RT Journal Article
SR Electronic
T1 Ligand Binding and Activation of Rat Nicotinic α4β2 Receptors Stably Expressed in HEK293 Cells
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 58
OP 66
DO 10.1124/mol.55.1.58
VO 55
IS 1
A1 Sabey, Kimberley
A1 Paradiso, Ken
A1 Zhang, Jessie
A1 Steinbach, Joe Henry
YR 1999
UL http://molpharm.aspetjournals.org/content/55/1/58.abstract
AB HEK293 cells were stably transfected with rat neuronal nicotinic α4 and β2 subunits. Binding of tritiated cytisine and nicotine to cell homogenates revealed the presence of a single class of high-affinity sites (dissociation constants 0.1 nM and 0.4 nM, respectively). Activation of nicotinic receptors was studied using whole-cell patch clamp methods, and acetylcholine, nicotine, dimethylphenylpiperazinium, and cytisine all produced a conductance increase. Responses desensitized to prolonged applications, at both positive and negative membrane potentials. The conductance was strongly rectifying, and outward currents were essentially absent. Responses were maximal at about 2 mM external calcium ion concentration and were reduced by about one-half at either nominally 0 or 10 mM external calcium. Di-hydro-β-erythroidine blocked physiological responses to acetylcholine and nicotine (IC50, 2.5 nM), and reduced cytisine binding in a competitive manner (K i20 nM). Physostigmine enhanced the response to low concentrations of acetylcholine or nicotine. The anesthetic steroid (+)-3α-hydroxy-5α-androstane-17β-carbonitrile blocked responses to acetylcholine (IC50, 1.3 μM), but had no effect on cytisine binding at a concentration of 30 μM. The binding properties of the receptors are those expected for rat neuronal nicotinic receptors composed of α4 and β2 subunits. The pharmacological properties indicate that the responsiveness of the receptors may be allosterically enhanced or inhibited.