Functional Asymmetry of Agonist Binding in Fetal and Adult Muscle Acetylcholine Receptors

We recorded currents through the cell membrane at single nAChR molecules, held at ACh or Epibatidine (Ebd) concentrations of 0.01, 0.1, 1, 10 or 100 μM. The measured current amplitudes had an absolutely fixed value of 15 pA. This was valid for different agonists at all concentrations. Binding an agonist at one or both sites in the ring of subunits allowed to open the channel, the site that initiated the opening determined the duration of the final opening of the channel. In addition, the current flow was continuously interrupted by < 3 μs shut times. The resolution of our records was optimized to reach 5 μs, but was insufficient to resolve an unknown proportion of shorter shut times. Therefore, measured durations of openings are overestimated, and cited in brackets: τo1 (3 μs) elicited by agonist-binding at the δ-site, τo2 and τo3 (40 and 183 μs) by binding at the ϵ-site, and τo4 (752 μs) by binding at the δ- and ϵ-site. Mono-liganded nAChRs trigger short bursts of 0.6 ms duration. Bi-liganded nAChRs generate long bursts that at low agonist concentrations last 12 ms. Above 10 μM ACh, long bursts are shortened, with 100 μM ACh, to 5 ms, and further at higher concentrations. While ACh was the main agonist, Ebd bound more effectively than ACh to the ϵ-site.

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Efficiency measures the conversion of agonist binding energy into receptor conformational change

The Journal of General Physiology ◽

10.1085/jgp.201812215 ◽

2019 ◽

Vol 151 (4) ◽

pp. 465-477 ◽

Cited By ~ 6

Author(s):

Tapan K. Nayak ◽

Ridhima Vij ◽

Iva Bruhova ◽

Jayasha Shandilya ◽

Anthony Auerbach

Keyword(s):

Activation Energy ◽

Binding Energy ◽

Conformational Change ◽

Acetylcholine Receptors ◽

Single Channel ◽

Equilibrium Constants ◽

Bk Channels ◽

Agonist Binding ◽

Aryl Hydrocarbon ◽

Efficiency Measures

Receptors alternate between resting↔active conformations that bind agonists with low↔high affinity. Here, we define a new agonist attribute, energy efficiency (η), as the fraction of ligand-binding energy converted into the mechanical work of the activation conformational change. η depends only on the resting/active agonist-binding energy ratio. In a plot of activation energy versus binding energy (an “efficiency” plot), the slope gives η and the y intercept gives the receptor’s intrinsic activation energy (without agonists; ΔG0). We used single-channel electrophysiology to estimate η for eight different agonists and ΔG0 in human endplate acetylcholine receptors (AChRs). From published equilibrium constants, we also estimated η for agonists of KCa1.1 (BK channels) and muscarinic, γ-aminobutyric acid, glutamate, glycine, and aryl-hydrocarbon receptors, and ΔG0 for all of these except KCa1.1. Regarding AChRs, η is 48–56% for agonists related structurally to acetylcholine but is only ∼39% for agonists related to epibatidine; ΔG0 is 8.4 kcal/mol in adult and 9.6 kcal/mol in fetal receptors. Efficiency plots for all of the above receptors are approximately linear, with η values between 12% and 57% and ΔG0 values between 2 and 12 kcal/mol. Efficiency appears to be a general attribute of agonist action at receptor binding sites that is useful for understanding binding mechanisms, categorizing agonists, and estimating concentration–response relationships.

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