Identification of threonine 422 in transmembrane domain αM4 of the nicotinic acetylcholine receptor as a possible site of interaction with hydrocortisone

Abstract

The modulatory effects exerted by the glucocorticoid hydrocortisone (HC) on the nicotinic acetylcholine receptor (AChR) were studied in mutants of the α subunit M4 transmembrane region. Based on the photoaffinity labeling of αM4 412 with the steroid promegestone this position was mutated to different residues to explore the properties of side-chain volume, hydrophobicity, and charge on AChR-steroid interactions. All mutants showed channel kinetics indistinguishable from those of the wild-type AChR, both in the absence and presence of HC (200 and 400 μM), in single-channel recordings at different acetylcholine (ACh) concentrations. An alanine-substituted quadruple mutant of four putative lipid-exposed residues in αM4 (L411, M415, C418 and T422) exhibited less inhibition by HC than that observed in wild-type AChR. When we dissected the quadruple mutant into four individual alanine-substituted receptors, we found that the T422 mutant AChR behaved like the quadruple mutant, whereas the other three were indistinguishable from the wild-type. We conclude that T422, a residue close to the extracellular-facing membrane hemilayer in αM4, has direct bearing on the changes in HC sensitivity and propose its involvement in the steroid-AChR interaction site.