Identification of tryptophan 55 as the primary site of [3H]nicotine photoincorporation in the gamma-subunit of the Torpedo nicotinic acetylcholine receptor.
[3H]nicotine has been used as a photoaffinity agonist to identify amino acids within the Torpedo nicotinic acetylcholine receptor (nAChR) gamma-subunit that contributes to the structure of the agonist binding site. UV irradiation (254 nm) of nAChR-rich membranes equilibrated with [3H]nicotine results in covalent incorporation into alpha- and gamma-subunits that is inhibitable by agonists and competitive antagonists, but not by non-competitive antagonists (Middleton, R.E. and Cohen, J.B. (1991) Biochemistry 30, 6887-6897). To identify sites of specific incorporation, SDS-PAGE and reversed-phase HPLC were used to isolate proteolytic fragments of [3H]nicotine-labeled gamma-subunit. Amino-terminal sequence analysis identified gammaTrp-55 as the major site of [3H]nicotine photoincorporation in gamma-subunit. Thus gammaTrp-55 is the first amino acid within a non-alpha-subunit to be identified by affinity labeling in direct contact with a bound agonist.
Chiara
D C
DC
Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
Middleton
R E
RE
Cohen
J B
JB
D000818:Animals; D001665:Binding Sites; D009538:Nicotine / Q000378:metabolism*; D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium; D014364:Tryptophan / Q000737:chemistry*
D019920:Photoaffinity Labels; D011978:Receptors, Nicotinic; D014316:Tritium; D009538:Nicotine; D014364:Tryptophan