Date of Award:

5-1-2005

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

Peter C. Ruben

Committee

Peter C. Ruben

Committee

Edmund D. Brodie Jr.

Committee

Daryll B. DeWald

Committee

Paul G. Wolf

Committee

Noelle E. Cockett

Abstract

The focus of this project was the identification of physiological and molecular genetic mechanisms of tetrodotoxin resistance in the common garter snake, Thamnophis sirtalis. Tetrodotoxin, a neurotoxin, specifically blocks the activity of voltage-gated sodium channels. Some populations of garter snake have evolved geographically variable resistance to tetrodotoxin in a coevolutionary arms race with their tetrodotoxic prey, newts of the genus Taricha. Variation in tetrodotoxin resistance among snake populations is extreme and spans three orders of magnitude. Here I describe a physiological mechanism of whole-animal tetrodotoxin resistance in four populations of snake that represent two independent evolutionary origins of resistance and span the range of variation observed across all populations. By recording action potentials from snake skeletal muscle fibers, I showed that tetrodotoxin-resistant sodium channels are expressed in this tissue in tetrodotoxin resistant snakes and that differences in tetrodotoxin binding affinity of these channels are responsible for adaptive differences in resistance among populations. These data demonstrate that both individual and population differences in the ability of snake skeletal muscle fibers to function in tetrodotoxin are closely correlated with the whole-animal measures of the tetrodotoxin resistant phenotype. Additionally, I describe the molecular and genetic mechanisms of tetrodotoxin resistance in the same four populations of garter snake. I identify novel changes in the molecular structure of the sodium channel expressed in snake skeletal muscle, tsNaV1.4, that are responsible for differences in tetrodotoxin resistance among the four snake populations. The pore sequence of tsNaV1.4 differs among the three tetrodotoxin-resistant populations with the least conservative and largest number of amino acid substitutions in the pore sequence of a snake with the most extreme tetrodotoxin resistance. By recording currents from cloned tsNaV1.4 and human/snake chimeric channels expressed in Xenopus oocytes, I demonstrated that differences in the amino acid sequence of the channel affect tetrodotoxin binding and impart different levels of resistance in the four snake populations. These results indicate that the evolution of a physiological trait has occurred through a series of unique changes in a region of the channel that is important for channel function and is highly conserved in other vertebrates.

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