THE HISTRIONICOTOXIN-SENSITIVE ETHIDIUM BINDING-SITE IS LOCATED OUTSIDE OF THE TRANSMEMBRANE DOMAIN OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR- A FLUORESCENCE STUDY

A novel, relatively photostable, long-wavelength fluorescent membrane probe, N-(Texas Red sulfonyl)-5(and 6)-dodecanoylamine (C-12-Texas Red ), was synthesized and used as an electronic energy acceptor for Forst er fluorescence resonance energy transfer (FRET) between ethidium boun d to a histrionicotoxin-sensitive binding site on the Torpedo nicotini c acetylcholine receptor (AChR) and the lipid membrane surface. FRET f rom membrane-partitioned 5-(N-dodecanoylamino)fluorescein (C-12-fluore scein) to the membrane-partitioned C-12-Texas Red was also determined with a parallel set of cuvettes to (1) compare FRET results with a don or in a known position in the membrane and (2) assess the surface dens ity of the membrane-partitioned C-12-Texas Red. Stern-Volmer analysis of the FRET results showed that C-12-Texas Red quenched membrane-parti tioned C-12-fluorescein fluorescence 2.9 times more effectively than i t quenched the receptor-bound ethidium fluorescence even though the Fo rster critical distances for the two donor-acceptor pairs were very si milar (49.9 and 54.3 Angstrom respectively). Analysis of the ethidium to C-12-Texas Red FRET as a function of acceptor surface density with the assumptions that the donor is attached along the major axis of sym metry of a cylindrical protein embedded perpendicularly into the membr ane (On-Axis FRET model) suggested that the distance of closest approa ch between the receptor-bound ethidium and the membrane surface was si milar to 52 Angstrom. Because the minimum distance between the surface of the lipid-membrane domain and the major symmetry axis of the AChR is similar to 28 Angstrom the FRET results strongly suggest that the e thidium binding site is not located near the entrance of the luminal t ransmembrane domain is generally assumed. These results for the first time provide direct evidence for an histrionicotoxin-sensitive NCI bin ding site that is located outside the transmembrane domain of the dese nsitized nicotinic acetylcholine receptor.