Specific binding of labeled spider toxin to the rat brain

$139 SPECIFIC BINDIN G OF LABELED SPIDER TOXI~ T~ THE RAT BRAIN. KUNIKO SBIMAZAKII~ NOBUFUMI KAWAI~ t AND TERUMI NAKAJIMA*~ r ±Department of N e u r o b i o l o g y , Tokyo e t r o p o l i t a n Institute for N e u r o s c i e n c e s w 2-6 Musashidai, Fuchu t T o k y o 183 and Department of A n a l y t i c a l C h e m i s t r y t F a c u l t y of P h a r m a c e u t i c a l Sciences r U n i v e r s i t y of T o k y o r 113, Japan. W e s t u d i e d t h e s p e c i f i c b i n d i n g o f a s p i d e r t o x i n , J S T X , a b l o c k e r of g l u t a m a t e receptors, to rat brain. S y n t h e s i z e d Joro spider t o x i n (JSTX-3) was c o n j u g a t e d w i t h b i o t i n w h i l e p r e s e r v i n g its t o x i c i t y . B i o t i n y l a t e d JSTX-3 b~nding sites were characterized a n d m a p p e d in W i s t a r r a t b r a i n . B i n d i n g of [JH]biotinylated JSTX-3 on a hippocampal membrane fraction {P2) has a d i s s o c i a t i o n c o n s t a n t of a p p r o x i m a t e l y 1 0 ~ M and a B~ax of 360 p m o l l m g protein. [ ~ H ] b i o t i n y l a t e d JSTX-3 b i n d i n g to the P2 fraction was 87% inhibited by Im M J S T X . In c o n t r a s t , there was no significant inhibition by Ash-Cad-Put, a s t r u c t u r a l l y r e l a t e d analou~ of JSTX-3 w h i c h is devoid of b l o c k i n g a c t i v i t y for g l u t a m a t e receptors. The |JB]biotinyl JSTX b i n d i n g was inhibited by glutamate, kainate and quisgualate, though much less than that caused by JSTX-3. Histochemical study using avidin-biotin-complex r e v e a l e d s t r o n g b i n d i n g of b i o t i n y l JSTX to the h i p p o c a m p u s and cerebellum, regions of d e n s e glutamatergic innervation. The CA3 region of the h i p p o c a m p u s was m o r e h e a v i l y stained than the CAI r e g i o n . In c e r e b e l l u m , b i n d i n g s i t e s w e r e c o n c e n t r a t e d specifically in Purkinje cells and dendrites where parallel fibers terminate. These results suggest that JSTX binds to n o n - N M D A type receptors in m a m m a l i a n brain.