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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We examined functional consequences of intrasubunit contacts in the nicotinic receptor ␣ subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues ␣ K145, ␣ D200, and ␣ Y190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations ␣ D200N and ␣ K145Q impair channel gating to the same extent as either single mutation, while ␣ K145E counteracts the impaired gating due to ␣ D200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, ␣ K145 and ␣ D200 form a salt bridge associated with the closed state of the channel. When ACh binds, ␣ Y190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches ␣ K145, which in turn loosens its contact with ␣ D200. The positional changes of ␣ K145 and ␣ D200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of ␤ -strand 7, which harbors ␣ K145 and is part of the signature Cys-loop, and the second is of ␤ -strand 10, which harbors ␣ D200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel.

Mukhtasimova et al. describe experimental modifications of the patch clamp technique that improve temporal resolution of currents through single acetylcholine receptor channels. The study not only distinguishes between the priming and gating steps, but it also reveals how rate and equilibrium constants change as a function of agonist occupancy.

Binding of neurotransmitter triggers gating of synaptic receptor channels, but our understanding of the structures that link the binding site to the channel is just beginning to develop. Here, we identify an intersubunit triggering element required for rapid and efficient gating of muscle nicotinic receptors using a structural model of the Torpedo receptor at 4 Å resolution, recordings of currents through single receptor channels, measurements of inter-residue energetic coupling, and functional consequences of disulfide trapping. Mutation of the conserved residues, ␣Tyr 127, Asn 39, and ␦Asn 41, located at the two subunit interfaces that form the agonist binding sites, markedly attenuates acetylcholine-elicited channel gating; mutant cycle analyses based on changes in the channel gating equilibrium constant reveal strong energetic coupling among these residues. After each residue is substituted with Cys, oxidizing conditions that promote disulfide bond formation attenuate gating of mutant, but not wild-type receptors. Gating is similarly attenuated when the Cys substitutions are confined to either of the binding-site interfaces, but can be restored by reducing conditions that promote disulfide bond breakage. Thus, the Tyr-Asn pair is an intersubunit trigger of rapid and efficient gating of muscle nicotinic receptors.

Gating of the muscle-type acetylcholine receptor (AChR) channel depends on communication between the ACh-binding site and the remote ion channel. A key region for this communication is located within the structural transition zone between the ligand-binding and pore domains. Here, stemming from β-strand 10 of the binding domain, the invariant αArg209 lodges within the hydrophobic interior of the subunit and is essential for rapid and efficient channel gating. Previous charge-reversal experiments showed that the contribution of αArg209 to channel gating depends strongly on αGlu45, also within this region. Here we determine whether the contribution of αArg209 to channel gating depends on additional anionic or electron-rich residues in this region. Also, to reconcile diverging findings in the literature, we compare the dependence of αArg209 on αGlu45 in AChRs from different species, and compare the full agonist ACh with the weak agonist choline. Our findings reveal that the contribution of αArg209 to channel gating depends on additional nearby electron-rich residues, consistent with both electrostatic and steric contributions. Furthermore, αArg209 and αGlu45 show a strong interdependence in both human and mouse AChRs, whereas the functional consequences of the mutation αE45R depend on the agonist. The emerging picture shows a multifaceted network of interdependent residues that are required for communication between the ligand-binding and pore domains.

Different agonists activate the muscle AChR with different efficacies. Mukhtasimova and Sine show that oxidative cross-linking of proximal residues alters the ability of some agonists but not others to open the AChR channel. The findings show that, in opening the channel, agonists with differing efficacy elicit distinct structural changes.

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