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 (K(D)) from the wild type's closed state to be 4.1 +/- 0.5 mM (and that of ACh to be 106 +/- 6 microM). 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.

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 (K(B) congruent with 12 mM in the presence of approximately 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 ( approximately 5.5 A 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 approximately 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.

Sodium channel blockade may contribute to the analgesic efficacy of antidepressants.

UNLABELLED: Sodium channel blockers such as lidocaine, lamotrigine, and carbamazepine can be effective in the treatment of neuropathic pain. Though not approved for neuropathic pain indications, tricyclic antidepressants are often considered first-line treatment for conditions such as post-herpetic neuralgia and diabetic neuropathy. Several tricyclic antidepressants have been shown to block peripheral nerve sodium channels, which may contribute to their antihyperalgesic efficacy. In this study, we compared the sodium channel-blocking potency of a number of antidepressants, including tricyclic antidepressants and selective serotonin reuptake inhibitors. All compounds tested inhibited Na(V)1.7 in a state- and use-dependent manner, with affinities for the inactivated state ranging from 0.24 micromol/L for amitriptyline to 11.6 micromol/L for zimelidine. The tricyclic antidepressants were more potent blockers of Na(V)1.7. Moreover, IC(50)s for block of the inactivated state for amitriptyline, nortriptyline, imipramine, desipramine, and maprotiline were in the range of therapeutic plasma concentrations for both the treatment of depression as well as neuropathic pain. By contrast, fluoxetine, paroxetine, mianserine, and zimelidine had IC(50)s for Na(V)1.7 outside their therapeutic concentration ranges and generally were not efficacious against post-herpetic neuralgia or diabetic neuropathy. These results suggest that block of peripheral nerve sodium channels may contribute to the antihyperalgesic efficacy of certain antidepressants. PERSPECTIVE: Tricyclic antidepressants are often considered first-line treatment for neuropathic pain. Some tricyclic antidepressants block sodium channels, which may contribute to their antihyperalgesic efficacy. In the current study, we compared the potency of peripheral sodium channel blockade for several tricyclic antidepressants and selective serotonin reuptake inhibitors with their therapeutic efficacy.