Modulation of Nicotinic Acetylcholine Receptor Conformational State by Free Fatty Acids and Steroids
2008; Elsevier BV; Volume: 283; Issue: 31 Linguagem: Inglês
10.1074/jbc.m800345200
ISSN1083-351X
AutoresGaspar A. Fernández Nievas, Francisco J. Barrantes, Silvia S. Antollini,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoSteroids and free fatty acids (FFA) are noncompetitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is purportedly located at the lipid-AChR interface, but their exact mechanism of action is still unknown. Here we studied the effect of structurally different FFA and steroids on the conformational equilibrium of the AChR in Torpedo californica receptor-rich membranes. We took advantage of the higher affinity of the fluorescent AChR open channel blocker, crystal violet, for the desensitized state than for the resting state. Increasing concentrations of steroids and FFA decreased the KD of crystal violet in the absence of agonist; however, only cis-unsaturated FFA caused an increase in KD in the presence of agonist. This latter effect was also observed with treatments that caused the opposite effects on membrane polarity, such as phospholipase A2 treatment or temperature increase (decreasing or increasing membrane polarity, respectively). Quenching by spin-labeled fatty acids of pyrene-labeled AChR reconstituted into model membranes, with the label located at the γM4 transmembrane segment, disclosed the occurrence of conformational changes induced by steroids and cis-unsaturated FFA. The present work is a step forward in understanding the mechanism of action of this type of molecules, suggesting that the direct contact between exogenous lipids and the AChR transmembrane segments removes the AChR from its resting state and that membrane polarity modulates the AChR activation equilibrium by an independent mechanism. Steroids and free fatty acids (FFA) are noncompetitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is purportedly located at the lipid-AChR interface, but their exact mechanism of action is still unknown. Here we studied the effect of structurally different FFA and steroids on the conformational equilibrium of the AChR in Torpedo californica receptor-rich membranes. We took advantage of the higher affinity of the fluorescent AChR open channel blocker, crystal violet, for the desensitized state than for the resting state. Increasing concentrations of steroids and FFA decreased the KD of crystal violet in the absence of agonist; however, only cis-unsaturated FFA caused an increase in KD in the presence of agonist. This latter effect was also observed with treatments that caused the opposite effects on membrane polarity, such as phospholipase A2 treatment or temperature increase (decreasing or increasing membrane polarity, respectively). Quenching by spin-labeled fatty acids of pyrene-labeled AChR reconstituted into model membranes, with the label located at the γM4 transmembrane segment, disclosed the occurrence of conformational changes induced by steroids and cis-unsaturated FFA. The present work is a step forward in understanding the mechanism of action of this type of molecules, suggesting that the direct contact between exogenous lipids and the AChR transmembrane segments removes the AChR from its resting state and that membrane polarity modulates the AChR activation equilibrium by an independent mechanism. Ligand-gated ion channels belong to a superfamily of proteins of which the muscle nicotinic acetylcholine receptor (AChR) 2The abbreviations used are: AChR, nicotinic acetylcholine receptor; Carb, carbamoylcholine; CrV, crystal violet; FFA, free fatty acids; sFFA, saturated FFA; cis-FFA, cis-unsaturated FFA; HC, hydrocortisone; NCA, non-competitive antagonist; N-PyrM, N-(1-pyrenyl)maleimide; PLA2, phospholipase A2; 5-SLFA, 5-spin labeled fatty acid; TM, transmembrane; FRET, Förster-type resonance energy transfer; GP, generalized polarization. is the best-characterized member. It is an integral membrane protein deeply embedded in the postsynaptic region, with two agonist binding sites and a central ion pore (1Karlin A. Nat. Rev. Neurosci. 2002; 3: 102-114Crossref PubMed Scopus (785) Google Scholar). A variety of high affinity noncompetitive antagonists (NCAs) of the AChR exert their action mainly through the ion pore. Some bind to the resting state of the AChR (tetracaine and 3-trifluoromethyl-3-(m-iodophenyl)diazirine), whereas others (e.g. phencyclidine, ethidium, chlorpromazine) preferentially bind to the desensitized form at equilibrium. Compounds such as these are predominantly hydrophilic molecules, their site of action being located in the extracellular part of the protein (for review, see Ref. 2Arias H.R. Mol. Membr. Biol. 1996; 13: 1-17Crossref PubMed Scopus (45) Google Scholar). A group of highly hydrophobic compounds (free fatty acids (FFA) and steroids) behave as low affinity NCAs of the AChR. 