Endogenous neurosteroids and their synthetic analogues-neuroactive steroids-have been found to bind to muscarinic acetylcholine receptors and allosterically modulate acetylcholine binding and function. Using radioligand binding experiments we investigated their binding mode. We show that neuroactive steroids bind to two binding sites on muscarinic receptors. Their affinity for the high-affinity binding site is about 100 nM. Their affinity for the low-affinity binding site is about 10 µM. The high-affinity binding occurs at the same site as binding of steroid-based WIN-compounds that is different from the common allosteric binding site for alcuronium or gallamine that is located between the second and third extracellular loop of the receptor. This binding site is also different from the allosteric binding site for the structurally related aminosteroid-based myorelaxants pancuronium and rapacuronium. Membrane cholesterol competes with neurosteroids/neuroactive steroids binding to both high- and low-affinity binding site, indicating that both sites are oriented towards the cell membrane..

Department of Experimental and Clinical Pharmacology University of Minnesota College of Pharmacy Minneapolis MN 55455 USA

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Prague Czech Republic

Institute of Physiology Czech Academy of Sciences Prague Czech Republic

References provided by Crossref.org

Muscarinic Receptors in Cardioprotection and Vascular Tone Regulation

Modulation of Muscarinic Signalling in the Central Nervous System by Steroid Hormones and Neurosteroids

Allosteric Modulation of Muscarinic Receptors by Cholesterol, Neurosteroids and Neuroactive Steroids