Identification of threonine 422 in transmembrane domain alpha M4 of the nicotinic acetylcholine receptor as a possible site of interaction with hydrocortisone.

The modulatory effects exerted by the glucocorticoid hydrocortisone (HC) on the nicotinic acetylcholine receptor (AChR) were studied in mutants of the alpha subunit M4 transmembrane region. Based on the photoaffinity labeling of alpha M4 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 microM), in single-channel recordings at different acetylcholine (ACh) concentrations. An alanine-substituted quadruple mutant of four putative lipid-exposed residues in alpha M4 (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 alpha M4, has direct bearing on the changes in HC sensitivity and propose its involvement in the steroid-AChR interaction site.