Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ

H. R. Arias, S. Alonso-Romanowski, E. A. Disalvo, F. J. Barrantes

Research output: Contribution to journalArticle

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Abstract

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 (Kdapp 30 μM) 10-fold higher than that in liposomes (Kdapp 290 μM). 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 (Kdim) 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.

Original languageEnglish
Pages (from-to)393-401
Number of pages9
JournalBBA - Biomembranes
Volume1190
Issue number2
DOIs
StatePublished - 23 Mar 1994
Externally publishedYes

Fingerprint

Electric Organ
Nicotinic Receptors
Cholinergic Receptors
Liposomes
Benzocaine
Membranes
Lipids
Binding Sites
Tissue
Temperature
Dimerization
Local Anesthetics
Titration
Absorption spectroscopy
merocyanine dye
Spectrum Analysis
Experiments
Crystalline materials
Molecules
Liquids

Keywords

  • Cholinergic receptor
  • Cyanine dye
  • Lipid annulus
  • Local anaesthetic
  • Torpedinidae

Cite this

Arias, H. R. ; Alonso-Romanowski, S. ; Disalvo, E. A. ; Barrantes, F. J. / Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ. In: BBA - Biomembranes. 1994 ; Vol. 1190, No. 2. pp. 393-401.
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Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ. / Arias, H. R.; Alonso-Romanowski, S.; Disalvo, E. A.; Barrantes, F. J.

In: BBA - Biomembranes, Vol. 1190, No. 2, 23.03.1994, p. 393-401.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ

AU - Arias, H. R.

AU - Alonso-Romanowski, S.

AU - Disalvo, E. A.

AU - Barrantes, F. J.

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N2 - 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 (Kdapp 30 μM) 10-fold higher than that in liposomes (Kdapp 290 μM). 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 (Kdim) 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.

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