The nicotinic acetylcholine receptor is found at neuromuscular junctions, where it functions to depolarize the postsynaptic membrane, triggering muscle contraction. Depolarization occurs by channel opening which is induced by the binding of two acetylcholine molecules. Neurotoxins inhibit the binding of acetylcholine by binding to a large portion of the receptor's a-subunit, blocking at least in part the acetylcholine binding site. The best structure for the receptor is at a low resolution level of 9 _, using a combination of electron microscopy and x-ray diffraction. NMR spectroscopy for this large membrane bound protein complex is clearly beyond current technological capabilities. However, peptides corresponding to the portion of the receptor near the acetylcholine binding site have been found to bind a-bungarotoxin with about the same affinity as the intact a-subunit. We propose here (1) to determine the rates of exchange and activation energies between R- and S-disulfide bond rearrangement and between a cis-trans amide bond interconversion in an eight-membered cysteinyl-cysteine ring, C192-C193 found in the active sight of the a-subunit; (2) to determine the active conformation of the cysteinyl-cysteine ring in peptide fragments derived from the AChR's a-subunit; (3) to determine the structure of the N-terminal extracellular region of the a-subunit that binds acetylcholine, agonists and antagonists in its free, agonist-bound and antagonist-bound states.Some of the peptides for this study have already been synthesized. We shall need to make a variety of different peptides whose syntheses have not been completely worked out. Products and reactions will have to be verified by mass spectrometry, which can be performed locally at UCSF. Once the desired products are made we shall pursue the structures and dynamics of these peptides in free and bound states by multi-dimensional multinuclear magnetic resonance techniques. The objective of this research is to determine whether or not the rare structural motif of a cysteinyl-cysteine ring may act as a molecular switch in opening and closing the ion channel of the AChR. From the molecular details of AChR ligand interactions with their binding site as well as the structural changes of the receptor binding site, we hope to shed light on AChR's highly specific recognition, mechanism and control.
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