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

Abstract

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl--dependent, and Cl--independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl--dependent 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 (Vmax of 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 Cl- 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., Ki values of 5.7 .+-. 1.4 .mu.M and 119 .+-. 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl--dependent site was unaffected by AP4 but was blocked by QUIS (Ki=14.2 .+-. 4.8 .mu.M).