ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 194, No. 2, May, pp. 468-480, 1979

Effects TERRENCE Department

of Phospholipase A, on the Binding and Ion Permeability Control Properties of the Acetylcholine Receptor J. ANDREASEN, of Biochemistry Received

DANIEL

and Biophysics, October

R. DOERCE, University

24, 1978; revised

AND MARK

of California,

January

Davis,

C. McNAMEE

Cal$omia

95616

3, 1979

An acidic phospholipase A, (EC 3.1.1.4) isolated from Naja naja siamensis venom blocks acetylcholine receptor function in excitable post synaptic membrane vesicles from ToTedo califomica electroplax. Specifically, the phospholipase acts catalytically to prevent the large increase in sodium efflux induced by carbamylcholine. The efflux inhibition can be correlated with specific hydrolysis of phospholipids in the membrane. During the time course of inhibition, the binding affinity of the receptor for carbamylcholine increases lo-fold, a phenomenon associated with receptor desensitization. Prolonged treatment of the membranes with phospholipase A, causes nonspecific lysis of the vesicles. Incorporation of unsaturated fatty acids or lysophosphatidylcholine into Torpedo membranes also blocks carbamylcholine-induced sodium efflux. The fatty acids have no effect on the binding affinity of the receptor, and lysophosphatidylcholine causes a small decrease in receptor affinity for carbamylcholine. Lysophosphatidylethanolamine and most saturated fatty acids have no direct effect on sodium efflux, but the lysophosphatides cause vesicle lysis. All of the inhibitory effects of the phospholipase and the fatty acids can be reversed and/or prevented by treatment of the vesicles with bovine serum albumin.

The binding of acetylcholine to the acetylcholine receptor (AChR)’ at neuromuscular end plates leads to increased cation permeability at the postsynaptic membrane and, eventually, to muscle contraction (1). The molecular mechanisms underlying the coupling of transmitter binding to permeability increases are imperfectly understood, although considerable progress has been made in purifying and characterizing nicotinic receptors from muscle and from fish electric organs. Several recent reviews provide a complete discussion of the current state of receptor research (2, 3). Most studies indicate that the AChR from

electroplax consists of four kinds of subunits, but the stoichiometry and architecture of these subunits in their quaternary structure are still uncertain. The smallest of the four subunits (M, = 40,000) has been found to contain all of the agonist and antagonist binding capacity of AChR. The roles of the three larger subunit types in AChR function are as yet unknown, but could provide the permeation mechanism (i.e., the ion channel) or a mechanism for coupling the channel to the binding subunits (2). The discovery that functional membrane vesicles enriched in AChR can be prepared from the electric tissue of Torpedo species has made it possible to study the structural and functional properties of the AChR in a membrane environment. The Torpedo membranes respond to receptor activators, such as carbamylcholine, by a selective increase The in Na+, K+, and Ca’+ permeability. increases are blocked by nicotinic receptor antagonists, such as d-tubocurare and snake a-neurotoxins. Prolonged exposure of the

Torpedo califomica

1 Abbreviations used: PLA,, Phospholipase A,; AChR, acetylcholine receptor; MBTA, maleimidobenzyltrimethylammonium iodide; ‘*51-~-Bgt, 1z51-iodinated+bungarotoxin; SDS, sodium dodecyl sulfate; PC, phosphatidylcholine; PE, phosphatidylethanolamine; carb, carbamylcholine chloride; BSA, bovine serum albumin. CM, carboxymethyl; ME, methyl; AC, acetyl; LPC, L-a-lysophosphatidylcholine; EGTA, ethylene glycol bis(&aminoethyl ether)N, N’-tetraacetic acid. 0003-9861/79/060468-13$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

