BEHAVIORAL AND NEURAL BIOLOGY 5 5 , 1 3 7 - 1 4 0
(1991)
RAPID COMMUNICATION Phosphatidylserine Increases the Affinity of the AMPA/Quisqualate Receptor in Rat Brain Membranes MICHEL BAUDRY, G u Y MASSICOTTE, 1 AND STEPHANIE HAUGE 2
Program in Neural, Informational, and Behavioral Sciences, University of Southern California, Los Angeles, California 90089-2520 We investigated the effects of phospholipids and cholesterol on the binding of [3H]-AMPA to rat telencephalic membranes. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cholesterol were without effect at concentrations up to 1.5 mg/mg protein. Only phosphatidylserine increased [3H]-AMPA binding in a dose-dependent manner. This effect was due to an increase in the affinity of the low affinity component of [3H]-AMPA binding. These results indicate that the distribution of phosphatidylserine in membranes modulates the properties of the AMPA/quisqualate receptor. © 199t AcademicPress, Inc.
At least two classes of glutamate receptors participate in synaptic transmission at excitatory synapses, and they have been defined on the basis of agonist preference as the N-methyl-o-aspartate (NMDA) and the AMPA (a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)/quisqualate (A/Q) receptors. While the A / Q receptor is involved in producing fast epsps, the NMDA receptor has been shown to trigger various forms of plasticity and to participate in some forms of neuronal degeneration (Cotman, Bridges, Taube, Clark, Geddes, & Monaghan, 1989). One type of synaptic plasticity that has been extensively studied is the long-term potentiation (LTP) of synaptic transmission that occurs in hippocampus and other telencephalic structures after brief bursts of electrical stimulation (Lynch & Baudry, 1987). LTP is triggered by the activation of NMDA receptors but is maintained through a modification of the responses elicited Present address: Department of Biological Chemistry, University of Quebec, Trois Rivirres, Quebec, Canada. 2 This work was supported by NSF Grant BNS 96284 to Michel Baudry and the "Fonds de la Recherche en Sante du Quebec" (Guy Massicotte). The authors thank Shelli Sedlak for her excellent secretarial assistance. Please address all correspondence and reprint requests to Michel Baudry at HNB 311, University of Southern California, University Park Campus, Los Angeles, CA 90089-2520. 137 0163-1047/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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by the A / Q receptors (Muller, Joly, & Lynch, 1988). We have recently reported that treatment of telencephalic membranes with phospholipase C or A2 produces an increased affinity of the A / Q receptor for their agonists (Massicotte & Baudry, in press). Our results also indicated that this effect was likely due to an interaction between the lipid microenvironment of the receptor and the receptor configuration. We now report the effects of different phospholipids and cholesterol on the binding of [3H]-AMPA, an agonist of the A / Q receptor, to rat telencephalic membranes. Only phosphatidylserine produced an increased [3H]-AMPA binding which was due to an increased affinity to the A / Q receptors. Experiments were performed on telencephalic membranes from adult male Sprague-Dawley rats (200-250 g) prepared as previously described (Massicotte & Baudry, in press). Membranes were pretreated with Triton X-100 (0.2%) in order to eliminate transport mechanisms and endogenous glutamate, and kept frozen at -70°C until the day of the experiment, when they were thawed at room temperature. Different amounts of phospholipids or cholesterol (Sigma, MO) in chloroform solution were evaporated to dryness in a speed vac centrifuge and membrane suspensions were added to the dry residues. The tubes were sonicated for 10 s and the suspensions were incubated at 35°C for 30 min. Membranes were washed by centrifugation and the final pellets were resuspended in 100 mM Tris/acetate, pH 7.4, containing 50/zM EGTA. [3H]-AMPA (NEN, sp act 29.3 Ci/mmole) binding was performed as previously described (Massicotte & Baudry, in press) with the nonspecific binding defined as the amount of binding measured in the presence of 0.2 mM quisqualate. Proteins were determined with the Bio-Rad reagent using ~/-globulin as a standard. The effects of different concentrations of the various classes of phospholipids and cholesterol on [3H]-AMPA binding to rat telencephalic membranes are shown in Fig. 1. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cholesterol did not modify [3H]-AMPA binding at concentrations up to 1.5 mg/mg protein. Only phosphatidylserine (PS) increased [3H]-AMPA binding in a dose-dependent manner. The effect was maximal at a concentration of about 1 mg/mg protein and it represented about a 75% increase. To determine whether the effect of PS on [3H]-AMPA binding was due to a change in Bma~ or in Kd, equilibrium kinetics of [3H]-AMPA were performed in control membranes and in membranes treated with 1.5 mg/mg protein PS. Control membranes exhibited both high and low affinity sites for [3H]-AMPA with a Kd of 10 ----- 2 and 940 ___ 80 nM, and a Bm~ of 0.35 _ 0.10 and 23.0 +__ 2.2 pmol/mg protein, respectively. Membranes treated with PS also exhibited high and low affinity sites with a Kd of 6 __- 2 and 430* _+ 40 nM and a Bm~x of 0.25 --- 0.08 and 20.6 --- 1.6 pmol/mg protein (means _ SEM of three experiments; *p < .01, Student's t test). Thus PS treatment
EFFECT OF PHOSPHATIDYLSERINE ON AMPA RECEPTORS
139
200 0
XT 175
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.
