Effect of Membrane Lipid Composition on the Conformational Equilibria of the Nicotinic Acetylcholine Receptor
2000; Elsevier BV; Volume: 275; Issue: 2 Linguagem: Inglês
10.1074/jbc.275.2.777
ISSN1083-351X
AutoresJohn E. Baenziger, Mary-Louise Morris, Tim E. Darsaut, Stephen E. Ryan,
Tópico(s)Photoreceptor and optogenetics research
ResumoThe effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590–24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity. The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590–24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity. nicotinic acetylcholine receptor carbamylcholine desensitized resting dioleoylphosphatidylcholine cholesterol dioleoylphosphatidic acid egg phosphatidylcholine Fourier transform infrared attenuated total reflectance The nicotinic acetylcholine receptor (nAChR)1 fromTorpedo is a large multisubunit integral membrane protein that has been used extensively as a model for studying the mechanisms of lipid-protein interactions (1.Miyazawa A. Fujiyoshi Y. Stowell M. Unwin N. J. Mol. Biol. 1999; 288: 765-786Crossref PubMed Scopus (430) Google Scholar, 2.McNamee M.G. Fong T.M. Aloia R.C. Curtain C.C. Gordon L.M. Lipid Domains and the Relationship to Membrane Function. Alan R. Liss, Inc., New York1988: 43-62Google Scholar). In native membranes, the nAChR transiently gates open a cation-selective ion channel across the postsynaptic membrane in response to the binding of agonists such as acetylcholine and carbamylcholine (Carb). Prolonged exposure to either agonist or a variety of noncompetitive antagonists leads to the stabilization of a channel inactive/desensitized (D) state. In reconstituted membranes, the ability of the nAChR both to conduct cations across the membrane and to undergo the resting to desensitized (R→D) conformational transition is highly sensitive to the composition of the surrounding lipid membrane. The molecular details of how lipids modulate the ability of the nAChR to undergo agonist-induced conformational change, however, remain unclear. The original studies of Fong and McNamee (3.Fong T.M. McNamee M.G. Biochemistry. 1986; 26: 3871-3880Crossref Scopus (111) Google Scholar) suggested that although the nAChR reconstituted into a dioleoylphosphatidylcholine (DOPC) membrane is not functional, the addition of both cholesterol (Chol) and an anionic lipid, such as dioleoylphosphatidic acid (DOPA), to the reconstituted DOPC membrane restores the ability of the nAChR both to conduct cations and undergo agonist-induced desensitization. The recovery of function in the presence of Chol and DOPA was attributed to both the formation of a lipid bilayer with an optimal membrane fluidity and a specific structural requirement of the nAChR for each lipid. The latter was proposed to result from the binding of each to distinct sites on the nAChR with the consequent formation of specific secondary structural features (3.Fong T.M. McNamee M.G. Biochemistry. 1986; 26: 3871-3880Crossref Scopus (111) Google Scholar, 5.Butler D.H. McNamee M.G. Biochim. Biophys. Acta. 1993; 1150: 17-24Crossref PubMed Scopus (40) Google Scholar, 6.Bhushan A. McNamee M.G. Biophys. J. 1993; 64: 716-723Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 7.Fernandez-Ballester G. Castresana J. Fernandez A.M. Arrondo J.L.R. Ferragut J.A. Gonzalez-Ros J.M. Biochemistry. 1994; 33: 4065-4071Crossref PubMed Scopus (69) Google Scholar). Subsequent work has led to contradictory conclusions regarding the additional lipids that are required in a reconstituted DOPC membrane for the nAChR to adopt a functional conformation. McCarthy and Moore (8.McCarthy M.P. Moore M.A. J. Biol. Chem. 1992; 267: 7655-7663Abstract Full Text PDF PubMed Google Scholar) proposed that anionic lipids are sufficient to stabilize a functional nAChR based on chemical labeling and α-bungarotoxin rate binding studies of the nAChR in membranes composed of egg phosphatidylcholine (EPC) and DOPA. Their work also found that when reconstituted into membranes composed of either EPC/Chol or EPC alone the nAChR adopts a predominantly D conformation (8.McCarthy M.P. Moore M.A. J. Biol. Chem. 1992; 267: 7655-7663Abstract Full Text PDF PubMed Google Scholar). In contrast, the binding kinetics of ethidium bromide suggest that mixtures of DOPA/Chol support a functional nAChR whereas in either DOPC/DOPA or DOPC alone the nAChR is