Monoclonal antibody technology affords the opportunity to generate site specific probes, often high affinity ligands, which can define a number of antigenic determinants on the molecule they recognize. The nicotinic acetylcholine receptor (AChR) has been characterized on both the biochemical and structural level. We have developed an extensive library of monoclonal antibodies (mAbs) which can be used to selectively ligate a specific antigenic determinant and assess resultant changes in receptor function. It is the aim of the proposed investigation to examine the electrophysiological effects of monoclonal antibodies directed against the AChR at the single channel level using extracellular patch clamping techniques. The mAbs to be used have been demonstrated to effect not only the binding of cholinergic agonists of antagonists and neurotoxins (see Project 1) but also the ability of agonist to induce ion permeation (Project 2). These mAbs also affected the ability of the membrane bound AChR to undergo reversible sensitization-desensitization affinity transitions. Experimentally, we will approach the question of the specificity of mAb epitopes in effecting receptor function using (1) purified and reconstituted AChRs from Torpedo electroplax and (2) in situ AChRs from chick skeletal muscle myotubes and Xenopus embryonic myotomal muscle cells maintained in tissue culture. We will integrate electrophysiological data to be obtained in the macroscopic miniature endplate potential and current (mepp and mepc) experiments of Project 4 with the whole-cell tight-seal voltage clamp data in intact muscle cells. Kinetic rate constants governing the gating behaviour of AChR channels will be examined in both the native and reconstituted AChR with the goal of identifying receptor subunits and localizing functional regions within the subunit molecule which determine channel closing.
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