Biochemistry of the locust excitatory neuromuscular synapse

• Main Author: James, Richard W.
• Published: University of Bath 1977
• Subjects: 573.8
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.460669

Some of the parameters of the process of chemical neuro- transmission at the locust excitatory neuromuscular synapse have been investigated. This has involved the application of two distinct procedures for purifying membrane proteins which exhibit pharmacological properties consistent with a neuroreceptor function. The first technique used chloroform/methanol mixtures to extract protein/lipid complexes (proteolipids) from the muscle. The second procedure adapted conventional subcellular fractionation techniques to yield a membrane fraction in an aqueous environment. Distinct high and low affinity binding sites for L-glutamate were detected in preparations obtained using both techniques. The chloroform/methanol extract was resolved by a chromatographic procedure and was shown to contain proteolipids which bound L-glutamate with high affinity (Kd = 8 muM) and low affinity (Kd = 50 muM) respectively. Competition studies with the high affinity site showed that binding of L-glutamate could be inhibited by L-aspartate and 2-amino-4-phosphonobutyric acid, whilst D-glutamate was without effect. The proteolipids which bound L-glutamate were further characterised by analysis of their associated phospholipids and unesterified fatty acids. The aqueous membrane preparation exhibited two L-glutamate- binding populations which could be defined by their sensitivities to sodium ions - a sodium-ion insensitive high affinity site (Kd = 0.53 muM) and a sodium-ion sensitive low affinity site (Kd = 21.6 muM). Binding of L-glutamate to the low affinity site was susceptible to freezing, sonicating and osmotically-shocking the membrane preparation. Binding of L-glutamate to the high affinity site was unaffected by such treatments. Competition studies indicated that only chlorpromazine, L-glutamyl-beta-hydroxamate and ouabain inhibited binding of L-glutamate at the low affinity site. These ligands were without effect at the high affinity site, where a wide range of ligands inhibited binding of L-glutamate, Kainie acid, quisqualic acid, hrmine hydrochloride and D,L-2-amino-4-phosphonobutyric acid were particularly potent inhibitors. L-aspartate was a potent inhibitor of binding of L-glutamate to both sites. It is concluded that the high affinity sites of both preparations are representative of neuroreceptor proteins for L-glutamate and as such supplement the larger body of electrophysiological evidence that supports the suggestion that transmission at the locust excitatory synapse is mediated by the amino acid. It is suggested that the low affinity sites represent uptake sites. They may function to remove neurally-released transmitter (L-glutamate) from the region of the synapse.