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Recently, we demonstrated that both exogenous and endogenous FFA and steroids localize inside the membrane, binding to common sites at the lipid-AChR interface (26Fernández Nievas G.A. Barrantes F.J. Antollini S.S. Biochemistry. 2007; 46: 3503-3512Crossref PubMed Scopus (19) Google Scholar). These molecules are likely to modulate the AChR function by a mechanism of action different from that exerted by hydrophilic inhibitors. For ligands acting at the lipid-protein interface, essentially two mechanisms of AChR inhibition can be considered, (a) displacement of an essential lipid from the protein-lipid interface or (b) changes in the physical properties of the lipid bilayer. The muscle AChR is a pentamer composed of four different but homologous subunits in the stoichiometry α2βγδ. Each subunit contains a relatively large extracellular domain and four hydrophobic segments referred to as M1-M4, proposed to be membrane-spanning segments, and ends with a short extracellular carboxyl-terminal domain. Three concentric rings can be distinguished in the AChR transmembrane (TM) region (27Barrantes F.J. Curr. Opin. Drug Discov. Devel. 2003; 6: 620-632PubMed Google Scholar, 28Barrantes F.J. Brain Res. Brain Res. Rev. 2004; 47: 71-95Crossref PubMed Scopus (149) Google Scholar). The M2 TM segments of all subunits outline the inner ring and form the walls of the ion channel proper; M1 and M3 constitute the middle ring, and the M4 segments form the outer ring, which is in closest contact with the AChR lipid microenvironment. Thus, an important portion of the AChR surface is exposed to the TM region, in contact with the surrounding lipids, establishing cross-talk between lipids and the protein moiety (27Barrantes F.J. Curr. Opin. Drug Discov. Devel. 2003; 6: 620-632PubMed Google Scholar, 28Barrantes F.J. Brain Res. Brain Res. Rev. 2004; 47: 71-95Crossref PubMed Scopus (149) Google Scholar). Subunit interactions, which are crucial for AChR activation, have been postulated to involve not only the AChR extracellular domain but also TM domains (29Hansen S.B. Taylor P. J. Mol. Biol. 2007; 369: 895-901Crossref PubMed Scopus (104) Google Scholar). Marsh and Barrantes (30Marsh D. Barrantes F.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4329-4333Crossref PubMed Scopus (170) Google Scholar) identified a layer of immobilized lipids surrounding the AChR distinct from the bulk lipids and postulated this layer as a possible site of pharmacological action. Criado et al. (31Criado M. Vaz W.L. Barrantes F.J. Jovin T.M. Biochemistry. 1982; 21: 5750-5755Crossref PubMed Scopus (43) Google Scholar) followed the kinetics of agonist-induced state transitions and showed that AChR function is sensitive to its lipid environment, postulating that the full function requires the presence of cholesterol or analogs. Sunshine and McNamee (32Sunshine C. McNamee M.G. Biochim. Biophys. Acta. 1992; 1108: 240-246Crossref PubMed Scopus (73) Google Scholar), reconstituting the AChR in different lipid environments, concluded that the lipid composition is more important than the bulk membrane fluidity in determining the AChR ion channel function; however, they subsequently indicated that the specific molecular structure of cholesterol was not key for sustaining AChR function and pointed to the hydrophobicity of the neutral lipids as the major factor in supporting AChR activity (33Sunshine C. McNamee M.G. Biochim. Biophys. Acta. 1994; 1191: 59-64Crossref PubMed Scopus (103) Google Scholar). They also found a marked lipid dependence on the rate of agonist-induced desensitization, suggesting that the membrane lipid environment can directly affect the conformational changes responsible for desensitization. Different combinations of lipids appear to induce the same, final desensitized conformation of the AChR as produced by prolonged exposure to agonist (34McCarthy M.P. Moore P.A. J. Biol. Chem. 1992; 267: 7655-7663Abstract Full Text PDF PubMed Google Scholar). Narayanaswami et al. (35Narayanaswami V. Kim J. McNamee M.G. Biochemistry. 