Synaptic receptors respond to neurotransmitters by opening an intrinsic ion channel in the final step in synaptic transmission. How binding of the neurotransmitter is conveyed over the long distance to the channel remains a central question in neurobiology. Here we delineate a principal pathway that links neurotransmitter binding to channel gating by using a structural model of the Torpedo acetylcholine receptor at 4-A resolution, recordings of currents through single receptor channels and determinations of energetic coupling between pairs of residues. We show that a pair of invariant arginine and glutamate residues in each receptor alpha-subunit electrostatically links peripheral and inner beta-sheets from the binding domain and positions them to engage with the channel. The key glutamate and flanking valine residues energetically couple to conserved proline and serine residues emerging from the top of the channel-forming alpha-helix, suggesting that this is the point at which the binding domain triggers opening of the channel. The series of interresidue couplings identified here constitutes a primary allosteric pathway that links neurotransmitter binding to channel gating.

In the course of synaptic transmission in the brain and periphery, acetylcholine receptors (AChRs) rapidly transduce a chemical signal into an electrical impulse. The speed of transduction owes in large part to rapid ACh association and dissociation, implying a binding site relatively non-selective for small cations; selective transduction has been supposed to originate from the ability of ACh, over that of other organic cations, to trigger the subsequent channel opening step. However transitions to and from the open state were shown to be similar for agonists with widely different efficacies.1,2,3 Here, by studying mutant AChRs, we find that the ultimate closed to open transition is agonist-independent and preceded by two primed closed states; the first primed state elicits brief openings, whereas the second elicits long-lived openings. Long-lived openings and the associated primed state are detected in the absence and presence of agonist, and exhibit the same kinetic signatures under both conditions. By covalently locking the agonist binding sites in the bound conformation, we find that each site initiates a priming step. Thus a change in binding site conformation primes the AChR for channel opening in a process that enables selective activation by ACh while maximizing speed and efficiency of the biological response.

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Three approaches are comparatively evaluated for the use of tris(2,2'-bipyridyl)ruthemum(II), Ru(bpy)33+, as a chemiluminescent reagent in flowing streams: (1) external generation of the reactive Ru(bpy)33+ oxidation state followed by contact with the analyte, (2) in situ generation of the Ru(bpy)33+ species from a solution mixture of the analyte and the Ru(bpy)32+ species as it passes through the reaction/observation cell, and (3) in situ generation of the Ru(bpy)33+ species from the Ru(bpy)32+ species immobilized within the observation cell. Oxalate and proline were used as representative analytes for comparison of these three modes with respect to the influence of experimental variables (reagent concentration, flow rate, pH) and resulting analytical performance (detection limit, working range, measurement precision). Additionally, a comparison was made of the relative ECL intensities obtained for a variety of analytes including oxalate, amino acids, aliphatic amines, peptides, and NADH. We find that each approach has its unique set of strengths and weaknesses. The external generation mode yields the most intense emission, especially for simple aliphatic amines, but working curves have poor linearity, and emission intensities have a large dependence on solution flow rate. The in situ immobilized approach results in lower intensities but yields the widest linear dynamic ranges, is most conservative of reagent, and has a particular sensitivity advantage for proline and NADH determinations. The in situ solution mode is superior for the detection of amino acids such as tryptophan, 5-hydroxytryptophan, and histidine and has time, convenience, and reliability advantages.In recent years, tris(2,2'-bipyridyl)ruthenium(III) electrogenerated chemiluminescence (ECL) has received considerable attention in chemical analysis because of its utility for detection of alkylamines, amino acids, and NADH without derivatization.Several research groups have been actively investigating the ECL mechanism of Ru(bpy)32+ and its analytical applications. Bard and co-workers have reported a series of investigations on the