essentially "locked" in an R conformation (9; see also Ref. 10.Raines D.E. Krishnan N.S. Biochim. Biophys. Acta. 1998; 1374: 83-93Crossref PubMed Scopus (6) Google Scholar). Fourier transform infrared (FTIR) difference spectra are consistent with the data of McCarthy and Moore (8.McCarthy M.P. Moore M.A. J. Biol. Chem. 1992; 267: 7655-7663Abstract Full Text PDF PubMed Google Scholar) in that they suggest that the nAChR in EPC alone is desensitized (11.Ryan S.E. Demers C.N. Chew J.P. Baenziger J.E. J. Biol. Chem. 1996; 271: 24590-24597Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). In contrast to both studies, the FTIR data show that the presence of small amounts of either neutral or anionic lipids in an EPC membrane is sufficient to stabilize a fraction of the nAChRs in a functional conformation that is capable of undergoing agonist-induced conformational change. The FTIR data led to the suggestion that lipids modulate the relative number of nAChRs in the R and D states in the absence of agonist. The apparent ability of a variety of structurally diverse neutral and anionic lipids to modulate the equilibrium between the R and D conformations suggests further that lipids influence nAChR conformational equilibria via an indirect effect on some physical property of the membrane. Mixtures of a variety of structurally diverse neutral and anionic lipids in DOPC membranes all support nAChR cation flux (12.Sunshine C. McNamee M.G. Biochim. Biophys. Acta. 1994; 1191: 59-64Crossref PubMed Scopus (103) Google Scholar). Recent FTIR studies have also been unable to detect any of the changes in nAChR secondary structure which were reported previously in the presence of neutral and/or anionic lipids (13.Méthot N. Demers C.N. Baenziger J.E. Biochemistry. 1995; 34: 15142-15149Crossref PubMed Scopus (49) Google Scholar). The contradictory conclusions in the literature regarding the specific lipid requirements of the nAChR may reflect a variety of factors including the fact that the functional status of the nAChR in most studies has been assessed in reconstituted DOPC or EPC membranes with the additional lipid of interest found at a single molar percentage of the total membrane lipids (usually 25% or less). In many cases, functional data have also been interpreted in terms of the stabilization of either a fully functional or a nonfunctional conformation. Both approaches ignore the possibility that lipids modulate the equilibria between different conformational states. The relative level of a given lipid in a reconstituted phosphatidylcholine membrane may be an important factor in determining the relative percentage of nAChRs stabilized in a functional conformation and/or the kinetics of nAChR conformational change. To gain a more conclusive picture of the specific lipid requirements of the nAChR and to test the above noted hypothesis regarding a lipid-dependent modulation of nAChR conformational equilibria, we have examined the ability of the nAChR to undergo the Carb-induced R→D conformational change in EPC membranes with varying levels of either DOPA or Chol. We find that increasing levels of either lipid in an EPC membrane increasingly stabilizes a larger proportion of nAChRs in a conformation(s) that is(are) capable of undergoing Carb-induced desensitization. However, only high levels of DOPA were found to stabilize a structure of the nAChR which is fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. These results suggest that the presence of either DOPA or Chol in a reconstituted EPC membrane can influence the equilibrium between the R and D conformational states but that anionic lipids are required for the nAChR to adopt a fully functional conformation. EPC and DOPA were purchased from Avanti Polar lipids, Inc. (Alabaster, AL), and the Chol was from Sigma. FrozenTorpedo californica electric tissue was from Marinus (Long Beach, CA). The [13C]acetylcholine was synthesized from choline bromide and [13C]acetylchloride (both from Sigma) and purified according to Damle et al. (14.Damle V.N. McLaughlin M. Karlin A. Biochem. Biophys. Res. Commun. 1978; 84: 845-851Crossref PubMed Scopus (109) Google Scholar). The infrared spectrum of the synthesized [13C]acetylcholine was superimposable on a similar spectrum of commercially available [13C]acetylcholine (15.Williamson P.T. Grobner G. Spooner P.J. Miller K.W. Watts A. Biochemistry. 