468

PHOSPHOLIPASE

A, AND ACETYLCHOLINE

vesicles to activators also blocks the permeability increases, a reversible phenomenon known as desensitization (2). We recently reported that an acidic phospholipase A, (PLA,) purified from Naja naja siumensis venom could completely block the activator-induced increases in cation efflux from Torpedo californica membrane vesicles (4). The inhibitory activity was clearly associated with the enzymic activity of the phospholipase and did not result from competitive binding to the identified AChR binding sites. Hanley has also reported that several basic phospholipase A,s, including one of the subunits of crotoxin, could block the agonist-induced increases in ion permeability in Torpedo membranes (5). He showed that hydrolyzed lipid extracts from Torpedo could also inhibit the permeability increases. We report and discuss here the results of an investigation into the mechanism by which phospholipase A, affects the binding and ion permeability control properties of the acetylcholine receptor in Torpedo californica membrane vesicles. MATERIALS

AND METHODS

Torpedo membrane vesicles. Membrane vesicles were prepared from Torpedo ca2@-nica electroplax by homogenization and sucrose density gradient centrifugation as previously described (4). Live Torpedo fish were obtained from Pacific Biomarine (Venice, Calif.), and the excised tissue was used immediately or after storage in liquid nitrogen. Typically, 500 g of tissue was used in a preparation, and the final membrane suspension was kept frozen in liquid nitrogen for up to 6 months. Assays for AChR binding. Three different types of assays were used to measure the binding properties of AChR. The MBTA-affinity labeling assay of Karlin el al. (6) was used to determine the number of specific receptor binding sites in the membranes. The [3H]MBTA (maleimidobenzyltri[3H]methyl ammonium iodide) was prepared as described by Karlin using [3H]methyl iodide from New England Nuclear (Boston, Mass.) for the final methylation step (7). The reaction time for the methylation was decreased to 2 h (from 3 days) since in trial runs with unlabeled methyl iodide the reaction appeared to be complete within that time. The specific activity of the final product was approximately 3000 cpmpmol. The binding of a-neurotoxins was used to provide an independent estimate of the total number of receptor binding sites. Both tritiated Naja nuja siamensis a-toxin and iodinated a-bungarotoxin (see

RECEPTOR. FUNCTION

469

below) were used in the gel filtration assay procedure of McNamee et al. (8). The relative affinity of the membrane-bound receptor for carbamylcholine was determined by measuring the effects of carbamylcholine on the rate of binding of lZ51-labeled a-bungarotoxin to receptor (9, 10). Purified a-bungarotoxin was a gift from Michael Hanley (Chemical Biodynamics Lab, University of California, Berkeley, Calif.), and 200 pg was iodinated by the iodine monochloride procedure described by Vogel et al. (ll), and the monoiodo derivative was isolated by ion-exchange chromatography on Whatman CM52 cellulose as described by Lukasiewicz et al. (12). Carrier free sodium [1Z51]iodide (5 mCi) was obtained from New England Nuclear. The specific activity of the mono[L’51]iodo-n-bungarotoxin (L2”I-cuBgt) was 1 x lo5 cpmipmol. The rate of the reaction between ‘s51-cu-Bgt and membrane-bound receptor was measured using DEAEcellulose filters to separate free toxin from toxin-AChR complexes. A typical reaction mixture contained 1 ml of 7.1 nM toxin, 100 mM NaCl, and 10 mM sodium phosphate, at pH 7.4 and 25°C. The reaction was started by addition of 2-10 nM a-toxin sites (final concentration). At various times, IOO-~1 samples were filtered through two Whatman DE-81 filters (24 mm) under low vacuum and then washed with 15 ml of 10 mM sodium phosphate buffer (pH 7.4) containing 0.1% Triton X-100. The filters were air dried and counted in 10 ml of PCS (AmershamiSearle) by liquid scintillation. Rates were calculated from the linear portion of the reaction (first 5 min). Sodium ef$ux assay. The permeability of the vesicles to sodium ions was measured by the “Naefflux Millipore filtration assay as described below. The vesicle dilution buffer contained: 255 mM KCI, 1.5 mM sodium phosphate, 4 mM CaCl,, and 2 mM MgCl, (pH 7.0). Torpedo membrane vesicles were incubated overnight at 4°C in vesicle dilution buffer with 22Na+. Typical incubation mixtures contained 8 mgiml protein and 50 &i/ml 2zNa+. For experiments involving preincubation with phospholipases (or other water soluble agents) lo-20 parts of the “2Na+-loaded vesicle suspension were mixed with 1 part of the phospholipase solution in vesicle dilution buffer. Control suspensions were always, diluted in the same manner. For preincubation with fatty acids, an appropriate amount of the fatty acid in chloroform and/or methanol was dried as a thin film on the bottom of a glass test tube under a stream of nitrogen. The vesicle suspension was then added and mixed gently during the preincubation. At time 0, an aliquot of the vesicle suspension was diluted 50- to lOO-fold with ice-cold vesicle dilution buffer, and l-ml samples were then removed at various times and filtered through HAWP 2400 Millipore filters using a Hoefer filtration manifold. The filters were washed three times with 2.5 ml of ice-cold dilution buffer and then transferred to glass scintillation vials and counted in 10 ml of

