ELSEVIER Biochimica et Biophysica Acta 1190 (1994) 393-401 Btl Biochi~mic~a et Biophysica Acta Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ H.R. Arias a, S. Alonso-Romanowski b, E.A. Disalvo b,,, F.J. Barrantes a a Instituto de Investigaciones Bioqu[micas de Bahia Blanca, CONICET/Universidad Nac. del Sur, 8000 Bahia Blanca, Argentina b Area de Biomembranas, Instituto de Investigaciones Fisicoqu{micas Tedricas y Aplicadas (INIFTA), Universidad Nac. de La Plata, 1900 La Plata, Argentina (Received 5 July 1993) Abstract Interactions between merocyanine 540 (MC540) and nicotinic acetylcholine receptor (AChR) have been studied by visible absorption spectroscopy using native receptor-rich membranes from Discopyge tschudii electric tissue and liposomes obtained by aqueous dispersion of endogenous lipids extracted from the same tissue. The fact that merocyanine partitions into the membrane when this is in the liquid-crystalline state, exhibiting a characteristic peak at 567 nm, was exploited to obtain quantitative information about the physical state of the AChR-rich membrane. Spectra of MC540 revealed that this molecule was preferentially incorporated into AChR-rich membranes, with an affinity (Kdapp 30 /~M) 10-fold higher than that in liposomes (Kdapp 290 /zM). Changes were observed in the equilibrium dissociation constant of MC540 at different temperatures: the two-fold higher affinity at 8°C than at 23°C can be rationalized in terms of a higher value of the overall dimerization constant (Kdi m) at the lower temperature. The local anaesthetic benzocaine competed for MC540 binding sites with higher potency in AChR-rich native membranes than in liposomes made with endogenous lipids. This competition was found to be AChR concentration-dependent, whereas in liposomes the displacement was constant at different lipid/MC540 molar ratios. Titration experiments yielded an apparent dissociation constant for benzocaine of 0.6 mM and 0.7 mM for liposomes and AChR-rich membranes, respectively. The possible location of the benzocaine binding site is deduced from the competition experiments to be at the lipid annulus surrounding the nicotinic AChR protein. Key words: Cholinergic receptor; Local anaesthetic; Lipid annulus; Torpedinidae; Cyanine dye 1. Introduction Merocyanine 540 (MC540) is a cyanine dye that changes its optical properties, such as absorption and emission, upon binding to lipid bilayer membranes [1-4], a variety of biomembranes from different origin [1,3,5] and surfactant micelles [6]. Such spectroscopic changes have been ascribed principally to the physical state of the lipids in the membrane and to the electri- cal potential at the interface or across the membrane [1,7]. For this reason, MC540 has been widely used to measure cell membrane potentials in different ex- citable tissues [8-11]. * Corresponding author. Present address: Qulmica General e Inor- ganica Facultad de Farmacia y Bioqu~mica, Universidad de Buenos Aires, Junin 956, 2 ° Pisolll3, Buenos Aires, Argentina. Fax: +54 1 9625341. 0005-2736/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0005-2736(93)E0350-J The mechanism by which MC540 responds to changes in the membrane has been the subject of several investigations, and plausible models have emerged [2,12-15]. In essence, these hypotheses postu- late that MC540 is present in the membrane in a monomer-dimer equilibrium, with differing optical properties for each species. When the membrane is in the fluid state, MC540 exhibits a peak at 570 nm; in the presence of membranes in the gel state, the spectrum is comparable to that of MC540 in water without lipids, i.e., it shows peaks at 500 and 530 nm. Hence, the increase in the 570 nm peak with respect to that at 500 nm can be related to the gel-fluid transition of the membrane. The absorption at 570 nm is ascribed to the presence of monomer in the membrane in equilibrium to monomers and dimers in the adjacent aqueous solu- tion. In addition, MC540 exhibits a mechanism of intramembranous dimerization coupled to a rate-limit- ing reorientation of the dye in the membrane [2].