Steroid inhibition of rat neuronal nicotinic alpha4beta2 receptors expressed in HEK 293 cells.

Steroids, in addition to regulating gene expression, directly affect a variety of ion channels. We examined the action of steroids on human embryonic kidney 293 cells stably transfected to express rat alpha4beta2 neuronal nicotinic receptors. Each steroid that was tested inhibited acetylcholine responses from these receptors, with slow kinetics requiring seconds for block to develop and recover. The action of one steroid [3alpha,5alpha, 17beta-3-hydroxyandrostane-17-carbonitrile (ACN)] was studied in detail. Block showed enantioselectivity, with an IC(50) value of 1.5 microM for ACN and 4.5 microM for the enantiomer. Inhibition curves had Hill slopes larger than 1, indicating more than one binding site per receptor. Block did not require intracellular compounds containing high-energy phosphate bonds and was not affected by analogs of GTP, suggesting that the mechanism does not require the activation of second messengers. Block did not appear to be strongly selective between open and closed channel states or to involve changes in desensitization. A comparison of different steroids showed that a beta-orientation of groups at the 17 position produced more block than alpha-orientated diastereomers. The stereochemistry at the 3 and 5 positions was less influential for block of alpha4beta2 nicotinic receptors, despite its importance for potentiation of gamma-aminobutyric acid(A) receptors. The ability of steroids to block neuronal nicotinic receptors correlated with their ability to produce anesthesia in Xenopus tadpoles, but the concentrations required for inhibition are generally greater. Similarly, the concentrations of endogenous neurosteroids required to inhibit receptors are larger than estimates of brain concentrations.

Ligand binding and activation of rat nicotinic alpha4beta2 receptors stably expressed in HEK293 cells.

HEK293 cells were stably transfected with rat neuronal nicotinic alpha4 and beta2 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-beta-erythroidine blocked physiological responses to acetylcholine and nicotine (IC50, 2.5 nM), and reduced cytisine binding in a competitive manner (Ki 20 nM). Physostigmine enhanced the response to low concentrations of acetylcholine or nicotine. The anesthetic steroid (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile blocked responses to acetylcholine (IC50, 1.3 microM), but had no effect on cytisine binding at a concentration of 30 microM. The binding properties of the receptors are those expected for rat neuronal nicotinic receptors composed of alpha4 and beta2 subunits. The pharmacological properties indicate that the responsiveness of the receptors may be allosterically enhanced or inhibited.