1993; 32: 12413-12419Crossref PubMed Scopus (29) Google Scholar) postulated that relatively fluid layers of lipid surrounding the AChR, probably with an optimal fluidity, are responsible for facilitating AChR conformational changes. Studies from our laboratory indicated the presence of distinct sites at the AChR lipid interface for cholesterol and phospholipids (36Antollini S.S. Barrantes F.J. Biochemistry. 1998; 37: 16653-16662Crossref PubMed Scopus (69) Google Scholar). In this paper we studied the effect of structurally different FFA and steroids on the AChR conformational equilibrium in native Torpedo californica membranes using a fluorescent dye that has a higher affinity for the desensitized state of the AChR in an attempt to understand the effect of these two types of hydrophobic molecules on the modulation of the AChR and to discriminate between direct and indirect modes of action. The results indicate that there are two levels of AChR conformational modulation. First, the mere presence of FFA or steroids can directly drive the AChR out of its native-resting-conformation; second, perturbations of membrane polarity prevent the AChR from reaching the agonist-induced desensitized state. T. californica specimens obtained from the Pacific coast of California were killed by pithing, and the electric organs were dissected and stored at -70 °C until further use. Laurdan and pyrene-maleimides were purchased from Molecular Probes (Eugene, OR). Phospholipase A2 (PLA2) was obtained from Roche Applied Science. Affi-Gel® 10 Gel and dithiothreitol were obtained from Bio-Rad. Synthetic lipids and 5-spin labeled fatty acid (5-SLFA) were from Avanti Polar Lipids, Inc. (Birmingham, AL). Crystal Violet (CrV) and all the others drugs were obtained from Sigma-Aldrich. Preparation of AChR-rich Membranes—Membrane fragments rich in AChR were prepared from the electric tissue of T. californica as described previously (37Barrantes F.J. Hucho F. Neuroreceptors. Walter de Gruyter & Co., Berlin1982: 315-328Crossref Google Scholar). Briefly, electric tissue was chopped into small pieces, homogenized under controlled conditions using a Virtis homogenizer, and submitted to a series of centrifugation steps ending in a high speed sucrose gradient centrifugation. Specific activities on the order of 2.0–2.8 nmol of α-bungarotoxin sites/mg of protein were obtained in the middle, AChR-rich membrane fraction (37Barrantes F.J. Hucho F. Neuroreceptors. Walter de Gruyter & Co., Berlin1982: 315-328Crossref Google Scholar). The orientation of AChR in the membrane vesicles was determined as described by Hartig and Raftery (38Hartig P.R. Raftery M.A. Biochemistry. 1979; 18: 1146-1150Crossref PubMed Scopus (45) Google Scholar) by comparing the total toxin binding sites in the presence of Triton X-100 and the right-side-out toxin binding sites in the absence of detergent as in previous work from our laboratory (39Gutierrez-Merino C. Bonini de Romanelli I.C. Pietrasanta L.I. Barrantes F.J. Biochemistry. 1995; 34: 4846-4855Crossref PubMed Scopus (19) Google Scholar). For the fluorescence measurements, AChR-rich membranes and N-PyrM-AChR were suspended in buffer A (150 mm NaCl, 0.25 mm MgCl2, and 20 mm HEPES buffer, pH 7.4) at a final concentration of 100 μg of protein/ml (0.4 μm) or 20 μg/ml, respectively. The optical density of the membrane suspension was kept below 0.1 to minimize light scattering. Preparation of Steroid and Free Fatty Acid Solutions—Cortisone and hydrocortisone (HC) were dissolved in (1:1) ethanol: DMSO. FFAs were dissolved in ethanol. In all cases the amount of organic solvent added to the samples was kept below 0.5%. After each steroid or FFA addition, samples were kept at 25 °C for 30 min to allow equilibration of the added hydrophobic compounds with the membrane. Fluorescence Measurements—All fluorimetric measurements were performed in an SLM model 4800 fluorimeter (SLM Instruments, Urbana, IL) using a vertically polarized light beam from Hannovia 200-W mercury/xenon arc obtained with a Glan-Thompson polarizer (4-nm excitation and emission slits) and 1-ml quartz cuvettes. The temperature was set at 25 °C with a thermostatted circulating water bath (Haake, Darmstadt, Germany) except when indicated otherwise. CrV Fluorescent Measurements—CrV was dissolved in buffer A at three different stock concentrations (10, 50, and 350 μm) and stored at -20 °C for posterior titration experiments. For the fluorescence measurements, AChR-rich membranes were suspended in buffer A. The samples were titrated with CrV. After each addition, the samples were incubated for 15 min to allow equilibration, and