Characterization of L‐Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion‐Dependent Sites

Abstract

AbstractThe effects of various ions on L‐glutamate (L‐Glu) binding sites (Na+‐dependent, Cl−‐dependent, and CP−‐independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. CP−‐de‐pendent binding sites were over twofold higher in spinal cord (Bmax= 152 ± 34 pmol/mg protein) as compared to forebrain SPM (Bmax=64 ± 12 pmol/mg protein). Na+dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax= 74 ± 10 pmol/mg protein) compared to forebrain SPM (408 ± 26 pmol/mg protein). Uptake of L‐Glu (Na+‐dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmaxof 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+on L‐Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br−, I−, and NO3−were equivalent to C−in their capacity to stimulate L‐Glu binding, whereas F−and CO32−were less effective. Cl−‐dependent l‐Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl−‐dependent binding site in forebrain membranes [i.e., Kivalues of 5.7 ± 1.4μMand 119 ± 38 nMfor 2‐amino‐4‐phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other CP‐dependent site was unaffected by AP4 but was blocked by QUIS (Ki= 14.2 ± 4.8μM).