470

ANDREASEN,

DOERGE,

PCS. The effects of activators were measured by including carbamylcholine in the dilution buffer. Counts retained on the filters were used as a measure of the zsNa+ trapped within the vesicles. For studies in which the time course of fatty acid-induced inhibition was measured, it was necessary to preincubate the vesicles in separate tubes for each preincubation time point in order to avoid changes in total fatty acid concentration that would occur if aliquots were removed. Phospholipase A,purijication. The procedure below is adapted from the method described by Deems and Dennis (13) for the purification of Naja naja naja phospholipase A,. Lyophilized Nuja naja siamensis venom (200 mg; Miami Serpentarium) was dissolved in 10 ml of water and centrifuged at SOOOg for 10 min at 4°C to remove insoluble material. Four volumes of 6% perchloric acid were added to the clear supernatant at 4°C while stirring, and the precipitate was collected after 1 h by centrifugation at SOOOg for 30 min. The precipitate was dissolved in 5 ml of water and the pH was adjusted to 8.0 with 1 N NaOH. The solution was centrifuged at 80009 for 10 min to remove a small amount of undissolved material. The clear supernatant was passed through a 1.5 x 50-cm column of CM-25 Sephadex equilibrated with 5 mM NaPO, buffer, pH 7.5 at room temperature. The first protein peak eluted from the column was lyophilized and then redissolved in 2 ml of water to give an estimated buffer concentration of 50 mM sodium phosphate. The solution was then fractionated on a 1.5 x 50.cm Sephadex G-100 column equilibrated with 50 mM NaPO,, pH 7.5. One-milliliter fractions were collected and assayed for total protein [A,,, and Lowry (14)], phospholipase A, activity (see below), and polypeptide composition by SDS-urea gel electrophoresis. [Slab gels, 12.5%, in SDS and 8 M urea were prepared, run, and stained according to the general procedures of Ames (15).] Phospholipase A, activity was routinely monitored by the tic procedure described previously (4), using egg phosphatidylcholine as the substrate, except that reaction mixtures were quenched after 1 min by addition of 0.2 M EDTA. The specific activity of the purified enzyme was determined by using the pa-stat assay procedure of Roberts et al. (16); the substrate was 4 mM egg phosphatidylcholine in 40 mM Triton X-100 and 5 mM CaCl,. One unit corresponds to 1 Fmol of fatty acid (H+)liberated/min. In several experiments, two-dimensional tic plates were run to resolve all the major lipid components. Supelco Redi-Coat plates (20 x 20 cm) were used. The solvent systems were: first dimension: CHCl,:MeOH: concentrated NH, (65:35:5); second dimension: CHCl,: HAc:acetone:MeOH:H,O (30:10:40:10:1). Plates were developed with iodine or with Supelco Phospray. Other enzyme assays. Acetylcholinesterase activity was measured by the Ellman procedure (17), and

