The Role of Lipids in the Function of the Acetylcholine Receptor

Abstract

The composition of membranes containing acetylcholine receptor was altered in order to examine the possible role of lipids in receptor function. Polyethylene glycol was used to fuse AcChR-rich membranes with an excess of lipid vesicles of defined composition. By this procedure, the cholesterol composition was reduced to as little as 20% of that found in native membranes. Using a TI⁺ flux assay it was possible to measure receptor function in such altered membrane environments. The apparent K_d for carbamylcholine was found to decrease as the cholesterol content was reduced. This result was confirmed by measuring the agonist-induced fluorescence change of the covalently attached probe, 4-[N-(iodoacetoxy)-ethyl-N-methyl] amino-7-nitrobenz-2-oxa-1,3-diazole. When the phospholipid composition was manipulated by membrane fusion, ion flux was found to be optimal when the lipid composition resembled that of native receptor membranes. These results indicate that membrane lipids potentially play a role in the regulation of acetylcholine receptor function.     

Halothane-induced changes in acetylcholine receptor channel kinetics are attenuated by cholesterol

The single-channel recording technique was used to investigate the role of membrane lipids in the action of general anesthetics on ion channels. We examined the effects of halothane on acetylcholine receptor channels in Xenopus laevis myocytes in which the plasma membrane cholesterol level had been changed by pretreatment with cholesterol-rich or cholesterol-free liposomes. We found that the alteration in acetylcholine receptor channel kinetics, elicited in the presence of clinically-relevant concentrations of halothane, is attenuated when membrane cholesterol is increased and enhanced when membrane cholesterol concentration is decreased. These findings support the idea that general anesthetics interact with synaptic receptor channels indirectly through the lipid domains in which these synaptic proteins are embedded.     

Two pools of cholesterol in acetylcholine receptor-rich membranes from Torpedo

The acetylcholine receptor (AChR)-containing electroplax membranes from Torpedo californica have a relatively high cholesterol content. Reconstitution studies suggest that this cholesterol may be important in preserving or modulating the function of the acetylcholine receptor-channel complex. We have manipulated cholesterol levels in intact Torpedo AChR-rich membrane fragments using small, unilamellar phosphatidylcholine liposomes. Conditions have been established that allow further subfractionation of sucrose gradient purified Torpedo electroplax membranes into AChR-rich and ATPase-rich populations and that, at the same time, achieve cholesterol depletion without phospholipid back exchange or fusion. The incubation of membranes with excess liposomes could only achieve about a 50% reduction in the molar ratio of cholesterol to phospholipid. In no case was the number of cholesterol molecules per AChR oligomer reduced below 36. The remaining cholesterol could not be depleted either by longer incubations or by multiple, sequential depletions. Cholesterol depletion was accompanied by a significant increase in bulk membrane fluidity as measured by electron spin resonance spectroscopy, but the equilibrium binding parameters of acetylcholine to its receptor were unaltered. This suggests strongly that there exist two pools of cholesterol in the AChR-rich Torpedo electroplax membrane: an easily depleted fraction that influences bulk fluidity, and a tightly-bound fraction perhaps surrounding the AChR oligomer.     

Antibody-Induced Acetylcholine Receptor Clusters Inhabit Liquid-Ordered and Liquid-Disordered Domains

The distribution of nicotinic acetylcholine receptor (AChR) clusters at the cell membrane was studied in CHO-K1/A5 cells using fluorescence microscopy. Di-4-ANEPPDHQ, a fluorescent probe that differentiates between liquid-ordered (Lo) and liquid-disordered (Ld) phases in model membranes, was used in combination with monoclonal anti-AChR antibody labeling of live cells, which induces AChR clustering. The so-called generalized polarization (GP) of di-4-ANEPPDHQ was measured in regions of the cell-surface membrane associated with or devoid of antibody-induced AChR clusters, respectively. AChR clusters were almost equally distributed between Lo and Ld domains, independently of receptor surface levels and agonist (carbamoylcholine and nicotine) or antagonist (α-bungarotoxin) binding. Cholesterol depletion diminished the cell membrane mean di-4-ANEPPDHQ GP and the number of AChR clusters associated with Ld membrane domains increased concomitantly. Depolymerization of the filamentous actin cytoskeleton by Latrunculin A had the opposite effect, with more AChR clusters associated with Lo domains. AChR internalized via small vesicles having lower GP and lower cholesterol content than the surface membrane. Upon cholesterol depletion, only 12% of the AChR-containing vesicles costained with the fluorescent cholesterol analog fPEG-cholesterol, i.e., AChR endocytosis was essentially dissociated from that of cholesterol. In conclusion, the distribution of AChR submicron-sized clusters at the cell membrane appears to be regulated by cholesterol content and cytoskeleton integrity.     