1998; 37: 10854-10859Crossref PubMed Scopus (32) Google Scholar). 2J. E. Baenziger, K. W. Miller, and K. J. Rothschild, unpublished observations. The nAChR was affinity purified on a bromoacetylcholine bromide-derivatized Affi-Gel 102 column (Bio-Rad) and then reconstituted into membranes composed of EPC with varying levels of either DOPA or Chol, as described by McCarthy and Moore (8.McCarthy M.P. Moore M.A. J. Biol. Chem. 1992; 267: 7655-7663Abstract Full Text PDF PubMed Google Scholar). In all cases except for the 9:1 molar ratios of EPC/DOPA and EPC/Chol, each reconstitution was performed between two and five times. FTIR samples were prepared by spreading 250 μg of the nAChR protein in 1H2O buffer on the surface of a 50 × 20 × 2-mm germanium attenuated total reflectance (ATR) internal reflection element (Harrick; Ossining, NY). After evaporating the bulk solvent with a gentle stream of N2 gas, the ATR crystal was installed in an ATR liquid sample cell (also from Harrick) and the nAChR film re-hydrated with excess Torpedo Ringer buffer (250 mm NaCl, 5 mm KCl, 2 mmMgCl2, 3 mm CaCl2, and 5 mm Na2HPO4, pH 7.0). FTIR spectra were acquired using the ATR technique on an FTS-40 spectrometer equipped with a DTGS detector. Spectra were recorded at 8 cm−1 resolution using 512 scans, which took roughly 7 min/spectrum. For the difference measurements, two consecutive R state spectra of the nAChR film in the absence of Carb were recorded with TorpedoRinger buffer flowing continuously through the sample compartment of the ATR cell at a rate of ∼1.5 ml/min. The flowing solution was then switched to an identical one containing 50 μm Carb. After 1 min, a spectrum of the D state was recorded. The difference between both the two R state spectra (absence of Carb; control spectra) and the consecutive R and D (presence of Carb) state spectra were calculated, stored, and the flowing buffer switched back to buffer without Carb. After a 20-min washing period to remove Carb from the film and convert the nAChR back into the R conformation, the process was repeated many times. Each experiment was repeated on several new films prepared from each affinity purification/reconstitution. All difference spectra were base line corrected between 1800 and 1000 cm−1 and were interpolated to an effective resolution of 4 cm−1. For more details see Ryan and Baenziger (16.Ryan S.E. Baenziger J.E. Mol. Pharmacol. 1999; 55: 348-355Crossref PubMed Scopus (26) Google Scholar). The difference between infrared spectra of the nAChR recorded in the presence and absence of Carb (referred to as a Carb difference spectrum) exhibits a complicated pattern of positive and negative vibrational bands. These difference bands reflect changes in the vibrations of those amino acid residues in the nAChR whose structures and/or surrounding environments change upon Carb binding. The pattern of difference bands provides a spectral map of the Carb-induced structural changes that occur in the nAChR. Specifically, this map includes features indicative of: 1) the vibrations of Carb bound to the nAChR (identified by arrows in the top trace of Fig. 1 A); 2) vibrational changes associated with the formation of direct physical interactions between Carb and binding site residues (e.g. hydrogen bonds, cation π-electron interactions, etc.); and 3) vibrational changes associated with the R→D conformational change. The conformational change probed in a typical Carb difference spectrum is shown schematically in Fig. 1 B, scheme i. Carb difference spectra recorded from affinity-purified nAChR reconstituted into membranes composed of 3:1:1 EPC/DOPA/Chol, a membrane that gives rise to a strong Carb-induced cation flux (3.Fong T.M. McNamee M.G. Biochemistry. 1986; 26: 3871-3880Crossref Scopus (111) Google Scholar), are similar to those recorded from the nAChR in native membranes and thus illustrate the pattern of difference bands expected for a functional nAChR (top trace in Fig. 1 A). Carb difference spectra recorded from the nAChR reconstituted into EPC membranes lacking neutral and anionic lipids are similar but exhibit a number of band intensity variations (middle trace in Fig.1 A). These variations reflect subtle membrane-induced changes in the structure/conformation of the nAChR and include a marked decrease in the intensity of five positive bands centered near 1663, 1655, 1547, 1430 and 1059 cm−1. The decrease in intensity of a positive band near 1663 cm−1 gives rise to the apparent increase in intensity of the overlapping negative band near 1668 cm−1 (see Fig. 3 and text in Ref. 16.Ryan S.E. Baenziger J.E. Mol. Pharmacol. 