Student Theses and Dissertations

Date of Award

1971

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Frederick Dodge Jr.

Keywords

cobra toxin, nicotinic ACh receptors, irreversible inactivation, receptor protection, desensitization, frog neuromuscular junction

Abstract

Cholinergic transmission has been studied in the presence of a pure polypeptide toxin, of known amino-acid sequence, from cobra venom. For the frog sartorius myoneural junction, micropipette electrodes and on-line computer were used to show that the toxin specifically and irreversibly inactivates postsynaptic acetylcholine (ACh) receptors. Raja and Torpedo electroplaques also show nicotinic pharmacology and were similarly affected by the toxin. The toxin failed to affect the muscarinic receptors of the isolated frog heart or the receptors of Aplysia neurons (neither nicotinic nor muscarinic). These and other observations suggest that the toxin's pharmacological specificity is confined to nicotinic ACh receptors. Further studies were conducted with the frog myoneural junction. At concentrations greater than 10-8 M, the toxin irreversibly inactivated receptors with first-order kinetics. The rate constant for inactivation increased linearly with the toxin concentration near the receptors. Micro-ionophoretic application of two reversible agonists, ACh and carbamylcholine (Carb), focally protected receptors against bath-applied toxin. Bath-applied Carb and d-tubocurarine (dTC) (a reversible antagonist) also protected against bath-applied toxin. Since the reversible agonists and antagonists are known to bind to the ACh receptor, these observations suggest that cobra toxin also binds directly to the receptor. Choline protects only at high concentrations, in agreement with its known weak effects on ACh receptors. Quantitative studies of protection were evaluated according to a simple scheme of transitions among available, reversibly blocked, and irreversibly blocked receptors. All agonists cause desensitization, and each desensitized receptor was completely protected against the toxin. Agonists also caused departures from first-order kinetics of receptor inactivation; these are explained by the transition scheme and known rate constants for desensitization. Protection by dTC, however, was less than would be expected if curarized receptors were completely inaccesible to the toxin. Desensitized and curarized receptors therefore have different vulnerability to the toxin; this suggests that agonists and antagonists produce fundamentally different effects on ACh receptor structure.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

License and Reuse Information

Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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