Gupta et al. use single-channel electrophysiology to investigate the gating mechanism of acetylcholine receptor ion channels. They propose that channel opening starts at the M2–M3 linker and ligand-binding sites and proceeds through four brief intermediate conformations before ending with the collapse of a gate bubble.

Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits—the transmitter-binding sites and αM2–αM3 linkers in the membrane domain—have the highest ϕ-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate. Large gating-energy changes occur at the transmitter-binding sites, α-subunit interfaces, the αM1 helix and the gate. We hypothesize that rearrangements of the linkers trigger the global allosteric transition, and that the hydrophobic gate unlocks in three steps. The mostly local character of side-chain energy changes and the similarly high ϕ-values of separated domains, both with and without ligands, suggest that gating is not strictly a mechanical process initiated by the affinity change for the agonist.

A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αe (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔG B1 ). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔG B1 is ∼−2 kcal/mol more favorable at αγ compared with at αe and αδ. Only three of the aromatics contribute significantly to ΔG B1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔG B1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αe and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αe and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse.allosteric protein | ion channel | ligand binding sites | single-channel electrophysiology | synaptic maturation R eceptors at synapses respond to specific chemical signals in the extracellular environment because the active conformation of the protein has a higher affinity for the ligand compared with the resting conformation (1, 2). The active vs. resting difference in binding free energy increases the relative stability of the active state and, hence, the probability of a cellular response. In this report, we describe and distinguish sources of ligandbinding free energy in three kinds of agonist site present in mouse muscle nicotinic acetylcholine receptors (AChRs). Our goal was to use single-channel electrophysiology to assess the relative contribution of significant functional groups to the overall free energy generated by the affinity change at each type of site.At cholinergic synapses, the main chemical signals are ACh released from nerve terminals and choline, which is an ACh precursor, hydrolysis product, and stable component of serum (3). The muscle AChR has central pore surrounded by five subunits of composition α 2 βδe in adult-type and α 2 βδγ in fetal-type (Fig. 1A) (4). The fetal, γ, subunit is essential for proper synapse maturation, and the adult, e, subunit is necessary for proper function of mature synapses (5-7). Each AChR pentamer has two agonist binding sites in the extracellular domain, at αδ and either αe (adult) or αγ (fetal) subunit interfaces.The change in agonist affinity occurs within the global, resting↔active...

The temperature dependence of agonist binding and channel gating were measured for wild-type adult neuromuscular acetylcholine receptors activated by acetylcholine, carbamylcholine, or choline. With acetylcholine, temperature changed the gating rate constants (Q(10) ≈ 3.2) but had almost no effect on the equilibrium constant. The enthalpy change associated with gating was agonist-dependent, but for all three ligands it was approximately equal to the corresponding free-energy change. The equilibrium dissociation constant of the resting conformation (K(d)), the slope of the rate-equilibrium free-energy relationship (Φ), and the acetylcholine association and dissociation rate constants were approximately temperature-independent. In the mutant αG153S, the choline association and dissociation rate constants were temperature-dependent (Q(10) ≈ 7.4) but K(d) was not. By combining two independent mutations, we were able to compensate for the catalytic effect of temperature on the decay time constant of a synaptic current. At mouse body temperature, the channel-opening and -closing rate constants are ∼400 and 16 ms(-1). We hypothesize that the agonist dependence of the gating enthalpy change is associated with differences in ligand binding, specifically to the open-channel conformation of the protein.

Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (κ) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations, κ ∼ 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90; αG153) or lower (0.25; εP121). The results suggest that backbone rearrangements in loop B, loop C, and the non-α surface participate in both LA binding and the LA ↔ HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process.