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.
Paradiso
K
K
Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Sabey
K
K
Evers
A S
AS
Zorumski
C F
CF
Covey
D F
DF
Steinbach
J H
JH
D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004962:Estranes / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D009570:Nitriles / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism*; D012018:Reflex / Q000187:drug effects; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection; D014982:Xenopus laevis
D004962:Estranes; D018733:Nicotinic Antagonists; D009570:Nitriles; D011963:Receptors, GABA-A; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; C084683:3-hydroxy-5-estrane-17-carbonitrile