Block of Muscle Nicotinic Receptors by Choline Suggests that the Activation and Desensitization Gates Act as Distinct Molecular Entities

Ion channel block in muscle acetylcholine nicotinic receptors (AChRs) is an extensively reported phenomenon. Yet, the mechanisms underlying the interruption of ion flow or the interaction of the blocker with the channel's gates remain incompletely characterized. In this paper, we studied fast channel block by choline, a quaternary-ammonium cation that is also an endogenous weak agonist of this receptor, and a valuable tool in structure–function studies. Analysis of the single-channel current amplitude as a function of both choline concentration and voltage revealed that extracellular choline binds to the open-channel pore with millimolar apparent affinity (KB ≅ 12 mM in the presence of ∼155 mM monovalent and 3.5 mM divalent, inorganic cations), and that it permeates the channel faster than acetylcholine. This, together with its relatively small size (∼5.5 Å along its longest axis), suggests that the pore-blocking choline binding site is the selectivity filter itself, and that current blockages simply reflect the longer-lived sojourns of choline at this site. Kinetic analysis of single-channel traces indicated that increasing occupancy of the pore-blocking site by choline (as judged from the reduction of the single-channel current amplitude) is accompanied by the lengthening of (apparent) open interval durations. Consideration of a number of possible mechanisms firmly suggests that this prolongation results from the local effect of choline interfering with the operation of the activation gate (closure of blocked receptors is slower than that of unblocked receptors by a factor of ∼13), whereas closure of the desensitization gate remains unaffected. Thus, we suggest that these two gates act as distinct molecular entities. Also, the detailed understanding gained here on how choline distorts the observed open-time durations can be used to compensate for this artifact during activation assays. This correction is necessary if we are to understand how choline binds to and gates the AChR

Estimating Binding Affinities of the Nicotinic Receptor for Low-efficacy Ligands Using Mixtures of Agonists and Two-dimensional Concentration–Response Relationships

The phenomenon of ligand-induced ion channel gating hinges upon the ability of a receptor channel to bind ligand molecules with conformation-specific affinities. However, our understanding of this fundamental phenomenon is notably limited, not only because the changes in binding site structure and ligand conformation that occur upon gating are largely unknown but, also, because the strength of these ligand–receptor interactions are experimentally elusive. Both high- and low-efficacy ligands pose a number of analytical and experimental challenges that can render the estimation of their conformation-specific binding affinities impossible. In this paper, we present a novel assay that overcomes some of the hurdles presented by weak agonists of the muscle nicotinic receptor and allows the estimation of their closed-state affinities. The method, which we have termed the “activation-competition” assay, consists of a single-channel concentration–response assay performed in the presence of a binary mixture of ligands of widely different efficacies. By plotting the channel response (i.e., the open probability) as a function of the concentration of each agonist in the mixture, interpreting the observed response in the framework of a plausible kinetic scheme, and fitting the open probability surface with the corresponding function, the affinities of the closed receptor for the two agonists can be simultaneously extracted as free parameters. Here, we applied this methodology to estimate the closed-state affinity of the muscle nicotinic receptor for choline (a very weak agonist) using acetylcholine (ACh) as the partner in the mixture. We estimated the dissociation equilibrium constant of choline (KD) from the wild type's closed state to be 4.1 ± 0.5 mM (and that of ACh to be 106 ± 6 μM). We also discuss the use of accurate estimates of affinities for low-efficacy agonists as a tool to discriminate between binding and gating effects of mutations, and in the context of the rational design of therapeutic drugs

Desensitization Contributes to the Synaptic Response of Gain-of-Function Mutants of the Muscle Nicotinic Receptor

Although the muscle nicotinic receptor (AChR) desensitizes almost completely in the steady presence of high concentrations of acetylcholine (ACh), it is well established that AChRs do not accumulate in desensitized states under normal physiological conditions of neurotransmitter release and clearance. Quantitative considerations in the framework of plausible kinetic schemes, however, lead us to predict that mutations that speed up channel opening, slow down channel closure, and/or slow down the dissociation of neurotransmitter (i.e., gain-of-function mutations) increase the extent to which AChRs desensitize upon ACh removal. In this paper, we confirm this prediction by applying high-frequency trains of brief (∼1 ms) ACh pulses to outside-out membrane patches expressing either lab-engineered or naturally occurring (disease-causing) gain-of-function mutants. Entry into desensitization was evident in our experiments as a frequency-dependent depression in the peak value of succesive macroscopic current responses, in a manner that is remarkably consistent with the theoretical expectation. We conclude that the comparatively small depression of the macroscopic currents observed upon repetitive stimulation of the wild-type AChR is due, not to desensitization being exceedingly slow but, rather, to the particular balance between gating, entry into desensitization, and ACh dissociation rate constants. Disruption of this fine balance by, for example, mutations can lead to enhanced desensitization even if the kinetics of entry into, and recovery from, desensitization themselves are not affected. It follows that accounting for the (usually overlooked) desensitization phenomenon is essential for the correct interpretation of mutagenesis-driven structure–function relationships and for the understanding of pathological synaptic transmission at the vertebrate neuromuscular junction