(1991)
RAPID COMMUNICATION Phosphatidylserine Increases the Affinity of the AMPA/Quisqualate Receptor in Rat Brain Membranes MICHEL BAUDRY, G u Y MASSICOTTE, 1 AND STEPHANIE HAUGE 2
Program in Neural, Informational, and Behavioral Sciences, University of Southern California, Los Angeles, California 90089-2520 We investigated the effects of phospholipids and cholesterol on the binding of [3H]-AMPA to rat telencephalic membranes. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cholesterol were without effect at concentrations up to 1.5 mg/mg protein. Only phosphatidylserine increased [3H]-AMPA binding in a dose-dependent manner. This effect was due to an increase in the affinity of the low affinity component of [3H]-AMPA binding. These results indicate that the distribution of phosphatidylserine in membranes modulates the properties of the AMPA/quisqualate receptor. © 199t AcademicPress, Inc.
At least two classes of glutamate receptors participate in synaptic transmission at excitatory synapses, and they have been defined on the basis of agonist preference as the N-methyl-o-aspartate (NMDA) and the AMPA (a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)/quisqualate (A/Q) receptors. While the A / Q receptor is involved in producing fast epsps, the NMDA receptor has been shown to trigger various forms of plasticity and to participate in some forms of neuronal degeneration (Cotman, Bridges, Taube, Clark, Geddes, & Monaghan, 1989). One type of synaptic plasticity that has been extensively studied is the long-term potentiation (LTP) of synaptic transmission that occurs in hippocampus and other telencephalic structures after brief bursts of electrical stimulation (Lynch & Baudry, 1987). LTP is triggered by the activation of NMDA receptors but is maintained through a modification of the responses elicited Present address: Department of Biological Chemistry, University of Quebec, Trois Rivirres, Quebec, Canada. 2 This work was supported by NSF Grant BNS 96284 to Michel Baudry and the "Fonds de la Recherche en Sante du Quebec" (Guy Massicotte). The authors thank Shelli Sedlak for her excellent secretarial assistance. Please address all correspondence and reprint requests to Michel Baudry at HNB 311, University of Southern California, University Park Campus, Los Angeles, CA 90089-2520. 137 0163-1047/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
138
BAUDRY, MASSICOTTE, AND HAUGE
by the A / Q receptors (Muller, Joly, & Lynch, 1988). We have recently reported that treatment of telencephalic membranes with phospholipase C or A2 produces an increased affinity of the A / Q receptor for their agonists (Massicotte & Baudry, in press). Our results also indicated that this effect was likely due to an interaction between the lipid microenvironment of the receptor and the receptor configuration. We now report the effects of different phospholipids and cholesterol on the binding of [3H]-AMPA, an agonist of the A / Q receptor, to rat telencephalic membranes. Only phosphatidylserine produced an increased [3H]-AMPA binding which was due to an increased affinity to the A / Q receptors. Experiments were performed on telencephalic membranes from adult male Sprague-Dawley rats (200-250 g) prepared as previously described (Massicotte & Baudry, in press). Membranes were pretreated with Triton X-100 (0.2%) in order to eliminate transport mechanisms and endogenous glutamate, and kept frozen at -70°C until the day of the experiment, when they were thawed at room temperature. Different amounts of phospholipids or cholesterol (Sigma, MO) in chloroform solution were evaporated to dryness in a speed vac centrifuge and membrane suspensions were added to the dry residues. The tubes were sonicated for 10 s and the suspensions were incubated at 35°C for 30 min. Membranes were washed by centrifugation and the final pellets were resuspended in 100 mM Tris/acetate, pH 7.4, containing 50/zM EGTA. [3H]-AMPA (NEN, sp act 29.3 Ci/mmole) binding was performed as previously described (Massicotte & Baudry, in press) with the nonspecific binding defined as the amount of binding measured in the presence of 0.2 mM quisqualate. Proteins were determined with the Bio-Rad reagent using ~/-globulin as a standard. The effects of different concentrations of the various classes of phospholipids and cholesterol on [3H]-AMPA binding to rat telencephalic membranes are shown in Fig. 1. Phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and cholesterol did not modify [3H]-AMPA binding at concentrations up to 1.5 mg/mg protein. Only phosphatidylserine (PS) increased [3H]-AMPA binding in a dose-dependent manner. The effect was maximal at a concentration of about 1 mg/mg protein and it represented about a 75% increase. To determine whether the effect of PS on [3H]-AMPA binding was due to a change in Bma~ or in Kd, equilibrium kinetics of [3H]-AMPA were performed in control membranes and in membranes treated with 1.5 mg/mg protein PS. Control membranes exhibited both high and low affinity sites for [3H]-AMPA with a Kd of 10 ----- 2 and 940 ___ 80 nM, and a Bm~ of 0.35 _ 0.10 and 23.0 +__ 2.2 pmol/mg protein, respectively. Membranes treated with PS also exhibited high and low affinity sites with a Kd of 6 __- 2 and 430* _+ 40 nM and a Bm~x of 0.25 --- 0.08 and 20.6 --- 1.6 pmol/mg protein (means _ SEM of three experiments; *p < .01, Student's t test). Thus PS treatment
EFFECT OF PHOSPHATIDYLSERINE ON AMPA RECEPTORS
139
200 0
XT 175
rO
150
m
.
