Phospholipase C Activity Affinity Purifies with the Torpedo Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function. Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function. IntroductionCys-loop receptors mediate fast transmission at chemical synapses by transiently opening an ion channel across the cell membrane in response to neurotransmitter binding (1.Dougherty D.A. Chem. Rev. 2008; 108: 1642-1653Crossref PubMed Scopus (94) Google Scholar, 2.Goetz T. Arslan A. Wisden W. Wulff P. Prog. Brain Res. 2007; 160: 21-41Crossref PubMed Scopus (73) Google Scholar, 3.Sine S.M. Engel A.G. Nature. 2006; 440: 448-455Crossref PubMed Scopus (421) Google Scholar, 4.Thompson A.J. Lummis S.C. Curr. Pharm. Des. 2006; 12: 3615-3630Crossref PubMed Scopus (174) Google Scholar, 5.Shen X.M. Deymeer F. Sine S.M. Engel A.G. Ann. Neurol. 2006; 60: 128-136Crossref PubMed Scopus (43) Google Scholar, 6.Karlin A. Nat. Rev. Neurosci. 2002; 3: 102-114Crossref PubMed Scopus (768) Google Scholar). The resultant influx of ions into the cell alters the membrane potential, leading to the generation or in some cases the inhibition of an action potential. Many endogenous and exogenous compounds influence the ability of Cys-loop receptors to flux ions, altering the efficiency of synaptic transmission. Cys-loop receptors play a dynamic role in communication within the nervous system.The ability of Cys-loop receptors to flux ions is sensitive to membrane lipid composition (7.Criado M. Eibl H. Barrantes F.J. J. Biol. Chem. 1984; 259: 9188-9198Abstract Full Text PDF PubMed Google Scholar, 8.Fong T.M. McNamee M.G. Biochemistry. 1986; 25: 830-840Crossref PubMed Scopus (261) Google Scholar, 9.Baenziger J.E. Morris M.L. Darsaut T.E. Ryan S.E. J. Biol. Chem. 2000; 275: 777-784Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10.Hamouda A.K. Sanghvi M. Sauls D. Machu T.K. Blanton M.P. Biochemistry. 2006; 45: 4327-4337Crossref PubMed Scopus (56) Google Scholar, 11.daCosta C.J. Baenziger J.E. J. Biol. Chem. 2009; 284: 17819-17825Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The effects of lipids on the function of the prototypical Cys-loop receptor, the Torpedo nicotinic acetylcholine receptor (nAChR), 4The abbreviations used are: nAChRnicotinic acetylcholine receptorPA1,2-diacyl-sn-glycero-3-phosphatePC1,2-diacyl-sn-glycero-3-phosphocholinePG1,2-diacyl-sn-glycero-3-phosphoglycerolPS1,2-diacyl-sn-glycero-3-[phospho-l-Serine]Carbcarbamylcholine chlorideDAGdiacylglycerol. have been intensely studied, particularly in reconstituted membranes of “well defined” lipid compositions. A mixture of both anionic and neutral lipids is optimal for the nAChR to adopt an agonist-activatable conformation (9.Baenziger J.E. Morris M.L. Darsaut T.E. Ryan S.E. J. Biol. Chem. 2000; 275: 777-784Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10.Hamouda A.K. Sanghvi M. Sauls D. Machu T.K. Blanton M.P. Biochemistry. 2006; 45: 4327-4337Crossref PubMed Scopus (56) Google Scholar, 11.daCosta C.J. Baenziger J.E. J. Biol. Chem. 2009; 284: 17819-17825Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Of the many anionic lipids, phosphatidic acid (PA) is particularly effective at stabilizing a large proportion of agonist-activatable resting nAChRs (10.Hamouda A.K. Sanghvi M. Sauls D. Machu T.K. Blanton M.P. Biochemistry. 