Interaction of Merocyanine-540 with Nicotinic Acetylcholine- Receptor Membranes from Discopyge-Tschudii Electric Organ

ARIAS, HR; ALONSOROMANOWSKI, S; DISALVO, EA; BARRANTES, FJ; EDGARDO ANIBAL DISALVO

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 1994 vol. 1190 p. 393 - 401

0005-2736

Interactions between merocyanine 540 (MC540) and nicotinic acetylcholine receptor (AChR) bave 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 ( <img height="13" border="0" style="vertical-align:bottom" width="19" alt="View the MathML source" title="View the MathML source" src="http://ars.els-cdn.com/content/image/1-s2.0-000527369490099X-si1.gif">app 30 ìM) 10-fold higher than that in liposomes ( <img height="13" border="0" style="vertical-align:bottom" width="19" alt="View the MathML source" title="View the MathML source" src="http://ars.els-cdn.com/content/image/1-s2.0-000527369490099X-si2.gif">app 290 ìM). 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 ( <img height="14" border="0" style="vertical-align:bottom" width="35" alt="View the MathML source" title="View the MathML source" src="http://ars.els-cdn.com/content/image/1-s2.0-000527369490099X-si3.gif">) 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.