Antibody-Induced Acetylcholine Receptor Clusters Inhabit Liquid-Ordered and Liquid-Disordered Domains

The distribution of nicotinic acetylcholine receptor (AChR) clusters at the cell membrane was studied in CHO-K1/A5 cells using fluorescence microscopy. Di-4-ANEPPDHQ, a fluorescent probe that differentiates between liquid-ordered (Lo) and liquid-disordered (Ld) phases in model membranes, was used in combination with monoclonal anti-AChR antibody labeling of live cells, which induces AChR clustering. The so-called generalized polarization (GP) of di-4-ANEPPDHQ was measured in regions of the cell-surface membrane associated with or devoid of antibody-induced AChR clusters, respectively. AChR clusters were almost equally distributed between Lo and Ld domains, independently of receptor surface levels and agonist (carbamoylcholine and nicotine) or antagonist (α-bungarotoxin) binding. Cholesterol depletion diminished the cell membrane mean di-4-ANEPPDHQ GP and the number of AChR clusters associated with Ld membrane domains increased concomitantly. Depolymerization of the filamentous actin cytoskeleton by Latrunculin A had the opposite effect, with more AChR clusters associated with Lo domains. AChR internalized via small vesicles having lower GP and lower cholesterol content than the surface membrane. Upon cholesterol depletion, only 12% of the AChR-containing vesicles costained with the fluorescent cholesterol analog fPEG-cholesterol, i.e., AChR endocytosis was essentially dissociated from that of cholesterol. In conclusion, the distribution of AChR submicron-sized clusters at the cell membrane appears to be regulated by cholesterol content and cytoskeleton integrity.     

Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces distinctive membrane lesions. We treated frog cutaneous and sartorius muscles with 0.04% filipin in a glutaraldehyde solution with or without prefixation with glutaraldehyde. Freeze- fracture of these muscles revealed numerous 19 to 38-nm protuberances and depressions (filipin-sterol complexes) in most areas of muscle, axon, and Schwann cell membranes. In the presynaptic membrane, however, these filipin-sterol complexes were absent from active zones consisting of ridges bordered with double rows of particles. In the postsynaptic membrane, filipin-sterol complexes were also virtually absent from the areas occupied by aggregates of large particles representing acetylcholine receptors. These results suggest that the membrane regions of active zones and acetylcholine receptor aggregates have a low cholesterol content.     

Exposure of acetylcholine receptor to the lipid bilayer

All four subunits of the acetylcholine receptor in membrane fragments isolated from T. californica have been labeled with a photolabile hydrophobic probe, [3H]adamantanediazirine, which selectively labels regions of integral membrane proteins in contact with the hydrocarbon core of the lipid bilayer. As all of the homologous subunits are exposed to the lipid bilayer, it is probable that they each interact with the surrounding membrane in a similar fashion.     

Lipid-protein interactions at the nicotinic acetylcholine receptor. A functional coupling between nicotinic receptors and phosphatidic acid-containing lipid bilayers.

The structural and functional properties of reconstituted nicotinic acetylcholine receptor membranes composed of phosphatidyl choline either with or without cholesterol and/or phosphatidic acid have been examined to test the hypothesis that receptor conformational equilibria are modulated by the physical properties of the surrounding lipid environment. Spectroscopic and chemical labeling data indicate that the receptor in phosphatidylcholine alone is stabilized in a desensitized-like state, whereas the presence of either cholesterol or phosphatidic acid favors a resting-like conformation. Membranes that effectively stabilize a resting-like state exhibit a relatively large proportion of non-hydrogen-bonded lipid ester carbonyls, suggesting a relatively tight packing of the lipid head groups and thus a well ordered membrane. Functional reconstituted membranes also exhibit gel-to-liquid crystal phase transition temperatures that are higher than those of nonfunctional reconstituted membranes composed of phosphatidylcholine alone. Significantly, incorporation of the receptor into phosphatidic acid-containing membranes leads to a dramatic increase in both the lateral packing densities and the gel-to-liquid crystal phase transition temperatures of the reconstituted lipid bilayers. These results suggest a functional link between the nicotinic acetylcholine receptor and the physical properties of phosphatidic acid-containing membranes that could underlie the mechanism by which this lipid preferentially enhances receptor function.     