2006; 45: 4327-4337Crossref PubMed Scopus (56) Google Scholar, 12.daCosta C.J. Medaglia S.A. Lavigne N. Wang S. Carswell C.L. Baenziger J.E. J. Biol. Chem. 2009; 284: 33841-33849Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar).Correlations between lipids and nAChR function are based on the assumption that the lipid composition of a reconstituted membrane is defined by the exogenous lipids supplied during affinity purification. Although this assumption holds in most cases, we show here that nAChR-reconstituted PC/PA membranes contain the unexpected lipid diacylglycerol (DAG). DAG appears during affinity purification of the nAChR as a result of the hydrolysis of PA. PA-specific phospholipase C activity co-purifies with the nAChR and is inhibited by vanadate but not N-ethylmaleimide. Also, the presence of DAG in a reconstituted membrane influences the ability of the nAChR to undergo agonist-induced conformational transitions. Our results support the novel hypothesis that nicotinic acetylcholine receptors influence cellular events by mechanisms that do not involve the flux of ions into the cell (13.Kihara T. Shimohama S. Sawada H. Honda K. Nakamizo T. Shibasaki H. Kume T. Akaike A. J. Biol. Chem. 2001; 276: 13541-13546Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 14.de Jonge W.J. Ulloa L. Br. J. Pharmacol. 2007; 151: 915-929Crossref PubMed Scopus (470) Google Scholar, 15.Sharma G. Vijayaraghavan S. J. Neurobiol. 2002; 53: 524-534Crossref PubMed Scopus (146) Google Scholar, 16.Suzuki T. Hide I. Matsubara A. Hama C. Harada K. Miyano K. Andrä M. Matsubayashi H. Sakai N. Kohsaka S. Inoue K. Nakata Y. J. Neurosci. Res. 2006; 83: 1461-1470Crossref PubMed Scopus (195) Google Scholar, 17.Blanchet M.R. Israël-Assayag E. Daleau P. Beaulieu M.J. Cormier Y. Am. J. Physiol. Lung Cell Mol. Physiol. 2006; 291: L757-L763Crossref PubMed Scopus (30) Google Scholar). The nAChR may be able to alter the lipid composition of its surrounding microenvironment in a manner that enhances its own function.DISCUSSIONIt is well established that nAChRs influence cellular activity by fluxing ions across the membrane in response to neurotransmitter binding. Increasing evidence suggests, however, that some nicotinic receptors play additional roles in cellular function not related to the flux of ions (13.Kihara T. Shimohama S. Sawada H. Honda K. Nakamizo T. Shibasaki H. Kume T. Akaike A. J. Biol. Chem. 2001; 276: 13541-13546Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 14.de Jonge W.J. Ulloa L. Br. J. Pharmacol. 2007; 151: 915-929Crossref PubMed Scopus (470) Google Scholar). Homomeric α7 nAChRs function in nonexcitable cells, such as endothelial cells, keratinocytes, and T cells (15.Sharma G. Vijayaraghavan S. J. Neurobiol. 2002; 53: 524-534Crossref PubMed Scopus (146) Google Scholar). Microglial α7 nAChRs couple with phospholipase C activation and Ca2+ mobilization from IP3-sensitive Ca2+ stores, leading to the suppression of neuroinflammation (16.Suzuki T. Hide I. Matsubara A. Hama C. Harada K. Miyano K. Andrä M. Matsubayashi H. Sakai N. Kohsaka S. Inoue K. Nakata Y. J. Neurosci. Res. 2006; 83: 1461-1470Crossref PubMed Scopus (195) Google Scholar). Nicotinic agonists exert anti-inflammatory effects on macrophages that can be countered by α7 nAChR-selective antagonists. Also, α3, α4, and α5 nAChR subunits associate with phosphatidylinositol 3-kinase in monocytes and macrophages leading to the stimulation of phospholipase C activity (17.Blanchet M.R. Israël-Assayag E. Daleau P. Beaulieu M.J. Cormier Y. Am. J. Physiol. Lung Cell Mol. Physiol. 