AND

McNAMEE

Na+-K+ ATPase activity was measured using a coupled enzyme assay procedure with pyruvate kinase and lactate dehydrogenase (18). Miscellaneous. L-a-Lysophosphatidylcholine (LPC) from egg yolk lecithin was obtained from Sigma and contained mainly stearoyl or palmitoyl fatty acid moieties. Bovine lysophosphatidylethanolamine was obtained from Applied Sciences Laboratories. Egg phosphatidylcholine was obtained from Sigma and was further purified by silicic acid gel chromatography in chloroform-methanol solvents (Unisil silicic acid, Clarkson Chemical Co.). P-Bungarotoxin was obtained from Boehringer-Mannheim. Crotalus adamanteus PLA,, Vipera russelli PLA,, and fatty acid-free bovine serum albumin were obtained from Sigma. [“H]Oleic acid [9, 10.“H(N), 5 CiimM] was obtained from New England Nuclear; [Wlstearic acid (carboryl14C, 55.5 nCi/mM) was obtained from International Chem Nuclear. RESULTS

Pur$ication of Phospholipase A2 from ’ Naja naja siamensis Venom

From 200 mg of venom, 10 mg of pure PLA, was obtained. The yield was similar to that reported by Deems and Dennis for the purification of PLA, from Naja nuja naja venom (13). The main protein fraction from the final G-100 Sephadex column was eluted at a volume corresponding to a molecular weight of 12,000. On 12.5% SDSurea polyacrylamide gels, the purified protein stained as a single band corresponding to a molecular weight of 16,000. The specific activity of the purified enzyme was 1340 units/mg at 37°C and pH 8.0 using the pHstat assay procedure of Roberts et al. (14). For the Nuja naja naja enzyme, the highest specific activity reported was 1550 unitslmg, and the molecular weight was 11,000. Using the thin layer chromatography procedure at 25°C with egg phosphatidylcholine as the substrate, the apparent PLA, activity was only 120 units/mg. In the latter assay approximately 50% of the substrate is hydrolyzed, whereas less than 5% of the substrate is hydrolyzed when initial rates are calculated from the pH-stat data. The reaction rate decreases significantly after -10% of the substrate is hydrolyzed in the tic assay. Using ToTedo membrane vesicles as a source of PLA, substrate, the apparent activities were 160 U/mg for phosphatidyl-

PHOSPHOLIPASE

A, AND ACETYLCHOLINE

3ooow

IO Efflux

Time

(mid

FIG. 1. Effect of Naja naja siamensis PLAp on sodium effiux from Z’ol-pedo membrane vesicles. Torpedo membranes preloaded with *2Na+ were treated with 5 Fgiml of PLA, for 10 min at 0°C or untreated. Sodium efflux was then measured in the presence and absence of 5 x 1O-4 M carbamylcholine as described under Materials and Methods. No PLA,, no carb (0); no PLA, +carb (0); +PLA, no carb (A); +PLA, +carb (A).

ethanolamine and 30 U/mg for phosphatidylcholine using the thin layer chromatography assay at 25°C and pH 7.0. The PLA, was also isolated from partially purified N. n. siamensis venom by two other procedures as reported previously (4). All three procedures gave a purified protein with identical catalytic properties, and all three proteins behaved identically on Sephadex G-100 columns and on SDS-urea polyacrylamide gels.

RECEPTOR

sodium ion (22Na+) efflux at 0°C from preloaded vesicles in the presence and absence of carbamylcholine (Fig. 1). Approximately 40% of the sodium ions trapped within the vesicles were released within 1 min when vesicles were diluted into buffer containing 5 x lop4 IM carbamylcholine. Preincubation of the vesicles with saturating amounts of N. n. sZamensis a-neurotoxin prevented the carbamylcholine-stimulated release (4). Preincubation of the 22Na+-loaded vesicles for 10 min at 0°C .with PLA, from Naja naja siamensis (5 pg/ml) resulted in complete inhibition of the carbamylcholineinduced increase in zzNa+ efflux (Fig. 1). After longer preincubation times, there was a slow increase in passive zzNa+ efflux, presumably due to nonspecific vesicle disruption (4). The effectiveness of N. n. siamensis PLA, in blocking the carbamylcholine response was concentration dependent (Table I). However, even at the TABLE INHIBITION

Naja

Typical preparations of Torpedo membrane vesicles contained 700- 1500 pmol toxinbinding sites/mg protein, using either iodinated a-bungarotoxin or tritiated Naja naja siamensis a-neurotoxin for the binding assays. Since purified receptor contains -8000 pmol toxin sites/mg protein, approximately lo-20% of the proteins in the vesicles were receptor molecules. The functional integrity of the vesicles was determined by measuring the rate of

