Acetylcholine receptors are pentameric ligand-gated ion channels that mediate rapid synaptic transmission. Each receptor possesses two agonist-binding sites, whose occupation leads to channel opening, but the precise molecular events that follow agonist binding are unclear. Here, Mukhtasimova et al. covalently lock receptors in conformations equivalent to agonist-bound states, and observe that each binding site initiates the formation of one of two 'primed' states, from which the channels are able to open and close. The acetylcholine receptor, which mediates rapid synaptic transmission, possesses two agonist binding sites, the occupation of which leads to channel opening; however, the precise molecular events that follow agonist binding are unclear. Here, the receptors are locked in conformations equivalent to agonist-bound states, and it is observed that each binding site initiates the formation of one of two 'primed' states from which the channels are able to open and close. 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 is facilitated by rapid ACh association and dissociation, suggesting a binding site relatively non-selective for small cations. Selective transduction has been thought 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 efficacies1,2,3. By studying mutant AChRs, we show here 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 an 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 the speed and efficiency of the biological response.

The synapse is a localized neurohumoral contact between a neuron and an effector cell and may be considered the quantum of fast intercellular communication. Analogously, the postsynaptic neurotransmitter receptor may be considered the quantum of fast chemical to electrical transduction. Our understanding of postsynaptic receptors began to develop about a hundred years ago with the demonstration that electrical stimulation of the vagus nerve released acetylcholine and slowed the heart beat. During the past 50 years, advances in understanding postsynaptic receptors increased at a rapid pace, owing largely to studies of the acetylcholine receptor (AChR) at the motor endplate. The endplate AChR belongs to a large superfamily of neurotransmitter receptors, called Cys-loop receptors, and has served as an exemplar receptor for probing fundamental structures and mechanisms that underlie fast synaptic transmission in the central and peripheral nervous systems. Recent studies provide an increasingly detailed picture of the structure of the AChR and the symphony of molecular motions that underpin its remarkably fast and efficient chemoelectrical transduction.

We examined functional consequences of intrasubunit contacts in the nicotinic receptor α 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 αK145, αD200, and αY190 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 αD200N and αK145Q impair channel gating to the same extent as either single mutation, while αK145E counteracts the impaired gating due to αD200K, 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, αK145 and αD200 form a salt bridge associated with the closed state of the channel. When ACh binds, αY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches αK145, which in turn loosens its contact with αD200. The positional changes of αK145 and αD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of β-strand 7, which harbors αK145 and is part of the signature Cys-loop, and the second is of β-strand 10, which harbors αD200 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.