1999; 55: 348-355Crossref PubMed Scopus (26) Google Scholar). Although the individual bands in the Carb difference spectra remain to be assigned to specific residues, it is significant that the above noted changes in intensity are all observed in Carb difference spectra recorded from the nAChR in 3:1:1 EPC/DOPA/Chol membranes maintained in continuous contact with desensitizing local anesthetics, such as dibucaine (Fig. 1 A, bottom trace). In addition, relatively low concentrations of the anesthetic tetracaine, which stabilize the nAChR in an R-like as opposed to a D conformation, lead to an increase, as opposed to a decrease, in the intensities of the same five difference bands. Both observations suggest that the five noted positive difference bands reflect changes in vibrational intensity which occur as a consequence of the R→D conformational transition itself (for a detailed discussion, see Ref. 16.Ryan S.E. Baenziger J.E. Mol. Pharmacol. 1999; 55: 348-355Crossref PubMed Scopus (26) Google Scholar). The absence of these five bands in difference spectra recorded from the nAChR reconstituted into EPC membranes indicates that the nAChR in this membrane environment does not undergo the R→D conformational transition upon the binding of Carb (Fig. 1 B, scheme ii). This result is consistent with photoaffinity labeling studies, which show that the nAChR in EPC is stabilized in the D state (8.McCarthy M.P. Moore M.A. J. Biol. Chem. 1992; 267: 7655-7663Abstract Full Text PDF PubMed Google Scholar). In addition to the marked band intensity changes discussed above, more subtle lipid-sensitive spectral changes may occur between 1750 and 1700 cm−1, 1580 and 1520 cm−1, and 1400 and 1100 cm−1. Changes in band intensity in the 1700–1750 cm−1 region are difficult to monitor because of the overlapping Carb vibration at 1720 cm−1 (Fig.2 A). To circumvent this problem, difference spectra were recorded using isotopically labeled acetylcholine (13C label on the carbonyl carbon) instead of Carb to induce the R→D conformational transition (Fig.2 B). The resulting [13C]acetylcholine difference spectra exhibit a clear window in the 1750–1700 cm−1 region for viewing underlying protein vibrations. [13C]Acetylcholine difference spectra recorded from the nAChR in EPC/DOPA/Chol exhibit both a weak negative and positive difference band centered near 1740 and 1720 cm−1, respectively, which could reflect changes in the vibrational intensity and/or frequency of either a protonated carboxyl or a lipid ester carbonyl. The 1740 cm−1 vibration is absent in [13C]acetylcholine difference spectra recorded from the nAChR reconstituted into EPC membranes (Fig. 2 C). Potential variations in intensity between 1520 and 1580 cm−1 and between 1400 and 1100 cm−1 are difficult to assess because of the possibility of temperature-sensitive base-line fluctuations that can occur in these regions and/or the relatively weak intensities of the difference bands. Comparisons of potential spectral changes in these regions with those observed in spectra recorded in the presence of desensitizing local anesthetics are also complicated because negative local anesthetic bands due to Carb-induced displacement of local anesthetics from the neurotransmitter binding site appear in the latter spectra (Fig.2 A, bottom trace). It is thus difficult to assess both whether or not these spectral changes are present and, if present, whether the putative spectral changes are associated with the R→D conformational change. Current discussion will thus focus mainly on the six lipid-sensitive bands that are noted above near 1740, 1663, 1655, 1547, 1430, and 1059 cm−1. Carb difference spectra recorded from the nAChR reconstituted into membranes composed of EPC with increasing molar proportions of the anionic lipid DOPA exhibit a DOPA-dependent increase in positive intensity at the five noted conformationally sensitive band frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm−1 (Fig.3). In general, the increases in intensity at these five frequencies are modest at low levels of DOPA up to the EPC/DOPA molar ratio of 3:1 whereas at higher levels of DOPA