Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a “gate” in the transmembrane domain (TMD). We used Φ-value analysis to probe the relative timing of the gating motions of α-subunit residues located near the ECD–TMD interface. Mutation of four of the seven amino acids in the M2–M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (Keq) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Φ-value for the whole linker was ∼0.64. One interpretation of this result is that the gating motions of the M2–M3 linker are approximately synchronous with those of much of M2 (∼0.64), but occur after those of the transmitter binding site region (∼0.93) and loops 2 and 7 (∼0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed Keq by 2800-, 10-, and 18-fold, respectively, and with an average Φ-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (≤0.51 kcal mol−1). The M2–M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an ∼16-Å border and involve about a dozen residues.

The gating isomerization of neuromuscular acetylcholine receptors links the rearrangements of atoms at two transmitter-binding sites with those at a distant gate region in the pore. To explore the mechanism of this reversible process, we estimated the gating rate and equilibrium constants for receptors with point mutations of alpha-subunit residues located between the binding sites and the membrane domain (N95, A96, Y127, and I49). The maximum energy change caused by a side-chain substitution at alphaA96 was huge (approximately 8.6 kcal/mol, the largest value measured so far for any alpha-subunit amino acid). A Phi-value analysis suggests that alphaA96 experiences its change in energy (structure) approximately synchronously with residues alphaY127 and alphaI49, but after the agonist molecule and other residues in loop A. Double mutant-cycle experiments show that the energy changes at alphaA96 are strongly coupled with those of alphaY127 and alphaI49. We identify a column of mutation-sensitive residues in the alpha-subunit that may be a pathway for energy transfer through the extracellular domain in the gating isomerization.

Muscle acetylcholine receptors are synaptic ion channels that "gate" between closed- and open-channel conformations. We used Phi-value analysis to probe the transition state of the diliganded gating reaction with regard to residues in the M3, membrane-spanning helix of the muscle acetylcholine receptor alpha-subunit. Phi (a fraction between 1 and 0) parameterizes the extent to which a mutation changes the opening versus the closing rate constant and, for a linear reaction mechanism, the higher the Phi-value, the "earlier" the gating motion. In the upper half of alphaM3 the gating motions of all five tested residues were temporally correlated (Phi approximately 0.30) and serve to link structural changes occurring at the middle of the M2, pore-lining helix with those occurring at the interface of the extracellular and transmembrane domains. alphaM3 belongs to a complex and diverse set of synchronously moving parts that change structure relatively late in the channel-opening process. The propagation of the gating Brownian conformational cascade has a complex spatial distribution in the transmembrane domain.

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The Cys-Loop gene super-family of ligand gated ion channels includes nicotinic acetylcholine (nACh), gamma aminobutyric acid (GABA A ), glycine (Gly), and serotonin (5-HT 3A ) receptors. Each receptor is either a homo-or heteropentamer made up of 5 identical or homologous subunits. Each subunit has an extracellular N-terminal domain, which houses the ligand binding site; a transmembrane domain which spans the membrane four times as a-helical segments (M1-M4); and a long loop between M3 and M4 that constitutes the intracellular domain. M2 lines the ion channel, whereas M1, M3 and M4 are abluminal. Muscle nAChRs consist of four different subunits with the clockwise arrangement agabd when viewed from the extracellular side. We used disulphide trapping between individually engineered Cys in the aM2 segments to investigate arrangement and flexibility of the upper part of aM2. Disulfide bond formation was monitored in a 2 bdg nAChR expressed in Xenopus laevis oocytes by two electrode voltage clamp experiments and Western blotting. In properly arranged Cys pairs disulfide bond formation can either occur spontaneously or it can be induced by oxidizing with copper phenanthroline (CuPhen). Cystine bond formation is reversible by reducing with dithiothreitol (DTT). To eliminate interference from the native vicinal disulphide present in the ligand binding site of the a-subunit, we utilized the background mutations aC192S-C193S. Position aE262C that was previously shown to face the channel formed DTT reducible dimers both spontaneously and upon application of the oxidizing agent CuPhen. Based on Unwin's 4-Å resolution model, the formation of disulfide bonds at this channel level would require substantial movement of the channel-lining M2 segments. No dimer formation was observed in aL263C. We are investigating a series of aM2 Cys mutants to determine which positions in M2 can form disulfide bonds.