then the emission spectra were collected. CrV was excited at 600 nm, and the fluorescence emission spectra were collected from 605 to 700 nm. A background spectrum (obtained from the same cuvette before CrV addition) was subtracted from the emission spectra obtained in the presence of CrV, and the maximum intensity (at 623–625 nm) was measured. To determine the CrV dissociation constants (KD), the value of CrV maximum fluorescence emission was plotted as a function of the logarithmic CrV concentration (molarity), the resulting sigmoid curve was fitted to the Boltzmann function, and the KD was calculated. To test the magnitude of the nonspecific drug binding (40Lurtz M.M. Pedersen S.E. Mol. Pharmacol. 1999; 55: 159-167Crossref PubMed Scopus (30) Google Scholar), we also undertook control experiments using membrane fractions from T. californica devoid of AChR. After obtaining the crude membrane fraction by differential centrifugation, an additional sucrose gradient centrifugation was performed, and three distinct membrane bands were obtained (37Barrantes F.J. Hucho F. Neuroreceptors. Walter de Gruyter & Co., Berlin1982: 315-328Crossref Google Scholar). The middle band is highly enriched in AChR ("AChR-rich membranes"), whereas the top band has less AChR protein, and the bottom band is devoid of AChR protein. Thus, the bottom band was used as a control for CrV unspecific labeling (see the supplemental figure). The nonspecific signal was found to be small and, therefore, does not introduce significant changes in the results showing the KD of CrV. PLA2 Treatment of AChR-rich Membranes—AChR-rich membranes were suspended at a final concentration of 1 mg/ml in phosphate buffer (10 mm sodium phosphate buffer containing 2.5 mm CaCl2 and 35 mm KCl, pH 8.0) in the presence of 1.5 enzymatic units of PLA2 (from Naja naja venom) and incubated at room temperature with gentle stirring. Aliquots were taken at different times to obtain different degrees of phospholipid degradation. The reaction was stopped by a 10-fold dilution with cold phosphate buffer (10 mm sodium phosphate buffer containing 3 mm EGTA, pH 7.4). Finally, the samples were centrifuged at 100,000 × g for 45 min. The resulting pellet was resuspended in buffer A and kept at -60 °C until use (26Fernández Nievas G.A. Barrantes F.J. Antollini S.S. Biochemistry. 2007; 46: 3503-3512Crossref PubMed Scopus (19) Google Scholar, 41Görne-Tschelnokow U. Strecker A. Kaduk C. Naumann D. Hucho F. EMBO J. 1994; 13: 338-341Crossref PubMed Scopus (100) Google Scholar). Generalized Polarization (GP) Determination—After AChR-rich membrane incubation with the exogenous hydrophobic compound, Laurdan was added to give a final probe concentration of 0.6 μm and incubated in the dark for 1 h (36Antollini S.S. Barrantes F.J. Biochemistry. 1998; 37: 16653-16662Crossref PubMed Scopus (69) Google Scholar, 42Antollini S.S. Soto M.A. Bonini de Romanelli I. Gutierrez-Merino C. Sotomayor P. Barrantes F.J. Biophys. J. 1996; 70: 1275-1284Abstract Full Text PDF PubMed Scopus (64) Google Scholar). GP values, obtained from emission spectra obtained with an excitation wavelength of 290 and 360 nm for Förster-type resonance energy transfer (FRET) or direct excitation, respectively, were calculated as GP = (I434 - I490)/(I434 + I490), where I434 and I490 are the emission intensities at the characteristic wavelength of the gel and liquid-crystalline phases, respectively (43Parasassi T. De Stasio G. d'Ubaldo A. Gratton E. Biophys. J. 1990; 57: 1179-1186Abstract Full Text PDF PubMed Scopus (630) Google Scholar, 44Parasassi T. De Stasio G. Ravagnan G. Rusch R.M. Gratton E. Biophys. J. 1991; 60: 179-189Abstract Full Text PDF PubMed Scopus (723) Google Scholar). Affinity Column Preparation and AChR Purification—T. californica crude membranes (2 mg/ml protein concentration) were solubilized in 1% sodium cholate for 45 min and then centrifuged for 1 h at 74,000 × g to discard the insoluble material. The AChR was purified by affinity chromatography in the presence of synthetic lipids (45daCosta C.J. Ogrel A.A. McCardy E.A. Blanton M.P. Baenziger J.E. J. Biol. Chem. 2002; 277: 201-208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 46daCosta C.J. Wagg I.D. McKay M.E. Baezinger J.E. J. Biol. Chem. 2004; 279: 14967-14974Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, the affinity column was prepared by coupling cystamine to Affi-Gel 10, reduction with dithiothreitol, and final modification with bromoacetylcholine bromide. The supernatant was applied to the affinity