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The properties and actions of a spider toxin (JSTX) on the glutamate synapse
S103
20 Neurotransmitters I Amino acids and others The PROPERTIES AND GLUTAMATE SYNAPSE.
ACTIONS
OF
A
SPIDER
TOXIN
(JSTX)
ON
THE
AKIKO MIWA*, NOBUFUMI KAWAI, MITSUYOSHI SAITO*, HIDEMITSU PAN-HOU*, MASANORI YOSHIOKA*, Dept. Neurobiol., Tokyo Metrop o l i t a n Inst. for N e u r o s c i . , 2-6 M u s a s h i d a i , Fuchu-city, Tokyo, and Fac. Pharmac. Sci., Setsunan Univ., 45-1, Nagaotoge, Hirakata-city, Osaka, 573-01, JAPAN T h e e f f e c t s o f J S T X - - a s p e c i f i c b l o c k e r of the g l u t a m a t e receptor ~ were studied using lobster neuromuscular synapses. J S T X w a s p u r i f i e d b y the p r o c e d u r e s of gel f i l t r a t i o n , and i o n exchange column chromatography followed by thin layer chromatography. The molecular w e i g h t of the t o x i n w a s ca. 500. Under further purification, JSTX was electrically neutral at
pH 7. The action of p u r i f i e d toxin on extracellularly recorded excitatory postsynapitc potential (EEPSP) was investigated at different membrane potentials. The amplitude of EEPSP was i n c r e a s e d l i n e a l l y by h y p e r p o l a r i z i n g the p o s t s y n a p t i c membrane, both in n o r m a l and in J S T X treated muscle, showing that the e f f e c t of the t o x i n w a s n o t v o l t a g e d e p e n d e n t . The time constant of t h e d e c a y p h a s e of E E P S P in p o i s o n e d m u s c l e w a s s h o r t e n e d b y membrane hyperpolarization in a s i m i l a r w a y to the n o r m a l m u s c l e . T h e r e s u l t s s u g g e s t t h a t J S T X i r r e v e r s i b l y b i n d s to t h e s i t e which is independent of the electric gate in the g l u t a m a t e receptor-ion channel complex.
EFFECTS OF HIGH-AFFINITY UPTAKE INHIBITORS ON DEPOLARIZATIONS INDUCED BY EXCITATORY AMINO ACIDS IN HIPPOCAMPAL NEURONS SATSUKI SAWADA*, MASATO HIGASHIMA* and CHOSABURO YAMAMOTO, Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa 920, Japan. The effects of dihydrokainate (DHKA) and 3-hydroxy-DL-aspartate (Hasp), inhibitors of high-affinity uptake for L-glutamate (Glu), were studied in vitro in thin transverse sections of the guinea pig hippocampus. The amplitudes of depolarizations induced by Glu and by L-aspartate (Asp) in CA3 neurons were markedly augmented by DHKA and Hasp administered iontophoretically, or by pressure. Depolarizations induced by D-homocysteate (DM) were unaffected by the inhibitors. Responses to L-homocysteate (LH) and to quisqualate (Quis) were slightly augmented by the inhibitors in about half of the neurons examined. Fast responses to kainate (KA) were augmented by the inhibitors, to a similar extent as were the Glu responses. Hasp was without effect on excitatory postsynaptic potentials elicited by stimulation of mossy fibers. These findings are in general agreement with the biochemical observations on amino acid uptake processes and are also consistent with the slow time-courses of depolarizations induced by DH, LH and Quis. Augmentation of the fast KA responses provides strong evidence for the hypothesis that a KA pulse causes a liberation of Glu and/or Asp from the tissue, and thereby induces fast KA responses in neurons nearby.
20 Neurotransmitters I Amino acids and others The PROPERTIES AND GLUTAMATE SYNAPSE.
ACTIONS
OF
A
SPIDER
TOXIN
(JSTX)
ON
THE
AKIKO MIWA*, NOBUFUMI KAWAI, MITSUYOSHI SAITO*, HIDEMITSU PAN-HOU*, MASANORI YOSHIOKA*, Dept. Neurobiol., Tokyo Metrop o l i t a n Inst. for N e u r o s c i . , 2-6 M u s a s h i d a i , Fuchu-city, Tokyo, and Fac. Pharmac. Sci., Setsunan Univ., 45-1, Nagaotoge, Hirakata-city, Osaka, 573-01, JAPAN T h e e f f e c t s o f J S T X - - a s p e c i f i c b l o c k e r of the g l u t a m a t e receptor ~ were studied using lobster neuromuscular synapses. J S T X w a s p u r i f i e d b y the p r o c e d u r e s of gel f i l t r a t i o n , and i o n exchange column chromatography followed by thin layer chromatography. The molecular w e i g h t of the t o x i n w a s ca. 500. Under further purification, JSTX was electrically neutral at
pH 7. The action of p u r i f i e d toxin on extracellularly recorded excitatory postsynapitc potential (EEPSP) was investigated at different membrane potentials. The amplitude of EEPSP was i n c r e a s e d l i n e a l l y by h y p e r p o l a r i z i n g the p o s t s y n a p t i c membrane, both in n o r m a l and in J S T X treated muscle, showing that the e f f e c t of the t o x i n w a s n o t v o l t a g e d e p e n d e n t . The time constant of t h e d e c a y p h a s e of E E P S P in p o i s o n e d m u s c l e w a s s h o r t e n e d b y membrane hyperpolarization in a s i m i l a r w a y to the n o r m a l m u s c l e . T h e r e s u l t s s u g g e s t t h a t J S T X i r r e v e r s i b l y b i n d s to t h e s i t e which is independent of the electric gate in the g l u t a m a t e receptor-ion channel complex.
EFFECTS OF HIGH-AFFINITY UPTAKE INHIBITORS ON DEPOLARIZATIONS INDUCED BY EXCITATORY AMINO ACIDS IN HIPPOCAMPAL NEURONS SATSUKI SAWADA*, MASATO HIGASHIMA* and CHOSABURO YAMAMOTO, Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa 920, Japan. The effects of dihydrokainate (DHKA) and 3-hydroxy-DL-aspartate (Hasp), inhibitors of high-affinity uptake for L-glutamate (Glu), were studied in vitro in thin transverse sections of the guinea pig hippocampus. The amplitudes of depolarizations induced by Glu and by L-aspartate (Asp) in CA3 neurons were markedly augmented by DHKA and Hasp administered iontophoretically, or by pressure. Depolarizations induced by D-homocysteate (DM) were unaffected by the inhibitors. Responses to L-homocysteate (LH) and to quisqualate (Quis) were slightly augmented by the inhibitors in about half of the neurons examined. Fast responses to kainate (KA) were augmented by the inhibitors, to a similar extent as were the Glu responses. Hasp was without effect on excitatory postsynaptic potentials elicited by stimulation of mossy fibers. These findings are in general agreement with the biochemical observations on amino acid uptake processes and are also consistent with the slow time-courses of depolarizations induced by DH, LH and Quis. Augmentation of the fast KA responses provides strong evidence for the hypothesis that a KA pulse causes a liberation of Glu and/or Asp from the tissue, and thereby induces fast KA responses in neurons nearby.