I ssNa+

OF CARBAMYLCHOLINE-INDUCED EFFLUX BY PHOSPHOLIPASE Apn

Source of PLA, naja

siamensis

Amount (k&d)

Inhibition* (%‘o)

0.1

38 48 64

0.3

1.0 4.0

10.0 Crotulus

adamanteus

Vipera

Effects of Phospholipase A, on Sodium EfJux from Torpedo Membrane Vesicles

471

FUNCTION

russelli

Bungarus

multicinctus

100 100

10.0

1.0

9

5.0

87 100

5.0

90

(p-bungarotoxin) n Vesicles preloaded with 22Na+ were preincubated with the indicated final concentrations of PLA, for 10 min at 0°C. Aliquots were then diluted, filtered, washed, and counted as described under Materials and Methods. The carb response is measured as the difference in 22Na+ cpm after 1 min of efflux at 0°C in the presence and absence of 5 x lo-” M carbamylcholine. * The percentage inhibition is defined as: 1_

i

carb response (+PLA) carb response (no PLA) i

x 100.

472

ANDREASEN,

DOERGE,

highest PLA, concentration used (10 pg/ml), there was a greater than lo-fold excess of toxin-binding sites over PLAz molecules. Thus, the PLA, could not be acting directly by competing with carbamylcholine for the ligand-binding sites. By contrast, the a-neurotoxins are fully effective at blocking the carbamylcholine response only when all of the available toxin sites are occupied (4). Many other PLA, enzymes, including those classified as either acidic or basic on the basis of ion-exchange properties, were able to block the carbamylcholine response, although the effectiveness of the enzymes varied. As shown in Table I, the PLAz from Crotalus adamanteus was relatively ineffective at 0°C even at high concentrations. At 25”C, the Crotalus PLA, did inhibit sodium efflux after prolonged incubation. Effect of Phospholipase A2 on LigandBinding Properties of AChR

AND

McNAMEE TABLE

II

EFFECT OF PLAl ON BINDING PROPERTIES OF THE ACETYLCHOLINE RECEPTOR” pm01 sitesimg protein PLA,

h.@nl) 0 1 10

N. n. siamensis [3H]MBTA 368 370 359

o-toxin 779 781 765

carbamylcholine0 5

x 10-G

5

x 10-T

-

L(Torpedo membranes were preincubated for 10 min at 0°C with various concentrations of N. n. siamensis PLA,. Aliquots were then assayed for 13H]MBTA labeling or N. 12.siamensis binding as described under Materials and Methods. DThe apparent dissociation constants for carbamylcholine (K,,,) were estimated for data presented in Fig. 2 according to the procedure of Quast et al. (10).

with PLA, for 10 min at 0°C resulted in a decrease in the rate of 1251-a-Bgt toxin binding to AChR in the presence of carbamylcholine when compared to untreated membranes. The PLA, had no effect on the binding of toxin to AChR in the absence of carbamylcholine. The effect of the PLA, was concentration dependent in the range of 0.1-10 @g/ml (Fig. 2). The effects of PLA, treatment were similar to the effects of preincubation of the membranes with carbamylcholine. It seems likely that the PLA, rapidly caused an increase in receptor affinity for agonists that was similar in nature to agonist-induced desensitization.