Fluidity of the lipids next to the acetylcholine receptor protein of torpedo membrane fragments. Use of amphiphilic reversible spin-labels.

Choline esters of spin-labeled fatty acids (long-chain acylcholines) were used to probe the hydrophobic environment of the acetylcholine receptor protein in membrane fragments from Torpedo marmorata. These spin-labels competitively inhibit the binding of [3H]acetylcholine to the receptor site. Their inhibition constants (KI) were close to 200 nM. At the high membrane concentration required for electron spin resonance (ESR) experiments, the apparent inhibition constants (KIapp) differed from KI determined by using dilute membrane concentration. This is due to the amphiphilic character of long-chain acylcholine. For most spin-labels used, only difference ESR spectroscopy provided reliable spectra corresponding to receptor-bound spin-labeled acylcholines. Acetylcholine receptor agonists and antagonists displaced the acylcholine from the receptor sites, whereas choline had only a weak effect. This produced a modification in the ESR spectra of the bound acylcholines and provided evidence that the acylcholines bound to the receptor sites in a specific manner. The interpretation of the spectra of receptor-bound spin-labels favored a strong barrier to the motion of the probe when attached to the middle of the acyl chain. However, when the probe was close to the methyl terminal of a stearoylcholine molecule a much greater fluidity was found. Short-range spin-spin interactions were created between spin-labels bound to the receptor site and spin-labels in a fluid phase. This indicates that lipids next to the receptor protein are not completely immobilized in spite of the semicrystalline organization of the proteins in the postsynaptic region.     

Changes of the membrane lipid organization characterized by means of a new cholesterol-pyrene probe

We synthesized 3beta-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): K(DOPC) > K(POPC) > K(DMPC) > K(DMPC/15 mol % Chol) > K(DMPC/30 mol % Chol). This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I(1)-I(5)) and excimer (I(E)) emission bands. In model membranes, I(1)/I(3) and I(E)/I(3) ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse delta-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse delta-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.     

Transmembrane topology of acetylcholine receptor subunits probed with photoreactive phospholipids

The domains of the acetylcholine receptor subunits that contact the lipid phase were investigated by hydrophobic photolabeling of receptor-rich membrane fragments prepared from Torpedo marmorata and Torpedo californica electric organs. The radioactive arylazido phospholipids used carry a photoreactive group, either at the level of the lipid polar head group (PCI) or at the tip of the aliphatic chain (PCII), and thus probe respectively the "superficial" and "deep" regions of the lipid bilayer. The four subunits of T. marmorata and T. californica acetylcholine receptor reacted with both the PCI and PCII probes and thus are all exposed to the lipid phase. Ligands known to stabilize different conformations of the acetylcholine receptor (nicotinic agonists, snake alpha-toxin, and noncompetitive blockers) did not cause any significant change in the labeling pattern. The acetylcholine receptor associated 43 000-dalton v1 protein did not react with any of the probes. A striking difference in labeling between T. marmorata and T. californica acetylcholine receptors occurred at the level of the alpha-subunit when the superficial PCI probe was used. An approximately 5-fold higher labeling of the alpha-subunit as compared to the beta-, gamma-, and delta-subunits was observed by using receptor-rich membranes from T. marmorata but not from T. californica. The same difference persisted after purification of the labeled receptors from the two species and was restricted to an 8000-dalton C-terminal tryptic peptide. The only mutation observed in this region of the complete alpha-subunit sequence of the two species is the substitution of cysteine-424 in T. marmorata by serine-424 in T. californica.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol modulates the organization of the gammaM4 transmembrane domain of the muscle nicotinic acetylcholine receptor

A 28-mer gammaM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor gamma-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gammaM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gammaM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low F?rster type resonance energy transfer efficiency from the Trp453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gammaM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gammaM4 peptide occupies approximately 30% of the patch area.     

Time-dependent binding of paramagnetic and fluorescent hydrophobic ions to the acetylcholine receptor from Torpedo

