Association of Spin-Labeled Local Anesthetics at the Hydrophobic Surface of Acetylcholine Receptor in Native Membranes from Torpedo marmorata

L. I. Horváth, H. R. Arias, H. O. Hankovszky, K. Hideg, F. J. Barrantes, D. Marsh

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43 Scopus citations

Abstract

The interactions between a series of spin-labeled local anesthetic analogues and the nicotinic acetylcholine receptor (AChR) have been investigated by means of electron spin resonance (ESR) and fluorescence spectroscopy. The paramagnetic local anesthetic analogues quenched the intrinsic tryptophan fluorescence of AChR-rich membranes in an agonist-dependent manner, demonstrating a direct interaction with the AChR. The quenching efficiency was greater for the benzocaine than for the thioprocaine analogue. The protein was found to restrict directly the molecular motion of the spin-labeled analogues, as seen by the appearance of a highly anisotropic component in the ESR spectrum. The relative affinity of the population of local anesthetic probes which interacts directly with the integral protein of the AChR-rich membranes was calculated on the basis of relative association constants, Kr, determined by ESR. By comparison with the relative association constant for spin-labeled phospholipid, Kro, it was possible to differentiate between local anesthetic analogues interacting with high (Kr/Kro > 2), intermediate (Kr/Kro = 1.6-1.9), and low (Kr/Kro ≤ 1.3) specificity and to calculate the fraction of protein-associated probe in each case. Differences were observed in the presence of agonist (0.1 mM carbamylcholine) with some, but not all, of the spin-labeled derivatives. The role of the protonatable diethylammonium group in the specificity of the interaction of the procaine and thioprocaine analogues was investigated. Only in the uncharged form, or in the charged form at high ionic strength, was there a preferential association of these two local anesthetic analogues. The specificity of the benzocaine derivative, which lacks the basic side chain, was unaffected by changes in pH or ionic strength.

Original languageEnglish
Pages (from-to)8707-8713
Number of pages7
JournalBiochemistry
Volume29
Issue number37
DOIs
StatePublished - 1 Sep 1990
Externally publishedYes

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