they are more substantial. The intensities of all five conformationally sensitive bands in the Carb difference spectra recorded from the nAChR in EPC/DOPA at both the 3:2 and 1:1 molar ratios approach those observed in difference spectra recorded from the nAChR in 3:1:1 EPC/DOPA/Chol membranes. In addition, the [13C]acetylcholine difference spectra recorded from the nAChR in 3:2 EPC/DOPA membranes exhibit a relatively strong negative and positive band near 1740 and 1720 cm−1, respectively. The difference spectra thus indicate that the nAChR in EPC/DOPA at both the 3:2 and 1:1 molar ratios recovers its ability to undergo the R→D conformational transition and thus must be stabilized predominantly in a functional R conformation. In terms of our technique, high levels of DOPA in an EPC membrane appear to be sufficient to stabilize the nAChR in a functional conformation that is equivalent to that found in 3:1:1 EPC/DOPA/Chol membranes, even in the absence of Chol. Assuming that the nAChR in EPC membranes is stabilized in a D state (see "Discussion"), our data are consistent with a gradual shift in the equilibrium toward the R state with increasing levels of DOPA. A close examination of the data reveals that the effects of DOPA may be more complex than the modulation of a simple two-state conformational equilibrium. The difference spectra recorded from the nAChR in both the EPC/DOPA 3:1 and 9:1 membranes exhibit large positive intensities near 1655 and 1430 cm−1 relative to the intensities of the bands in spectra recorded from the nAChR in EPC (see Figs.5 A and 6). In contrast, the intensities of the two vibrations near 1059 cm−1 and 1663 cm−1remain weak and are similar to the intensities of the two bands in the Carb difference spectra recorded from the nAChR in EPC membranes completely lacking DOPA. This pattern of band intensity variations suggests that a large percentage of the conformationally sensitive residues in the nAChR which contribute intensity to the difference band near 1655 and 1430 cm−1 adopt an R-like conformation in 3:1 EPC/DOPA membranes, whereas the majority of the residues that contribute intensity to the difference bands near 1663 and 1059 cm−1 adopt a D-like conformation. The EPC/DOPA 9:1 and 3:1 membranes thus appear to stabilize a conformation that is a structural intermediate between the R and D states. Note that this same intermediate pattern of band intensities has been observed in Carb difference spectra recorded from the nAChR in EPC/DOPA/Chol membranes, but while the nAChR is maintained in the presence of concentrations of desensitizing local anesthetics where binding occurs exclusively to the noncompetitive blocker site located near the ion channel pore (16.Ryan S.E. Baenziger J.E. Mol. Pharmacol. 1999; 55: 348-355Crossref PubMed Scopus (26) Google Scholar). A similar pattern of band intensities variations is also observed in Carb difference spectra recorded from the nAChR in both 3:1 EPC/phosphatidylserine and 3:1 EPC/squalene membranes (11.Ryan S.E. Demers C.N. Chew J.P. Baenziger J.E. J. Biol. Chem. 1996; 271: 24590-24597Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar).Figure 6Summary of the changes in intensity centered near 1668 and 1655 cm −1 in Carb difference spectra recorded from the nAChR reconstituted into EPC membranes with increasing levels of either DOPA (panel A) or Chol (panel B). In both panels, the 0% and 100% values correspond to the intensities of these two bands in Carb difference spectra recorded from the nAChR reconstituted into EPC and 3:1:1 EPC/DOPA/Chol membranes, respectively. The measured values are sensitive to both base-line fluctuations and spectral scaling. They are estimated to be accurate within less than ±10%. For a given percentage of either DOPA or Chol, the relative changes in intensity at the two reported frequencies have a much higher accuracy.