Phospholipase C activity affinity purifies with the Torpedo nicotinic acetylcholine receptor

Abstract

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.

FIGURE 1.

Characterization of lipids in reconstituted nAChR membranes. Thin layer chromatography shows that the lipid compositions of most nAChR reconstitutions are as expected based on the lipids supplied during affinity purification of the nAChR, except that some of the PA in PC/PA-nAChR is hydrolyzed to DAG. A, lanes 1, 3, 5, and 7 are lipid standards. Lanes 2, 4, 6, and 8 are lipid extracts from PC-nAChR, PC/PG-nAChR, PC/PS-nAChR, and PC/PA-nAChR. The TLC plate was developed using a chloroform:methanol:formic acid:H₂O (50:37.5:3.5:2, v/v/v/v) solvent system. B, the same as A except that the TLC plate was developed using a less polar solvent system (benzene:2-propanol:ethyl acetate:formic acid (72.5:3.5:22:2)) that resolves neutral lipids while phospholipids remain at the origin. C, lane 2, dipalmitin (a mixture of 1,2-DAG and 1,3-DAG with palmitoyl acyl chains); lane 3, 1-palmitoyl-2-oleoyl-DAG; lane 4, PC/PA-nAChR; lane 5, cholesterol (Chol); lane 6, cholesterol ester, lane 7, free fatty acid (FFA)-oleic acid. The same solvent system as in B was used. D, representative SDS-PAGE from two nAChR reconstitutions. Lane 1, standards; lane 2, PC-nAChR; lane 3, PC/PA-nAChR. The SDS-PAGE samples were from the purifications used for the lipid extractions in A–C.

FIGURE 2.

PA-specific phospholipase activity elutes with the nAChR from the bromoacetylcholine affinity column. A, affinity purification of the nAChR in the presence of PC. B, affinity purification of the nAChR in the presence of PC and PA. Panels i and ii are TLCs developed in chloroform:methanol:formic acid:H₂O (50:37.5:3.5:2, v/v/v/v) of lipid extracts from the control lipid solutions and the detergent-solubilized nAChR fractions eluted from the affinity column. In each case, Control 1 is an extract of the stock lipid A solution. Control 2 is an extract of the same lipid A solution after it has been washed through the affinity column while the nAChR is bound to the column. Fractions 1–9 were collected upon elution of the solubilized nAChR from each affinity column with Carb. Panels i, the lipid extractions were performed immediately after eluting the nAChR from the column. Panels ii, the lipid extractions were performed 1 week later (samples maintained at 4 °C under N₂). Panels iii are SDS-PAGE of each of the collected nAChR fractions. Panels iv, the A₂₈₀ of each of the eluted nAChR fractions. B, panel iv also shows a densitometric analysis of the DAG bands observed on the TLC in panel i of B.

FIGURE 3.

PA-specific phospholipase activity is associated with the nAChR. Affinity purification of the nAChR in the presence of PC and PA either without (A) or with (B) prior treatment of the solubilized nAChR with ∼8 μm α-bungarotoxin, the latter to diminish nAChR binding to the affinity resin. Panels i and ii are TLCs developed in chloroform:methanol:formic acid:H₂O (50:37.5:3.5:2, v/v/v/v) of lipid extracts from the control lipid solutions and the detergent-solubilized nAChR fractions eluted from the affinity column. The control lipids are extracts of the stock lipid A solution, as described for Fig. 2. Fractions 1–6 were collected upon elution of the solubilized nAChR from each affinity column with Carb. Panels i, the lipid extractions were performed immediately after running the column. Panels ii, the lipid extractions were performed 1 week later (samples maintained at 4 °C under N₂). Panels iii, SDS-PAGE of each of the collected nAChR fractions. Panels iv, A₂₈₀ of each of the eluted nAChR fractions.

FIGURE 4.

The effects of N-ethylmaleimide and vanadate on the nAChR-associated phospholipase activity. Affinity purification of the nAChR in the presence PC and PA in the absence of inhibitor (A) or in the presence of 4.2 mm N-ethylmaleimide (B) or 1 mm sodium vanadate (C). Panels i and ii are TLCs developed in chloroform:methanol:formic acid:H₂O (50:37.5:3.5:2, v/v/v/v) of lipid extracts from the control lipid solutions and the detergent-solubilized nAChR fractions eluted from the affinity column. In each case, Control 1 is an extract of the stock lipid A solution. Control 2 is an extract of the same lipid A solution after it has been washed through the affinity column with the column-bound nAChR. Fractions 1–6 were collected upon elution of the solubilized nAChR from each affinity column with Carb. Panels i, lipid extractions performed immediately after running the column. Panels ii, lipid extractions performed 1 week later (samples maintained at 4 °C under N₂). Panels iii are SDS-PAGEs of each of the collected nAChR fractions. Panels iv, the A₂₈₀ of each of the eluted nAChR fractions. Note that sodium vanadate absorbs at 280 nm, making the absolute A₂₈₀ readings somewhat unreliable.

FIGURE 5.

DAG influences the ability of the nAChR to undergo Carb-induced conformational transitions. The fluorescence emission of ethidium at 590 nm was monitored in the presence of the nAChR reconstituted into 3:2 PC/PA (trace i), PC (trace ii), 3:1 PC/DAG (trace iii), 3:2 PC/PS (trace iv), and 3:1:1 PC/PS/DAG membranes (trace v). A, at the indicated times ∼50 nm nAChR, 500 μm Carb, and 500 μm dibucaine (Dib) were added to a 0.3 μm ethidium solution. The sharp spikes in each fluorescence emission trace reflect the scattering of light upon insertion of the pipette into the cuvette. Note the large increase in fluorescence upon addition of 3:1 PC/DAG to ethidium is due primarily to vesicle scattering. B, dibucaine-displaceable ethidium fluorescence emission intensity at 590 nm in the presence (+) or absence (−) of Carb. The error bars are the standard errors.