ABSTRACT
A fast signaling mode of natural and synthetic steroids is exerted on some ion channels and cell-surface receptors. This activity contrasts with their classic mode of action, via intracellular receptors. Early studies from our laboratory demonstrated that spin-labeled androstanol and cholestane interact with the nicotinic acetylcholine receptor (AChR) and that lipid mobility at the lipid belt surrounding the AChR is reduced relative to that of the bulk membrane lipid. The occurrence of discrete and independent sites for phospholipids and sterols, both accessible to fatty acids, was subsequently disclosed in the native membrane. Synthetic and natural glucocorticoids were found to act as noncompetitive inhibitors of AChR function. The influence of different substituent groups in the cyclepentane perhydrophenanthrene ring on the channel-shortening potency of various steroids has also been assayed in muscle-type AChR, and we found a certain selectivity of this effect. Some organochlorine pesticides are xenoestrogens, that is, environmental agents capable of disrupting endocrine system signaling. We determined their effects on the AChR membrane using novel fluorescence techniques.
Subject(s)
Receptors, Nicotinic/physiology , Steroids/physiology , Animals , Cholesterol/metabolism , Estrogens/pharmacology , Genome , Humans , Lipid Metabolism , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/metabolism , Steroids/pharmacology , Xenobiotics/pharmacologyABSTRACT
The so-called generalized polarization (GP) of the fluorescent probe Laurdan and the steady-state fluorescence anisotropy of the probe diphenylhexatriene (DPH) and its phenylpropionic derivative (PA-DPH) were used to study the effects of several organochlorine insecticides of the chlorophenylethane, chlorinated cyclohexane and chlorinated cyclodiene families on the Torpedo nicotinic acetylcholine receptor (AChR)-rich native membrane. All insecticides, with the exception of Lindane, augmented Laurdan GP both in the native membrane and in model lipid systems. Most organochlorine compounds produced a concentration-dependent decrease of DPH and PA-DPH anisotropy in the AChR-rich membrane. These compounds exhibited a dual behavior vis-à-vis the native AChR-rich membrane, exerting disordering effects at the bilayer core while ordering and/or excluding water molecules from the lipid-protein interface region, as sensed by DPH anisotropy and Laurdan GP, respectively. Furthermore, all insecticides decreased the efficiency of fluorescence resonance energy transfer between the intrinsic protein and Laurdan, albeit to different extents. On the basis of all these observations, the existence of potential target sites for insecticides in the protein-lipid interface region is postulated.
Subject(s)
Insecticides/toxicity , Receptors, Nicotinic/drug effects , 2-Naphthylamine/analogs & derivatives , Animals , Anisotropy , Cholesterol/chemistry , Diphenylhexatriene/analogs & derivatives , Electric Organ/metabolism , Energy Transfer , Fluorescent Dyes , In Vitro Techniques , Laurates , Lipid Bilayers/chemistry , Membrane Lipids/metabolism , Membranes/chemistry , Membranes/metabolism , Receptors, Nicotinic/metabolism , Spectrometry, Fluorescence , TorpedoABSTRACT
Since the discovery of the fraction of immobilized lipid in contact with the nicotinic acetylcholine receptor (AChR), the lipid-belt region around this protein has become the focus of a variety of biophysical studies aimed at defining its properties. Here we summarize recent spectroscopic studies from our laboratory using Laurdan fluorescence to characterize distinct sites for lipids and to describe their effect on the AChR microenvironment.