In receptor-rich vesicles isolated from Torpedo, paramagnetic or fluorescent phosphonium ions bind to both the acetylcholine receptor (AcChR) and the receptor membrane. When added to receptor vesicles, two to three phosphoniums undergo a slow time-dependent binding to the AcChR. The presence of agonist increases the rate but not the extent of binding of the alkylphosphonium nitroxides. Approximately one phosphonium per receptor can be displaced by the addition of saturating concentrations of the high-affinity histrionicotoxin derivative isodihydrohistrionicotoxin or by the addition of phencyclidine or quinacrine mustard. In addition, preincubation of the receptor with these channel blockers prevents approximately one phosphonium from binding to the receptor. When a series of alkyltriphenylphosphonium ions was studied, it was found that the rate of phosphonium binding to the receptor decreased with increasing probe hydrophobicity. This appears to be a function of the partitioning of the probe between membrane and aqueous phases. The phosphonium ions used here promote desensitization of the receptor, as judged by the binding rate of the fluorescent agonist NBDA-C5-acylcholine or alpha-bungarotoxin. Preincubation of the receptor with isodihydrohistrionicotoxin virtually eliminates the phosphonium-mediated desensitization. The rates of the phosphonium-mediated desensitization also appear to be dependent upon the phase partitioning of the probe. These results strongly suggest that the binding sites for the phosphonium ion (and the high-affinity histrionicotoxin blocking site) are accessible only through the aqueous phase. The phosphonium binding and agonist-induced transitions observed here are not observed with a negative hydrophobic ion probe, or a negative surface amphiphile, indicating that modifications in membrane electrostatics do not contribute to the observed changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activatable pool: implications in the pathogenesis of a novel congenital myasthenic syndrome

Cholesterol modulates the plasmalemma's biophysical properties and influences the function and trafficking of membrane proteins. A fundamental phenomenon that remains obscure is how the plasmalemma's lipid composition regulates the activatable pool of membrane receptors. An outstanding model to study this phenomenon is the nicotinic acetylcholine receptor (nAChR), since the nAChR activatable pool has been estimated to be but a small fraction of the receptors present in the plasmalemma. Studies on the effect of cholesterol depletion in the function of the Torpedo californica nAChR, using the lipid-exposed nAChR mutation (alpha C418W) that produces a congenital myasthenic syndrome (CMS), demonstrated that cholesterol depletion causes a remarkable increase in the alpha C418W nAChR's macroscopic current whereas not in the wild-type (WT). A variety of approaches were used to define the mechanism responsible for the cholesterol depletion mediated-increase in the alpha C418W nAChR's macroscopic current. The present study suggests that a substantial fraction of the alpha C418W nAChRs is located in caveolin-1-positive domains, "trapped" in a non-activatable state, and that membrane cholesterol depletion results in the relocation of these receptors to the activatable pool. Co-fractionation and co-immunoprecipitation of the alpha C418W nAChR and the membrane raft protein caveolin-1 (cav1) support the notion that interactions at lipid-exposed domains regulate the partition of the receptor into membrane raft microdomains. These results have potential implications as a novel mechanism to fine-tune cholinergic transmission in the nervous system and in the pathogenesis associated to the alpha C418W nAChR.     

Phospholipase A2 hydrolysis of membrane phospholipids causes structural alteration of the nicotinic acetylcholine receptor

Thermal perturbation techniques have been used to probe structural alteration of the nicotinic acetylcholine receptor as a function of perturbations of its native membrane environment. Differential scanning calorimetry and a technique involving heat inactivation of the alpha-bungarotoxin-binding sites on the receptor protein reveal that there is a profound destabilization of the acetylcholine receptor structure when receptor-containing membranes are exposed to phospholipase A2. The characteristic calorimetric transition assigned to irreversible denaturation of the receptor protein and the heat inactivation profile of alpha-bungarotoxin-binding sites are shifted to lower temperatures by approx. 7 and 5 C degrees, respectively, upon exposure to phospholipase A2 at a phospholipase/neurotoxin binding site molar ratio of about 1:100. The effects of phospholipase A2 on receptor structure can be (i) reversed by using bovine serum albumin as a scavenger of phospholipase hydrolysis products of membrane phospholipids, and (ii) stimulated by incorporation into the membranes of free, polyunsaturated fatty acids. In particular, linolenic acid (18:3(n-3] causes detectable destabilization of the alpha-bungarotoxin binding sites on the receptor at free fatty acid/receptor molar ratios as low as 10:1. Furthermore, alteration of receptor structure by added phospholipase occurs very rapidly, which is consistent with the observation of rapid in situ phospholipase A2 hydrolysis of membrane phospholipids, particularly highly unsaturated phosphatidylethanolamine and phosphatidylserine. Based on previously published data on the inhibition of acetylcholine receptor cation-gating activity caused by the presence of either phospholipase A2 or free fatty acids (Andreasen T.J. and McNamee M.G. (1980) Biochemistry 19, 4719), we interpret our data as indicative of a correlation between structural and functional alterations of the membrane-bound acetylcholine receptor induced by phospholipase A2 hydrolysis products.     