Treatment of Torpedo membrane vesicles with N. n. siamensis PLA, (10 and 100 wg/ml) had no effect on the number of toxinbinding sites detected by iodinated cu-bungarotoxin or tritiated N. n. siamensis a-neurotoxin. Similarly, the PLA, had no effect on the number of sites that could be affinity-labeled by [3H]MBTA (Table II). The 2:l ratio of toxin sites to MBTA sites always observed for Torpedo AChR was also not altered by PLA, treatment. The MBTA assay involves a kinetically controlled reaction sequence, and any decrease in the rate of affinity labeling would be readily detected. However, none of the Effect of PLA, on Lipid Hydrolysis above assays would detect increases in the In parallel with the 22Na+ efllux studies, affinity of the receptor for activating ligands. the effects of several PLA,s on lipid hyThere is now substantial evidence that desensitization of receptor function is ac- drolysis were monitored by the tic assay. companied by an increase in the affinity of Figure 3 shows the rate of hydrolysis of the receptor for activating ligands such as PE and PC for two concentrations each of N. n. siamensis PLA, and C. adamanteus carbamylcholine (19). In order to determine if the PLA, was inhibiting efflux by de- PLA,. On Torpedo membranes at O”C, the N. n. siamensis PLA, was clearly more sensitizing the receptor, the kinetic toxinbinding assay of Weiland et al. (9) was active, since both enzymes were equally active against egg phosphatidylcholine in used. The effectiveness of carbamylcholine the tic assay. PE was hydrolyzed more in slowing down the rate of toxin binding increased as the affinity of the receptor for rapidly, and at low enzyme concentration carbamylcholine increased. only PE hydrolysis could be detected. For N. n. siamensis PLA2, complete inhibition Pretreatment of Torpedo membranes

PHOSPHOLIPASE

A, AND ACETYLCHOLINE

RECEPTOR

473

FUNCTION

TIME WIN,

TiME IMIN1

FIG. 3. Phospholipase A, hydrolysis of Torpedo membranes at 0°C was monitored by the thin layer chromatography method described under Materials and Methods. PE, (0); PC, (0).

2. Effect of carbamylcholine and PLA, on the of 1P51-~-Bgt binding to Torpedo AChR. Torpedo membrane vesicles were incubated with 0, 0.1, 1.0, or 10.0 pgiml of PLA, for 10 min at 0°C. Each membrane suspension was then diluted loo-fold in vesicle dilution buffer and the toxin-binding reaction was initiated by addition of aliquots (final concentration of a-toxin binding sites = 2 nM) to a mixture of 7.1 nM *9-cy-Bgt in buffer containing 100 mM NaCl and 10 mM Na-phosphate, (pH 7.4) with and without 5 pM carbamylcholine. The amount of bound toxin was determined at 1-min intervals by the DEAE-filtration procedure described under Materials and Methods. In the figure, solid symbols represent samples with no carbamylcholine and open symbols represent samples in the presence of 5 @M carbamylcholine. Concentrations of PLA,: 0.1 pgiml (+), (0); 1 pgiml, (A), (A); 10 &ml cm), (0); no PLA,, (01, (0). FIG.

Kinetics

of 22Na+ efflux was observed within 10 min at 1 pg/ml. Under these conditions, only PE hydrolysis was evident. For the Crotalus PLA2, lipid hydrolysis can be detected under conditions in which no inhibition takes place. In the absence of calcium ions [Ca”] < lop9 M), lipid hydrolysis was blocked, as expected, and the inhibitory effects of PLA, were also completely blocked (Table III). For the N. n. siamensis PLA2, the appearance of lyso PC, either after long times of incubation or at high PLA, concentrations,

appeared to be correlated with the increase in nonspecific leakiness of the vesicles. In addition to the major phospholipids, PC and PE, the Torpedo membranes also contain sphingomyelin, phosphatidylserine, and phosphatidylinositol. On two-dimensional thin layer chromatograms, it was possible to monitor the fate of the minor lipid components. As expected, the PLA, had no effect on sphingomyelin. The phosphatidylserine and phosphatidylinositol, however, TABLE

III

EFFECTOF Ca'* ON PLA, INHIBITION CARBAMYLCHOLINE-INDUCED **Na+ EFFLUX"

OF

Carb response’ [Ca’+]” (M)

No PLA

4 x 10-Z 1 x 10-e 0

4170 3250 3060

+PLA 0 120 3080

Inhibition (%o) 100 96 0

” Vesicles loaded with 22Na+ were preincubated with PLA, for 10 min at 0°C and diluted, filtered, washed, and counted as described under Materials and Methods. ’ The concentration of free calcium ions was controlled by using EGTA in the incubation and dilution buffers. The solution corresponding to [Ca2+] = 0 actually contained 3 mM EGTA and 1 pM Ca*+. The calculated free concentration of Ca*+ is