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Fluorescence energy transfer between cobra alpha-toxin molecules bound to the acetylcholine receptor.

1984; Elsevier BV; Volume: 259; Issue: 9 Linguagem: Inglês

10.1016/s0021-9258(18)91073-2

1083-351X

David A. Johnson, Judith G. Voet, Palmer Taylor,

Nicotinic Acetylcholine Receptors Study

An approach was developed with steady state fluorescence energy transfer measurements to examine the spatial relationship between the two a-toxins bound to the acetylcholine receptor.By taking advantage of the slow dissociation rates of a-toxins (Naja naja siamensis 3) from the receptor and of the equal probability with which a-toxins bind to the two a-toxin-binding sites, we derived an equation which allows prediction of a "true" efficiency of transfer based on the relationship between fractional site occupancy and the observed transfer efficiency ascertained from donor quenching.Using this approach, we examined the efficiency of energy transfer between two fluorescently labeled a-toxins, W-fluorescein isothiocyanate lysine 23 a-toxin and monolabeled tetramethylrhodamine isothiocyanate a-toxin bound to the receptor from the Torpedo californica electric organ.Significantly greater (32 versus 14%) energy transfer was observed with the membrane-associated than with the solubilized receptor, suggesting that transfer between fluorophores on separate receptor molecules is greater than that occurring intramolecularly between the two sites on the receptor.The magnitude of the distances calculated from the intrareceptor energy transfer efficiency combined with the considerable inter-receptor energy transfer indicate that the fluorophores would reside on the outer perimeter of the receptor molecule rather than near the central axis perpendicular to the plane of the membrane.The AChR' is a cation channel whose activation is elicited by acetylcholine.Owing to its relative abundance, the most thoroughly characterized AChR comes from the Torpedo californica electric organ (1-3).In receptor-rich membrane fragments from the Torpedo, the AChR exists as tightly packed, disc-shaped particles of 90 f 10 A in diameter, with a central 15-20-A cavity (4-6).The pentameric receptor is comprised of four different subunits, designated as az, p, y, and 6 (7,8).