2006; 291: L757-L763Crossref PubMed Scopus (30) Google Scholar).We report here the novel finding that phospholipase C activity co-purifies with affinity-purified nAChR from Torpedo. This phospholipase activity is specific for PA over other anionic (PS, PG, 1,2-diacyl-sn-glycero-3-phosphoinositol, and cardiolipin) and zwitterionic (PC) phospholipids. The phospholipase activity is inhibited by sodium vanadate but not N-ethylmaleimide. The phospholipase activity elutes from an acetylcholine-linked nAChR affinity column with the same profile as the nAChR. SDS-PAGE shows that the eluted fractions contain four subunits corresponding to the α, β, γ, and δ subunits of the nAChR. No species corresponding in molecular mass to either cytosolic phosphatidate phosphatases (∼100 kDa) or membrane-bound lipid phosphate phosphohydrolases (∼35 kDa) are observed (24.Reue K. Brindley D.N. J. Lipid Res. 2008; 49: 2493-2503Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 25.Simon M.F. Rey A. Castan-Laurel I. Grés S. Sibrac D. Valet P. Saulnier-Blache J.S. J. Biol. Chem. 2002; 277: 23131-23136Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 26.Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) (FIGURE 1, FIGURE 2, FIGURE 3), although we cannot rule out the existence of trace amounts of these proteins. We therefore conclude that either the nAChR itself or a protein that closely associates with the nAChR, and that binds tightly with the nAChR on the affinity resin, exhibits phospholipase C activity.The finding that either the nAChR itself or a tightly associated protein exhibits phospholipase activity provides further evidence that the nAChR can influence cellular function via mechanisms that do not involve the flux of ions. This phospholipase activity could generate DAG to influence cellular metabolism. The presence of DAG in the lipid microenvironment surrounding the nAChR could also directly influence nAChR function. It is interesting to note that discharge of Torpedo electric organ leads to the turnover of PA (29.Bleasdale J.E. Hawthorne J.N. Widlund L. Heilbronn E. Biochem. J. 1976; 158: 557-565Crossref PubMed Scopus (16) Google Scholar), possibly because of PA hydrolysis to DAG. The possibility that the nAChR can alter its lipid microenvironment in a manner that influences nAChR function with important biological consequences requires further investigation. IntroductionCys-loop receptors mediate fast transmission at chemical synapses by transiently opening an ion channel across the cell membrane in response to neurotransmitter binding (1.Dougherty D.A. Chem. Rev. 2008; 108: 1642-1653Crossref PubMed Scopus (94) Google Scholar, 2.Goetz T. Arslan A. Wisden W. Wulff P. Prog. Brain Res. 2007; 160: 21-41Crossref PubMed Scopus (73) Google Scholar, 3.Sine S.M. Engel A.G. Nature. 2006; 440: 448-455Crossref PubMed Scopus (421) Google Scholar, 4.Thompson A.J. Lummis S.C. Curr. Pharm. Des. 2006; 12: 3615-3630Crossref PubMed Scopus (174) Google Scholar, 5.Shen X.M. Deymeer F. Sine S.M. Engel A.G. Ann. Neurol. 2006; 60: 128-136Crossref PubMed Scopus (43) Google Scholar, 6.Karlin A. Nat. Rev. Neurosci. 2002; 3: 102-114Crossref PubMed Scopus (768) Google Scholar). The resultant influx of ions into the cell alters the membrane potential, leading to the generation or in some cases the inhibition of an action potential. Many endogenous and exogenous compounds influence the ability of Cys-loop receptors to flux ions, altering the efficiency of synaptic transmission. Cys-loop receptors play a dynamic role in communication within the nervous system.The ability of Cys-loop receptors to flux ions is sensitive to membrane lipid composition (7.Criado M. Eibl H. Barrantes F.J. J. Biol. Chem. 1984; 259: 9188-9198Abstract Full Text PDF PubMed Google Scholar, 8.Fong T.M. McNamee M.G. Biochemistry. 1986; 25: 830-840Crossref PubMed Scopus (261) Google Scholar, 9.Baenziger J.E. Morris M.L. Darsaut T.E. Ryan S.E. J. Biol. Chem. 2000; 275: 777-784Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10.Hamouda A.K. Sanghvi M. Sauls D. Machu T.K. Blanton M.P. Biochemistry. 