PROBING THE AGONIST BINDING POCKET IN THE NICOTINIC ACETYLCHOLINE-RECEPTOR - A HIGH-RESOLUTION SOLID-STATE NMR APPROACH

Acetylcholine, the agonist for the nicotinic acetylcholine receptor, h as been observed directly when bound specifically to its binding site in the fully functional receptor-enriched membranes from Torpedo nobil iana. High-resolution solid-state, magic angle spinning C-13 NMR metho ds have been used to observe selectively N+((CH3)-C-13)(3) acetylcholi ne bound in as few as 20 nmol of receptor binding sites, against a bac kground of natural abundance membrane resonances and excess acetylchol ine in free solution. The specificity of the binding has been demonstr ated to be pharmacologically significant through the use of the compet itive inhibitor alpha-bungarotoxin which selectively displaces and pre vents binding of acetylcholine to the membrane-bound receptor. The che mical shift assigned to N+((CH3)-C-13)(3) acetylcholine in solution an d crystalline solid is 53.9 +/- 0.04 ppm, and it changes by 1.6 ppm (p < 0.05) for agonist when bound specifically in the receptor binding s ite. Through the use of computer simulations of chemical shifts carrie d out on acetylcholine bound to the acetylcholinesterase, we propose t hat the cause for this change is the presence of aromatic side chains lining the receptor binding site. It is suggested that the binding of acetylcholine to the nicotinic acetylcholine receptor is mediated prim arily through the interaction of the quaternary ammonium group of the acetylcholine with the pi bonded systems in the aromatic side chains. Longitudinal relaxation time measurements show that the residency time for the acetylcholine observed in DDCP experiments is long (>200 ms) with respect to the longitudinal relaxation time of other assignable r esonances within the spectrum from the lipid and protein and confirms that the acetylcholine is protein-associated, and not free in solution or nonspecifically bound.