MECHANISTIC STUDIES OF THE ALLOSTERIC MODULATION OF MUSCARINIC RECEPTORS
Open Access
- Author:
- Stahl, Edward L
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 24, 2011
- Committee Members:
- John Ellis, Dissertation Advisor/Co-Advisor
John Ellis, Committee Chair/Co-Chair
Victor Alan Canfield, Committee Member
Robert G Levenson, Committee Member
Blaise Peterson, Committee Member - Keywords:
- G protein-coupled receptors
acetylcholine
muscarinic
allosteric
amiodarone - Abstract:
- This dissertation is the result of a detailed investigation into the effects of the antiarrhythmic drug amiodarone on muscarinic receptors. We have found that amiodarone interacts with muscarinic receptors in a specifically allosteric manner. In radioligand binding studies, amiodarone was only able to partially inhibit the binding of the orthosteric antagonist [3H]N-methylscopolamine (NMS). Additionally, amiodarone was able to alter the rate of dissociation of [3H]NMS from M1, M2, and M5 receptors. These findings suggest that NMS and amiodarone are able to bind to the receptor simultaneously. Furthermore, the pharmacology of the effect on NMS dissociation demonstrated that amiodarone does not interact with the “common” site at which gallamine, obidoxime, and many other muscarinic allosteric ligands are known to bind. In functional studies, amiodarone enhanced the response stimulated by an acetylcholine concentration that produced about 20% of maximal arachidonic acid release from the M5 receptor. However, under the same conditions, amiodarone did not enhance M1 arachidonic acid release. More detailed studies at M5 found that the effect of amiodarone was to enhance the efficacy of acetylcholine, without increasing its potency. This was the first demonstration of allosteric enhancement of efficacy at the M5 receptor, and the first demonstration of enhancement of efficacy but not potency at any muscarinic receptor. Amiodarone also enhanced the maximal level of agonist-stimulated release of arachidonic acid (AA) by the M3 muscarinic receptor; this enhancement was observed for acetylcholine and for the partial agonist pilocarpine. A similar effect of amiodarone was observed when pilocarpine was used to stimulate inositol phosphate (IP) metabolism, but not when acetylcholine was used. Subsequent studies showed that the IP response exhibited a much larger receptor reserve than the AA response, and reduction of that reserve by receptor alkylation unmasked amiodarone’s enhancement of the maximal IP response to acetylcholine. Modulating the receptor reserve also revealed acetylcholine’s greater affinity (KA) for the conformation of the receptor that mediates the AA response. The amiodarone analog Nethylamiodarone (NEA) did not alter the maximal agonist response stimulated by M3, but merely reduced agonist potency (that is, it appeared to be an antagonist). However, the action of NEA could be clearly distinguished from the action of the orthosteric antagonist NMS. Demonstration of this point was facilitated by an elaboration of Hall’s allosteric two-state model; this new model represents a system composed of two ligands that compete with each other at the orthosteric site and two ligands that compete with each other at the allosteric site. In conclusion, amiodarone competes with NEA at a novel, extracellular, allosteric site to enhance the maximal stimulation evoked by acetylcholine and pilocarpine in two different responses.