Steroid inhibition of rat neuronal nicotinic alpha 4 beta 2 receptors expressed in HEK 293 cells

Steroids, in addition to regulating gene expression, directly affect a vari ety of ion channels. We examined the action of steroids on human embryonic kidney 293 cells stably transfected to express rat alpha 4 beta 2 neuronal nicotinic receptors. Each steroid that was tested inhibited acetylcholine r esponses from these receptors, with slow kinetics requiring seconds for blo ck to develop and recover. The action of one steroid [3 alpha,5 alpha,17 be ta-3-hydroxyandrostane-17-carbonitrile (ACN)] was studied in detail. Block showed enantioselectivity, with an IC50 value of 1.5 mu M for ACN and 4.5 m u M for the enantiomer. Inhibition curves had Hill slopes larger than 1, in dicating more than one binding site per receptor. Block did not require int racellular compounds containing high-energy phosphate bonds and was not aff ected by analogs of GTP, suggesting that the mechanism does not require the activation of second messengers. Block did not appear to be strongly selec tive between open and closed channel states or to involve changes in desens itization. A comparison of different steroids showed that a beta-orientatio n of groups at the 17 position produced more block than alpha-orientated di astereomers. The stereochemistry at the 3 and 5 positions was less influent ial for block of alpha 4 beta 2 nicotinic receptors, despite its importance for potentiation of gamma-aminobutyric acid(A) receptors. The ability of s teroids 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 end ogenous neurosteroids required to inhibit receptors are larger than estimat es of brain concentrations.