column. To facilitate complete exchange of endogenous for defined lipids, the column was then washed with a linear gradient of defined lipids (POPC:POPA:cholesterol 3:1:1; where POPC is palmitoyl-oleoyl phosphatidylcholine, and POPA is palmitoyl-oleoyl phosphatidic acid) dissolved in dialysis buffer (100 mm NaCl, 10 mm Tris-HCl, 0.1 mm EDTA, 0.02% NaN3, pH 7.8) and containing 1% cholate, from 1.3 to 3.2 mm and then to 0.13 mm lipid concentration (45daCosta C.J. Ogrel A.A. McCardy E.A. Blanton M.P. Baenziger J.E. J. Biol. Chem. 2002; 277: 201-208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The AChR was then eluted from the column with a 0.13 mm lipid solution in 250 mm NaCl, 0.1 mm EDTA, 0.02% NaN3, 5 mm phosphate, pH 7.8, with 0.5% cholate and 10 mm carbamoylcholine (Carb). After elution from the column, the AChR was dialyzed against 1 liter of dialysis buffer with five buffer changes (every 12 h) at 4 °C. The AChR purification was checked by SDS-PAGE, and protein concentration was determined by the method of Lowry (47Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The samples were stored at -70 °C until use. N-(1-Pyrenyl)maleimide AChR Labeling—The labeling of TM AChR cysteines was performed according to Li et al. (48Li L. Schuchard M. Palma A. Pradier L. McNamee M.G. Biochemistry. 1990; 29: 5428-5436Crossref PubMed Scopus (40) Google Scholar) and Narayanaswami et al. (35Narayanaswami V. Kim J. McNamee M.G. Biochemistry. 1993; 32: 12413-12419Crossref PubMed Scopus (29) Google Scholar) with slight modifications. Briefly, purified AChR (1 mg/ml) solubilized in 1% cholate was incubated in the presence of N-PyrM final concentration of 1 mm for 1 h at room temperature with gentle shaking. The solution was centrifuged for 45 min at 70,000 × g to pellet aggregates. The supernatant was dialyzed against 1 liter of dialysis buffer with five buffer changes (every 12 h) at 4 °C and stored at -70 °C until use. N-PyrM-AChR samples were submitted to SDS-PAGE, and the N-PyrM label was visualized under a UV transilluminator showing that the band corresponding to the γ-subunit was predominantly labeled, as shown in previous work (35Narayanaswami V. Kim J. McNamee M.G. Biochemistry. 1993; 32: 12413-12419Crossref PubMed Scopus (29) Google Scholar). Fluorescence Quenching with Acrylamide and Spin-label Free Fatty Acid—Quenching experiments were developed with N-PyrM-AChR. Excitation wavelength was 345 nm, and the fluorescence emission scanning was from 360 nm up to 500 nm. The maximum pyrene fluorescence emission was recorded at 374 nm. To confirm that the pyrene label was at the TM segments of the AChR, a sample of N-PyrM-AChR was titrated with acrylamide, a water-soluble quencher, up to a concentration of 500 mm. To corroborate the correct quenching process, the intrinsic Trp emission was recorded at 330 nm on the same samples (290-nm excitation wavelength). N-PyrM-AChR samples (20 μg/ml) were first titrated with steroid (HC) or different FFA up to 600 or 60 μm, respectively, and control samples were obtained with the addition of only the vehicle of the exogenous compounds. For each sample, sequential additions of 5-SLFA dissolved in ethanol were performed. After each addition, samples were kept for 30 min at 25 °C before the fluorescence measurement to allow equilibration of the 5-SLFA with the membrane. From the fluorescence data, Stern-Volmer plots were obtained according to the equation Fo/F = 1 + Ksv[Q], where Fo and F correspond to the fluorescence emission of pyrene-labeled AChR in the absence and presence of 5-SLFA, respectively, [Q] is the concentration of the quencher, and Ksv is the Stern-Volmer constant (49Lakowicz R.L. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic/Plenum Publishers, New York1999: 239-243Google Scholar). Ksv is a measure of the quencher concentration in the vicinity of the fluorophore, which allowed us to obtain topological information on the transverse location of the labeled cysteine. Aminotriarylmethane dyes share structural similarities with many known NCA of the AChR: (a) the presence of aromatic groups, (b) tertiary or quaternary amines and (c) a net positive charge. Lurtz and Pedersen (40Lurtz M.M. Pedersen S.E. Mol. Pharmacol. 