Purification of AMPA type glutamate receptor by a spider toxin.
A glutamate receptor was purified from Triton X-100-solubilized bovine cerebellum membranes. The purification was carried out in two steps: affinity chromatography using a spider toxin (Joro spider toxin; JSTX) immobilized on a lysine-agarose column, and a Mono Q anion exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified active fraction showed a single band with Coomassie Blue staining, which migrated with a M(r) = 130,000. The specific [3H]amino-3-hydroxy-5-methyl-isoxazole propionate ([3H]AMPA) binding activity of the affinity-purified fraction was 2095-fold higher than that of the crude soluble fraction. Lineweaver-Burk plot analysis showed a Kd of 12.7 nM [3H]AMPA in the purified fraction. The purified fraction was examined with patch-clamp recording methods in reconstituted liposomes. A glutamate-activated channel was observed and was inhibited with JSTX. The rank order of potency of agonists inducing channel currents was AMPA = glutamate greater than quisqualate much greater than kainate greater than NMDA. Thus, there is strong evidence that the 130 kDa protein is a purified component of the native AMPA type glutamate channel of bovine cerebellum.
Shimazaki
K
K
Department of Physiology, Jichi Medical School, Tochigi-ken, Japan.
Robinson
H P
HP
Nakajima
T
T
Kawai
N
N
Takenawa
T
T
D000818:Animals; D002417:Cattle; D002531:Cerebellum / Q000737:chemistry; D018118:Chloride Channels; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid; D007051:Ibotenic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D007608:Kainic Acid / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000187:drug effects; D016202:N-Methylaspartate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism; D016318:Quisqualic Acid / Q000494:pharmacology; D018091:Receptors, AMPA; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D013111:Spider Venoms; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
D018118:Chloride Channels; D005971:Glutamates; C044936:JSTX spider toxin; D008081:Liposomes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018091:Receptors, AMPA; D017981:Receptors, Neurotransmitter; D013111:Spider Venoms; D007051:Ibotenic Acid; D018698:Glutamic Acid; D016202:N-Methylaspartate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D016318:Quisqualic Acid; D007608:Kainic Acid