Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

Abstract

BackgroundAlong with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites.ResultsComputer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates.ConclusionsInvestigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches.

Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4–α4 interface

“…Peculiarly, these populations are dependent on expression of b2, a4 or b4 subunits and their expression pattern resembles that of a4b2 receptors (Marks et al, 2010). Several speculations have been put forward to explain the existence of these low affinity a4 and b2 dependent sites including stoichiometry dependency (2a:3b vs. 3a:2b), state dependency (resting vs. desensitized), improperly assembled receptors or complex mixtures of receptors incorporating more than two different subunits (Person and Wells, 2011;Marks et al, 2007). The recent knowledge that 3a:2b receptors harbor an orthosteric aea interface suggests an alternative explanation.…”

“…Several potential explanations can be given but perhaps the most plausible one is that binding to the aea interface is unresolved under the assay conditions used. In a study by Person and Wells (2011) the caveats of detecting a low affinity a4b2 site in the presence of high affinity sites is addressed extensively with mathematical simulations. With the small fraction of total binding imposed by the low affinity site (maximally 1/3) and expected large difference in affinities, low affinity binding was shown to easily be mistaken for non-specific binding under standard conditions as those utilized in the current study.…”

Calculation and Visualization of Binding Equilibria in Protein Studies

“…This model is useful in the analysis of ligand binding specificity as a function of ligand concentration and in the interpretation of binding site competition experiments. 6,7 The model includes the calculation of the specificity factor…”

“…The two receptors can be either in the same protein or in two different proteins. This model is useful in the analysis of ligand binding specificity as a function of ligand concentration and in the interpretation of binding site competition experiments. , The model includes the calculation of the specificity factor as defined by Eaton et al It shows higher values of specificity at low ligand concentration but decreases when the ligand concentration increases, indicating loss of specificity (Figure ).…”