INTERACTION OF MEROCYANINE-540 WITH NICOTINIC ACETYLCHOLINE-RECEPTOR MEMBRANES FROM DISCOPYGE-TSCHUDII ELECTRIC ORGAN

Interactions between merocyanine 540 (MC540) and nicotinic acetylcholi ne receptor (AChR) have been studied by visible absorption spectroscop y using native receptor-rich membranes from Discopyge tschudii electri c tissue and liposomes obtained by aqueous dispersion of endogenous li pids extracted from the same tissue. The fact that merocyanine partiti ons into the membrane when this is in the liquid-crystalline state, ex hibiting a characteristic peak at 567 nm, was exploited to obtain quan titative information about the physical state of the AChR-rich membran e. Spectra of MC540 revealed that this molecule was preferentially inc orporated into AChR-rich membranes, with an affinity (K(d)app 30 muM) 10-fold higher than that in liposomes (K(d)app 290 muM). Changes were observed in the equilibrium dissociation constant of MC540 at differen t temperatures: the two-fold higher affinity at 8-degrees-C than at 23 -degrees-C can be rationalized in terms of a higher value of the overa ll dimerization constant (K(dim)) at the lower temperature. The local anaesthetic benzocaine competed for MC540 binding sites with higher po tency in AChR-rich native membranes than in liposomes made with endoge nous lipids. This competition was found to be AChR concentration-depen dent, whereas in liposomes the displacement was constant at different lipid/MC540 molar ratios. Titration experiments yielded an apparent di ssociation constant for benzocaine of 0.6 mM and 0.7 mM for liposomes and AChR-rich membranes, respectively. The possible location of the be nzocaine binding site is deduced from the competition experiments to b e at the lipid annulus surrounding the nicotinic AChR protein.