Interactions of Local Anesthetics and Cholinergic Ligands with Membrane-Bound Acetylcholine Receptor from Torpedo californica

Author: Blanchard, Steven Gerard

Year: 1980

Degree: Dissertation (Ph.D.)

Advisor: Raftery, Michael A.

Committee Member: Unknown, Unknown

Option: Chemistry

DOI: 10.7907/ptae-9r08

Abstract

A rapid method for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described in Chapter 1. This purification was achieved by sucrose-density-gradient centrifugation in a reorienting tube rotor. Further purification of these membranes was achieved by selective extraction of proteins at alkaline pH. The alkali treated membranes retained the functional properties of the untreated membranes and in addition contained essentially only the four polypeptides (molecular weights 40,000, 50,000, 60,000, and 65,000) characteristic of the receptor purified by affinity chromatography.

Chapter 2 describes the effects of local anesthetics on the membrane-bound acetylcholine receptor. The rate of the transition in receptor affinity (low to high) was determined by following the time-dependent increase in inhibition of iodinated α-bungarotoxin binding caused by 1 μM carbamylcholine. At concentrations below those that directly inhibited the binding of iodinated a-bungarotoxin, dibucaine increased the rate of the transition to a high-affinity state and tetracaine decreased this rate. A parallel was observed between the effectiveness of a compound in increasing or decreasing the rate of the agonist-induced transition in affinity and the change in its apparent inhibition constant in the presence of carbamylcholine measured by the displacement of trititated perhydrohistrionicotoxin. (Blanchard, S. G., Elliott, J., and Raftery, M. A. (1979) Biochemistry 18, 5880- 5885). This parallel could be explained by assuming (a) that local anesthetics bound directly to the specific histrionicotoxin binding site or (b) that they bound to a different site and the observed effects were caused indirectly by changes in receptor conformation.

The kinetics of local anesthetic binding to the receptor were examined using the fluorescent probe ethidium. The binding of tetracaine, dibucaine and lidocaine could not be described by a common mechanism and models which are qualitatively consistent with the data are discussed.

The preparation and characterization of the local anesthetic analog procaine amide azide are described in Chapter 3. When low concentrations of 3H-labeled procaine amide azide were photolyzed in the presence of acetylcholine receptor containing membrane fragments, two major labeled bands with apparent molecular weights of ~ 43,000 and 90,000 were seen after electrophoresis on sodium dodecyl sulfate/ polyacrylamide gels. When the length of the gels was increased, the labeled band of lower molecular weight was resolved into a major protein of 43,000 daltons and a minor one of 40,000 daltons. The radioactivity incorporated into the 40,000 dalton protein was due to [3H] procaine amide azide binding to cholinergic ligand binding sites, whereas the 43,000 and 90,000 molecular weight bands appeared to be unrelated to the acetylcholine receptor. Experiments with alkali-treated membrane preparations showed that the 60,000 and 65,000 dalton receptor subunits were also labeled by procaine amide azide.

Chapter 4 describes the alkylation of acetylcholine receptor enriched membrane fragments (following their reduction with low concentrations of dithiothreitol) by the fluorescent probe 5-iodoacetamido salicylic acid. This modification did not affect the equilibrium binding of carbamylcholine to the receptor. The fluorescence of bound 5-iodoacetamido salicylic acid was enhanced when the labeled membrane fragments were mixed with carbamylcholine. This increase in fluorescence was abolished by preincubation of the membrane fragments with excess α-bungarotoxin and was therefore specific for the acetylcholine receptor. Estimates of dissociation constants obtained from centrifugation experiments using radioactive ligand and from fluorescence titration data were in good agreement, showing that the observed fluorescence enhancement was an accurate reflection of receptor- carbamylcholine complex formation. The kinetics of carbamylcholine binding to labeled membrane fragments have been investigated over a wide range of ligand concentrations using stopped-flow fluorescence techniques. The kinetic signal was complicated and four distinct exponential phases were observed. A kinetic mechanism has been proposed to account for this behavior.

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