1999; 55: 159-167Crossref PubMed Scopus (30) Google Scholar) demonstrated that CrV, one such compound, displays higher affinity for the D conformation (in the presence of agonist) than for the R conformation (in the absence of agonist) of the AChR. We took advantage of these differences in affinity to monitor AChR conformational states in the presence of two types of low affinity NCA of the AChR; that is, steroids and FFA. These molecules share the characteristic of being highly hydrophobic compounds. We recently demonstrated that these lipids exert their action on the TM domain of the AChR (26Fernández Nievas G.A. Barrantes F.J. Antollini S.S. Biochemistry. 2007; 46: 3503-3512Crossref PubMed Scopus (19) Google Scholar), but the mechanism by which they cause AChR inhibition is not yet known. Steroids Induce a Conformational Change of the Resting to the Desensitized AChR State—To study whether the presence of steroids affects the AChR conformational equilibrium, AChR-rich membranes from T. californica were first incubated with increasing concentrations (100–600 μm) of steroid (cortisone or HC). Carb was subsequently added to a subset of these membranes, and then all samples were titrated with CrV (Fig. 1a). Remarkably, in the absence of agonist, the curves furnished in the presence of steroid were displaced toward those in the D state. In the presence of agonist, the curves obtained with or without steroid (control) were similar. In the absence of agonist, HC produced a concentration-dependent diminution of the KD of the R state, which approached that of the (control) KD of the D state (Fig. 1b). Free Fatty Acids Modify Both the Resting and the Desensitized State of the AChR—We next studied the effect of different exogenous FFA. AChR-rich membranes from T. californica were incubated with increasing concentrations of different cis-unsaturated FFA (cis-FFA) (arachidonic acid, cis-20:4; linolenic acid, cis-18:3; oleic acid, cis-18:1) followed by the addition of 1 mm Carb to a subset of samples (Fig. 2a). A diminution of the KD of CrV was observed when the membranes were treated with cis-18:1 in the absence of agonist. A statistically significant increase in the value of KD was observed in the presence of agonist. Similar results were obtained for cis-20:4 and cis-18:3 (data not shown). Experiments were also performed with saturated FFA (sFFA). AChR-rich membranes from T. californica were incubated in the presence of increasing concentrations of arachidic acid (20:0) or stearic acid (18:0). In contrast to what was observed with cis-FFA (Fig. 2a), the KD obtained showed no statistically significant changes either in the absence or presence of agonist (Fig. 2b). A third structural class of FFA, with trans isomerism, was also tested (Fig. 2c); elaidic acid (trans-18:1). The observed effects may be due to differential partitioning of the fatty acids into the membrane and not to the chemical properties of the different acyl chains. The magnitude of the incorporation of different FFA to AChR-rich membranes was extensively characterized in a previous work from our laboratory using the fluorescence probe ADIFAB (acrylodan-derivatized intestinal fatty acid binding protein) (50Antollini S.S. Barrantes F.J. J. Biol. Chem. 2002; 277: 1249-1254Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The calculated partition coefficient values allowed us to classify the FFA into three different groups; 1) highly hydrophobic FFA (such as 20:0 and 18:0), 2) less hydrophobic FFA (such as cis-18:1 and trans-18:1), and 3) more hydrophilic FFA (such as 18:0, 18:2, 18:3, 20:4, and 22:6). Here we used FFA from the three groups (20:0, cis and trans 18:1, cis-18:3, and cis-20:4), and the observed effects show no relation with the degree of FFA partition, since the most hydrophobic FFA (i.e. the one exhibiting the highest membrane partition coefficient) did not display any effect, and the two FFA with similar partition coefficients (cis and trans 18:1) showed very different effects. Free Fatty Acids and Steroids Together Modify Both Conformational States of the AChR—The combined effects of FFA and steroids were studied next. Toward this end steroid (HC) or FFA (oleic acid) were added to AChR-rich membranes from T. californica at saturating concentrations (cf. Figs. 1 and 2), and the second lipid was added subsequently, also at saturating concentration (Fig. 3). The order of addition of the two types of lipids did not matter; either combination resulted in similar final states of the AChR both in the presence or absence of agonist. Furthermore, when CrV was added in the presence of agonist, the KD values obtained from samples treated with both HC and oleic acid were similar to those obtained with oleic acid alone. Membrane Polarity Modulates the Transition of the AChR to the D St
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