Solubilization, partial purification, and reconstitution of glutamate- and N-methyl-D-aspartate-activated cation channels from brain synaptic membranes

Journal Article · Tue Apr 30 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00231a029· OSTI ID:5596697

Ly, A M; Michaelis, E K ^[1]

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1. Univ. of Kansas, Lawrence (USA)

L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. ({sup 14}C)Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L-Glutamate caused an increase in the rate of ({sup 14}C)methylamine influx into liposomes reconstituted with either solubilized membrane proteins or partially purified glutamate-binding proteins. Of the major glutamate receptor agonists, only N-methyl-D-aspartate activated cation fluxes in liposomes reconstituted with glutamate-binding proteins. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D-aspartate- or glutamate-induced influx of NA{sup +} led to a transient increase in the influx of the lipid-permeable anion probe S{sup 14}CN{sup {minus}}. These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the {approximately}69-kDa protein in the function of these ion channels.

• OSTI ID: 5596697
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:17; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALCOHOLS
AMINES
AMINO ACIDS
ANIMALS
ASPARTIC ACID
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOCHEMICAL REACTION KINETICS
BODY
BRAIN
CARBON 14 COMPOUNDS
CARBON COMPOUNDS
CARBOXYLIC ACIDS
CATIONS
CENTRAL NERVOUS SYSTEM
CHARGED PARTICLES
DOSE-RESPONSE RELATIONSHIPS
DRUGS
GLUTAMIC ACID
GLYCEROL
HYDROXY COMPOUNDS
IONS
KINETICS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
METHYLAMINE
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PROTEINS
PURIFICATION
RATS
REACTION KINETICS
RECEPTORS
RODENTS
VERTEBRATES