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 (C12-Texas Red), was synthesized and used as an electronic energy acceptor for Förster fluorescence resonance energy transfer (FRET) between ethidium bound to a histrionicotoxin-sensitive binding site on the Torpedo nicotinic acetylcholine receptor (AChR) and the lipid membrane surface. FRET from membrane-partitioned 5-(N-dodecanoylamino)fluorescein (C12-fluorescein) to the membrane-partitioned C12-Texas Red was also determined with a parallel set of cuvettes to (1) compare FRET results with a donor in a known position in the membrane and (2) assess the surface density of the membrane-partitioned C12-Texas Red. Stern-Volmer analysis of the FRET results showed that C12-Texas Red quenched membrane-partitioned C12-fluorescein fluorescence 2.9 times more effectively than it quenched the receptor-bound ethidium fluorescence even though the Förster critical distances for the two donor-acceptor pairs were very similar (49.9 and 54.3 A, respectively). Analysis of the ethidium to C12-Texas Red FRET as a function of acceptor surface density with the assumptions that the donor is attached along the major axis of symmetry of a cylindrical protein embedded perpendicularly into the membrane (On-Axis FRET model) suggested that the distance of closest approach between the receptor-bound ethidium and the membrane surface was approximately 52 A. Because the minimum distance between the surface of the lipid-membrane domain and the major symmetry axis of the AChR is approximately 28 A, the FRET results strongly suggest that the ethidium binding site is not located near the entrance of the luminal transmembrane domain is generally assumed.(ABSTRACT TRUNCATED AT 250 WORDS)
Johnson
D A
DA
Division of Biomedical Sciences, University of California, Riverside 92521-0121.
Nuss
J M
JM
D000664:Amphibian Venoms / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D004735:Energy Transfer; D004996:Ethidium / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D011487:Protein Conformation; D011789:Quantum Theory; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014101:Torpedo; D014966:Xanthenes / Q000138:chemical synthesis
D000664:Amphibian Venoms; D005456:Fluorescent Dyes; C088722:N-(Texas Red sulfonyl)-5-dodecanoylamine; C088723:N-(Texas Red sulfonyl)-6-dodecanoylamine; D011978:Receptors, Nicotinic; D014966:Xanthenes; D004996:Ethidium; C007948:histrionicotoxin