2006; 45: 4327-4337Crossref PubMed Scopus (56) Google Scholar, 11.daCosta C.J. Baenziger J.E. J. Biol. Chem. 2009; 284: 17819-17825Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The effects of lipids on the function of the prototypical Cys-loop receptor, the Torpedo nicotinic acetylcholine receptor (nAChR), 4The abbreviations used are: nAChRnicotinic acetylcholine receptorPA1,2-diacyl-sn-glycero-3-phosphatePC1,2-diacyl-sn-glycero-3-phosphocholinePG1,2-diacyl-sn-glycero-3-phosphoglycerolPS1,2-diacyl-sn-glycero-3-[phospho-l-Serine]Carbcarbamylcholine chlorideDAGdiacylglycerol. have been intensely studied, particularly in reconstituted membranes of “well defined” lipid compositions. A mixture of both anionic and neutral lipids is optimal for the nAChR to adopt an agonist-activatable conformation (9.Baenziger J.E. Morris M.L. Darsaut T.E. Ryan S.E. J. Biol. Chem. 2000; 275: 777-784Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10.Hamouda A.K. Sanghvi M. Sauls D. Machu T.K. Blanton M.P. Biochemistry. 2006; 45: 4327-4337Crossref PubMed Scopus (56) Google Scholar, 11.daCosta C.J. Baenziger J.E. J. Biol. Chem. 2009; 284: 17819-17825Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Of the many anionic lipids, phosphatidic acid (PA) is particularly effective at stabilizing a large proportion of agonist-activatable resting nAChRs (10.Hamouda A.K. Sanghvi M. Sauls D. Machu T.K. Blanton M.P. Biochemistry. 2006; 45: 4327-4337Crossref PubMed Scopus (56) Google Scholar, 12.daCosta C.J. Medaglia S.A. Lavigne N. Wang S. Carswell C.L. Baenziger J.E. J. Biol. Chem. 2009; 284: 33841-33849Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar).Correlations between lipids and nAChR function are based on the assumption that the lipid composition of a reconstituted membrane is defined by the exogenous lipids supplied during affinity purification. Although this assumption holds in most cases, we show here that nAChR-reconstituted PC/PA membranes contain the unexpected lipid diacylglycerol (DAG). DAG appears during affinity purification of the nAChR as a result of the hydrolysis of PA. PA-specific phospholipase C activity co-purifies with the nAChR and is inhibited by vanadate but not N-ethylmaleimide. Also, the presence of DAG in a reconstituted membrane influences the ability of the nAChR to undergo agonist-induced conformational transitions. Our results support the novel hypothesis that nicotinic acetylcholine receptors influence cellular events by mechanisms that do not involve the flux of ions into the cell (13.Kihara T. Shimohama S. Sawada H. Honda K. Nakamizo T. Shibasaki H. Kume T. Akaike A. J. Biol. Chem. 2001; 276: 13541-13546Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 14.de Jonge W.J. Ulloa L. Br. J. Pharmacol. 2007; 151: 915-929Crossref PubMed Scopus (470) Google Scholar, 15.Sharma G. Vijayaraghavan S. J. Neurobiol. 2002; 53: 524-534Crossref PubMed Scopus (146) Google Scholar, 16.Suzuki T. Hide I. Matsubara A. Hama C. Harada K. Miyano K. Andrä M. Matsubayashi H. Sakai N. Kohsaka S. Inoue K. Nakata Y. J. Neurosci. Res. 2006; 83: 1461-1470Crossref PubMed Scopus (195) Google Scholar, 17.Blanchet M.R. Israël-Assayag E. Daleau P. Beaulieu M.J. Cormier Y. Am. J. Physiol. Lung Cell Mol. Physiol. 2006; 291: L757-L763Crossref PubMed Scopus (30) Google Scholar). The nAChR may be able to alter the lipid composition of its surrounding microenvironment in a manner that enhances its own function.

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