Cholesterol depletion activates rapid internalization of submicron-sized acetylcholine receptor domains at the cell membrane

Novel effects of cholesterol (Chol) on nicotinic acetylcholine receptor (AChR) cell-surface stability, internalization and function are reported. AChRs are shown to occur in the form of submicron-sized (240-280 nm) domains that remain stable at the cell-surface membrane of CHO-K1/A5 cells over a period of hours. Acute (30 min, 37 degrees C) exposure to methyl-beta-cyclodextrin (CDx), commonly used as a diagnostic tool of endocytic mechanisms, is shown here to enhance AChR internalization kinetics in the receptor-expressing clonal cell line. This treatment drastically reduced ( approximately 50%) the number of receptor domains by accelerating the rate of endocytosis (t(1/2) decreased from 1.5-0.5 h). In addition, Chol depletion produced ion channel gain-of-function of the remaining cell-surface AChR, whereas Chol enrichment had the opposite effect. Fluorescence measurements under conditions of direct excitation of the probe Laurdan and of F?rster-type resonance energy transfer (FRET) using the intrinsic protein fluorescence as donor both indicated an increase in membrane fluidity in the bulk membrane and in the immediate environment of the AChR protein upon Chol depletion. Homeostatic control of Chol content at the plasmalemma may thus modulate cell-surface organization and stability of receptor domains, and fine tune receptor channel function to temporarily compensate for acute AChR loss from the cell surface.     

Probing the effects of membrane cholesterol in the Torpedo californica acetylcholine receptor and the novel lipid-exposed mutation alpha C418W in Xenopus oocytes

The effects of cholesterol on the ion-channel function of the Torpedo acetylcholine receptor (nAChR) and the novel lipid-exposed gain in function alpha C418W mutation have been investigated in Xenopus laevis oocytes. We found conditions to increase the cholesterol/phospholipid (C/P) molar ratio on the plasma membrane of Xenopus oocytes from 0.5 to 0.87, without significant physical damage or change in morphology to the oocytes. In addition, we developed conditions to deplete endogenous cholesterol from oocytes using a methyl-beta-cyclodextrin incubation procedure without causing membrane instability of the cells. Methyl-beta-cyclodextrin was also used to examine the reversibility of the inhibitory effect of cholesterol on AChR function. Depletion of 43% of endogenous cholesterol from oocytes (C/P = 0.3) did not show any significant change in macroscopic response of the wild type, whereas in the alpha C418W mutant the same cholesterol depletion caused a dramatic gain-in-function response of this lipid-exposed mutation in addition to the increased response caused by the mutation itself. Increasing the C/P ratio to 0.87 caused an inhibition of the macroscopic response of the Torpedo wild type of about 52%, whereas the alpha C418W mutation showed an 81% inhibition compared with the responses of control oocytes. The wild type receptor did not recover from this inhibition when the excess cholesterol was depleted to near normal C/P ratios; however, the alpha C418W mutant displayed 63% of the original current, which indicates that the inhibition of this lipid-exposed mutant was significantly reversed. The ability of the alpha C418W mutation to recover from the inhibition caused by cholesterol enrichment suggests that the interaction of cholesterol with this lipid-exposed mutation is significantly different from that of the wild type. The present data demonstrate that a single lipid-exposed position in the AChR could alter the modulatory effect of cholesterol on AChR function.     

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP

Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (M_f). The apparent diffusion coefficient (D_app) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a “membrane fence” model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more “fluid” and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of G_q/G₁₁ family.In conclusion: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R–G_q/G₁₁α protein coupling.     

Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 1. Equilibrium studies.

The interactions between the fluorescent probe ethidium and acetylcholine receptor enriched membranes from Torpedo californica are described. One class of saturable ethidium sites was blocked by alpha-bungarotoxin and therefore reflects direct binding to the receptor (Kd approximately 3 micrometers; stoichiometry--one ethidium site per two alpha-bungarotoxin sites). The second class of sites was nonsaturable and unaffected by alpha-toxin and was therefore considered nonspecific in nature. The increase in fluorescence intensity observed upon addition of cholinergic agonists and antagonists accurately reflects the dissociation constant and stoichiometry of the high-affinity receptor sites for these ligands. The effects of local anaesthetics are complex in nature and depend on the structure of the ligand. For carbamylcholine, the increase in flourescence intensity was due to an increase in the quantum yield of the dye bound to the membrane rather than a dye uptake. In general, ethidium appears not to strongly alter the properties of the membrane-bound acetylcholine receptor and can therefore be profitably used as a spectroscopic probe.