STRUCTURE OF THE NICOTINIC RECEPTOR ACETYLCHOLINE-BINDING SITE - IDENTIFICATION OF ACIDIC RESIDUES IN THE DELTA-SUBUNIT WITHIN 0.9-NM OF THE ALPHA-SUBUNIT-BINDING SITE DISULFIDE

In the nicotinic receptor, the quaternary ammonium group of acetylcholine (ACh) binds to a negative subsite at most 1 nm from a readily reducible disulfide formed between alpha-subunit residues Cys(192) and Cys(193). The cross-linker S-(2-[H-3]glycylamidoethyl)dithio-2-pyridine formed a disulfide bond with reduced alpha Cys(192)/Cys(193) and an amide bond with an acidic residue in the delta subunit (Czajkowski, C., and Karlin, A (1991) J. Biol. Chem. 266, 22603-22612). The fully extended cross-linking moiety -NRCH(2)CONHCH(2)CH(2)S- is 0.9 nm long. After the disulfide bond linking -NRCH(2)CONHCH(2)CH(2)S- to the alpha subunit was reduced, -NHCH2CONHCH2CH2SH remained linked to the delta subunit by an amide bond. The delta subunit was cleaved at Met residues, and the radioactive fragments were isolated and sequenced by automated Edman degradation. Additionally, the isolated radioactive fragments were further cleaved at Trp residues and sequenced. Peaks of release of radioactivity were obtained in the sequencing cycles corresponding to delta Asp(165), delta Asp(180) and delta Glu(182). The mutation of delta Asp(180) to Asn decreased the affinity of the receptor for ACh 100-fold, whereas the mutation of either delta Asp(165) delta Glu(182), Or 8 Other acidic residues in the same region of delta decreased the affinity by 3-fold or less (Czajkowski, C., Kaufmann, C., and Karlin, A. (1993) Proc. Natl. Acad. Sci. U. S. A 90, 6285-6289). Because delta Asp(180) both contributes to ACh binding and is suitably close to the binding site disulfide, it is likely to be part of the ACh-binding site formed in the interface between the alpha and the delta subunits.