that would further elucidate the roles of other genes would be to construct a microarray that has the genome of the snake on it. While it would require have the garter snake genome sequenced, a microarray would allow us to see which genes are turned on and off, and perhaps see if there are genes expressed in snakes that help confer this resistance. Subsequent knockouts of these hypothetical regulatory genes would allow us to further understand how TTX resistance is conferred.