This web page was produced as an assignment for an undergraduate course at Davidson College.
Review of Molecular Biology Paper
On this webpage, I will present and critically evaluate the results of a paper found in Nature entitled: Evolutionary diversification of TTX-resistant sodium channels in a predator-prey interaction. This webpage has nothing to do with my previous pages, yet is important in thinking critically about molecular biology. To be able to present and evaluate data is an important tool for all biologists. I will also suggest future research that might be done to further explain and/or support the thesis of this paper.
Goal of paper:
In this paper, the researchers sought to understand the genetic basis of adaptation. The researchers focused on the molecular genetics behind the coevolution of the garter snake Thamnophis sirtalis and the newt Taricha granulosa. The newt contains a toxin in its skin called tetrodotosin (TTX). This toxin binds to sodium channels blocking nerve and muscle fibre activity, thus causing paralysis and eventual death. The garter snake is a predator of the newt and has developed a tolerance for TTX. The amount of toxin a garter snake can tolerate is dependent on its geographic location. The researchers looked at four different garter snake populations to figure out the molecular basis of this adaptation. The researchers took a functional approach to the genes studied (a functional approach means they looked at the proteins encoded by the genes rather than the regulation of the genes).
Data and results:
The researchers made a cDNA library for a snake population called Benton that had an increased resistance to TTX. To find the TTX-resistant sodium channel they screened the library with probes to identify sequences of the skeletal and cardiac muscle sodium channel that matched other sequences of known sodium channels (Nav1.4 and Nav1.50). Within the sequence, an open reading frame (ORF) was found that encoded a skeletal muscle sodium channel (they call it tsNav1.4). As with all sodium channel sequences, a tyrosine residue was found in domain I of the sequence that is responsible for TTX binding. This residue was not the researchers' focus, but rather two amino acid substitutions that occured in the pore helix and beta strand of the domain IV outer pore. Both of these regions are involved in TTX binding and pore structure and thus, became the focus of the researchers.
The three other snake populations had identical sequences in the I, II, and III domains of tsNav1.4, but differed in sequence in the pore helix and beta strand sections (domain IV) of their tsNav1.4. Fig. 1 (I can't reproduce the figure due to copyright rules) shows the difference in sequence between the four populations and also shows the relatedness of the three populations in a phylogenetic tree. In the tree, the Willow Creek population is the most closely related to the root population (Illinois- no resistance) and has the most resistance to TTX. The next closest relative to Illinois is Bear Lake (no resistance) followed by the two most closely related populations Warrenton (less resistance than Benton) and Benton. Warrenton, Benton, and Willow Creek populations' sequences of tsNav1.4 are lined up as compared to Bear Lake. Warrenton has one difference in sequence from Bear Lake, and Benton and Willow Creek have 2 and 4 differences, respectively.
The next step the researchers took was to determine whether the difference in domain IV sequence of the tsNav1.4 determined TTX sensitivity. They tested seven different channels in Xenopus oocytes. A human-snake chimaera channel was constructed for each population. The chimaera consisted of a human Nav1.4 sequence that had the outer pore sequence replaced with each snake population's tsNav1.4 sequence in this domain. Each snake populations' tsNav1.4 channel was also tested in the oocytes.
The data from this experiment are represented in Fig. 2 of the paper. This figure has 3 graphs and a current recording. In the three graphs the X-axis represents the TTX concentration and the Y- axis represents the percentage of the channel not blocked by the TTX. The first graph (2a) is a control showing that the amount of channel blocked is the same for entirely snake sequences and chimaeric sequences. The second graph (2b) shows the amount unblocked channel for each chimaera of each population. The third graph (2c) shows the change in amount of blocking of the channel when a valine is changed to an isoleucine in the position 1561 of the pore helix. The current recording (2d) shows the amount of channel blocked in a Bear Lake and Willow Creek chimaeras. From Figure 2 a Table was constructed that showed the amount of blockage of the different chimaeras. They use a value called Kd, which represents the amount of TTX needed to block 50% of the sodium channel, to show the TTX sensitivity of each chimaera.
From figure 2 and table 1, the researchers conclude that domain IV is the most important in determining the TTX sensitivity of a sodium channel. They also say that the valine in position 1561 of the pore helix of domain IV is critical for TTX tolerance.
Figure 3 is a graph comparing the sensitivity of each snake population to TTX. The X-axis represents skeletal muscle Kd and the Y-axis represents cloned-channel Kd. As predicted, Willow Creek has the most tolerance followed by Benton, Warrenton, and Bear Lake.
Critical evaluation of data:
Figure 1 is a pertinent, helpful figure helping the reader visualize the relatedness of the four populations and show the differences in genetic sequence of the sodium channels. The most interesting thing about this figure is the fact that Willow Creek, the most TTX tolerant population, is the most related to Illinois, the root population with no tolerance of TTX. The authors could have mentioned this and maybe proposed a reason why Willow Creek has diverged genetically so much from the root population.
Figure 2 is a logical step from Figure 1 and the data helps support the author's claim that domain IV is responsible for determinig TTX sensitivity. An explanation of the S5-S6 linker in the different domains of the sodium channel would help to understand the chimaeras. Why did they choose the S5-S6 linkers for tsNav1.4 placement? The S5-S6 linkers are not mentioned prior to this point in the paper. The construction of the chimaeras is confusing without some info about the S5-S6 linkers.
Figure 3 should be placed right after Figure 1. Although this would give away the conclusion of the paper, Figure 3 is out of place at the end. The large error bars on Willow Creek is something that the reader should question and the authors should have explained .
Further research on topic:
Crystallization of TTX and domain IV would help to visualize the structure/function relationship between the two molecules. By showing an image of each molecule one could highlight the specific molecules responsible for binding and show how different residues would create different binding properties for the whole molecule.
The authors mention at the end of the paper a whole genomic study of each snake population may help explain the evolution of TTX tolerance better. This genomic study of each snake may be done through microarrays that show which genes for each population are expressed/ repressed when a snake is exposed to differing amounts of TTX. The results would probably show that more than one gene is activated during exposure to TTX and other genes may be responsible for elevated tolerance.
The yeast two hybid method could be used with TTX as the bait and different garter snake proteins as possible targets. This would further prove that TTX does or does not bind to the different version of the sodium channel, and could also show that proteins that interact with TTX.
Ruben C., Peter, S. G., E.F., E. B., E. B.2005. Evolutionary diversification of TTX-resistant sodium channels in a predator-prey interaction. Nature 434: 759-763.
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