Elsevier

Brain Research

Volume 959, Issue 1, 3 January 2003, Pages 98-102
Brain Research

Research report
Cytisine binds with similar affinity to nicotinic α4β2 receptors on the cell surface and in homogenates

https://doi.org/10.1016/S0006-8993(02)03733-2Get rights and content

Abstract

Cytisine and nicotine bound to specific sites in homogenates prepared from HEK 293 cells which stably express human neuronal nicotinic α4 and β2 subunits. The number of sites was the same for both ligands and nicotine was a full competitive inhibitor of cytisine binding. However, when binding was done to intact cells the number of binding sites per cell for nicotine was ∼4-fold the number of sites for cytisine. Nicotine fully blocked cytisine binding, but cytisine only partially blocked nicotine binding to intact cells. When cells were permeabilized with saponin, the number of sites for nicotine was unchanged, while the number of sites for cytisine was increased, and cytisine was able to fully block nicotine binding. These data indicate that cytisine binds only to surface receptors on intact cells. The apparent affinity of cytisine for surface receptors (Kd=0.8 nM) was not significantly different from that for receptors in the cell homogenate (0.3 nM).

Introduction

The binding of agonists to nicotinic receptors is the result both of the intrinsic affinity of the agonist for the ligand binding sites and the associated changes in the state of the receptor. At equilibrium, binding largely reflects the occupancy of sites on the high affinity, desensitized receptor [3], [14]. Accordingly, the apparent affinity of a population of receptors may be affected by the ability of the receptor to undergo state transitions and hence may depend on extrinsic factors including whether the measurement is made on an intact or a disrupted preparation. The question becomes of greater significance when an attempt is made to correlate the apparent affinities for a nicotinic agonist to affect several aspects of receptor function, for example activation, desensitization and upregulation.

It has been reported that the affinity of neuronal nicotinic α4β2 receptors for nicotine differs significantly (30-fold) depending on whether receptors were examined in intact cells (Xenopus oocytes) or in homogenates [5]. We set out to determine whether similar differences could be seen when receptors were expressed in a mammalian cell system. Our data indicate that nicotine is not a suitable ligand to use to assay affinity for surface receptors in our system. Cytisine binds to surface receptors in intact cells, and shows an insignificant difference in affinity between surface receptors and the receptors in cell homogenates.

Section snippets

Materials and methods

Unless otherwise noted, all chemicals were obtained from Sigma (St. Louis, MO). [3H]Nicotine (64 Ci/mmol) and [3H]cytisine (40 Ci/mmol) were obtained from Perkin Elmer Life Sciences.

The procedure followed that described earlier [13]. In brief, HEK293 cells (CRL-1573, ATCC, Gaithersburg, MD) were transfected by electroporation with expression constructs for the human α4 and β2 subunits. Transfected cells were selected by growth in medium containing G418 (450 μg/ml; Gibco), then repeatedly

Ligand binding to cell homogenates

Nicotine and cytisine bound to homogenates prepared from cells stably expressing human α4 and β2 nicotinic subunits (Fig. 1). The binding to homogenates from transfected cells was much greater than to homogenates from untransfected cells (Fig. 1), indicating the specificity of the binding. When the Hill equation was used to describe the data for specific binding, the values obtained were, for nicotine (n=4 binding experiments), maximal binding of 8500±1600 fmol/mg protein, Kd=1.1±0.4 nM and n

Discussion

We conclude from our observations that cytisine can be used to measure the binding properties of surface nicotinic receptors, while nicotine can not. We believe that the different behavior of nicotine and cytisine in binding to intact cells reflects the relative ease with which nicotine crosses the hydrophobic cell membrane. Octanol–water partition coefficients calculated by the Advanced Chemistry Development Software Solaris V4.67 (incorporated in the Sci-Finder Scholar web site) provided

Acknowledgements

This research was supported by NIH NS 22356. We thank Gustav Akk, John Bracamontes and Ken Paradiso for discussions and comments on the manuscript. J.H.S. is the Russell and Mary Shelden Professor of Anesthesiology.

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