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

<p>Abstract</p> <p>Background</p> <p>Along with high affinity binding of epibatidine (<it>K</it>_d1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (<it>K</it>_d2≈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.</p> <p>Results</p> <p>Computer 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.</p> <p>Conclusions</p> <p>Investigating 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.</p

Transmitter receptors and functional anatomy of the cerebral cortex

The currently available architectonic maps of the human cerebral cortex do not match the high degree of cortical segregation as shown by functional imaging. Such functional imaging studies have demonstrated a considerable number of functionally specialized areas not displayed in the architectonic maps. We therefore analysed the regional and laminar distribution of various transmitter receptors in the human cerebral cortex, because these signalling molecules play a crucial role in cortical functions. They may provide a novel and functionally more relevant insight into the regional organization of the cortex, which cannot be achieved by architectonic observations in cell body- or myelin-stained sections. Serial cryostat sections through whole human hemispheres were used for quantitative receptor autoradiography. The regional and laminar densities of numerous receptors of classic transmitter systems were analysed. Alternating sections were stained for comparisons based on cyto- or myeloarchitectonic criteria. Our results demonstrate that the regional distribution of transmitter receptors reflects well-established cyto- and myeloarchitectonically defined borders of cortical areas, but in addition enables the identification of more cortical areas than previously demonstrated. Moreover, the laminar distribution patterns of a given receptor type in different cortical areas as well as those of different receptor types in the same cortical area reveal novel and functionally relevant data concerning the intracortical organization in the human cerebral cortex