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Carb difference spectra recorded from the nAChR reconstituted into EPC membranes with increasing proportions of the neutral lipid Chol are similar to those recorded from the nAChR in EPC membranes with increasing levels of DOPA. There is a Chol-dependent increase in the intensities of four of the five noted conformationally sensitive bands near 1663, 1655, 1547, and 1430 cm−1 (Fig.4). The intensities of these four bands in the difference spectra recorded from the nAChR in 3:2 EPC/Chol approach the intensities of those recorded from the nAChR in 3:1:1 EPC/DOPA/Chol, suggesting that the nAChR has, for the most part, adopted an R-like conformation. The 3:1 EPC/Chol membranes also appear to stabilize a conformational intermediate between the R and D states as indicated by the relatively large positive intensity near 1655 cm−1 versus the relatively weak intensity near 1663 and 1059 cm−1 (Fig.5 B). There are, however, subtle variations between the Carb difference spectra recorded in the presence of increasing proportions of DOPA and Chol which suggest differences in the abilities of these two lipids to modulate conformational equilibria of the nAChR. First, at equivalent levels of either DOPA or Chol in the EPC membranes, the presence of DOPA leads to a greater intensity of the conformationally sensitive bands near 1663, 1655, 1547, 1430, and 1059 cm−1, implying that DOPA is slightly more effective at shifting the equilibrium toward the R conformation (Fig.6). This result is in agreement with the labeling studies of McCarthy and Moore, which suggest that the nAChR in 3:1 EPC/Chol (25%) is predominantly in the D state, whereas in 3:1 EPC/DOPA it is predominantly in the R conformation (8.McCarthy M.P. Moore M.A. J. Biol. Chem. 1992; 267: 7655-7663Abstract Full Text PDF PubMed Google Scholar). Second, increasing proportions of Chol have weak if any effect on the intensity of the difference band centered near 1059 cm−1. At all levels of Chol, the intensity near 1059 cm−1remains essentially the same as the intensity of the band in difference spectra recorded from the nAChR in EPC alone. In contrast, the intensity of this band doubles in difference spectra recorded from the nAChR in either 3:1:1 EPC/DOPA/Chol, 3:2 EPC/DOPA, or 1:1 EPC/DOPA (right panel of Fig. 3). The presence of Chol also does not lead to difference spectra with negative intensity near 1740 cm−1 comparable to that observed in spectra recorded from the nAChR in both 3:1:1 EPC/DOPA/Chol and 3:2 EPC/DOPA membranes (Fig.2). The lack of an effect of Chol on the intensities of these two vibrations may indicate that there are subtle structural differences between the nAChR in EPC membranes either with or without anionic lipids (see "Discussion"). Finally, the difference spectra recorded from the nAChR reconstituted into 1:1 EPC/Chol are similar to those recorded from the nAChR in EPC/Chol 3:1 (Fig. 6). In other words, the pattern of increasing intensity at each of the conformationally sensitive difference bands near 1663, 1655, 1547, and 1430 cm−1 with increasing Chol is reversed at very high levels of Chol. This reversal in the pattern of difference band intensity changes suggests that the ability of Chol to stabilize the nAChR in an R-like conformation is weakened at very high levels of Chol. A similar reversal in trend is observed with the peptide 1H/2H exchange kinetics of the nAChR upon reconstitution into EPC membranes with increasing levels of Chol (23.Baenziger J.E. Darsaut T.E. Morris M.-L. Biochemistry. 1999; 38: 4905-4911Crossref PubMed Scopus (28) Google Scholar). The main goal of this work was to test the hypothesis that the nAChR requires neutral and anionic lipids in its surrounding membrane environment in order to adopt a functional conformation that will undergo agonist-induced conformational change. We have shown previously that the difference between FTIR spectra of the nAChR recorded in the presence and absence of Carb exhibits positive and negative bands that serve as markers of the ability of the nAChR to undergo the R→D conformational transition (11.Ryan S.E. Demers C.N. Chew J.P. Baenziger J.E. J. Biol. Chem. 1996; 271: 24590-24597Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 16.Ryan S.E. Baenziger J.E. Mol. Pharmacol. 1999; 55: 348-355Crossref PubMed Scopus (26) Google Scholar). These markers are absent in Carb difference spectra recorded from the nAChR reconstituted into EPC membranes. The absence of both neutral and anionic lipids from a reconstituted EPC membrane thus leads to a receptor that cannot undergo Carb-induced conformational change. In contrast, these markers are present with increasing intensity in Carb difference spectra recorded from the nAChR reconstituted into EPC membranes with increasing levels of either DOPA
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