Regulation of NMDA receptor properties by amino acids and cations : A biochemical and electrophysiological study

NMDA receptors are glutamate-regulated ion channels that arepermeable to Ca2+, Na+ and K+ andare sensitive to voltage-dependent Mg2+-block. Toinvestigate the modulatory effects of amino acids and cations on NMDAreceptors and the possible regional differences in the pharmacologyof NMDA receptors, I used mainly two techniques in this thesis:electrophysiological analysis of expressed NMDA receptors in Xenopusoocytes and biochemical receptor binding using [3H]MK-801in membrane preparations from the rat cerebral cortex and spinalcord. [3H]MK-801 binds selectively to the inside of thechannel pore in an agonist-dependent fashion. The results show that: 1. In the presence of Mg2+ or in Krebs buffer, glutamate and glycine decreased the affinity of[3H]MK-801 binding, which may correspond to a decrease in the affinity of the Mg2+block. In fact, the affinity of the Mg2+-block was reduced by glutamate and glycine inNMDA receptors expressed in Xenopus oocytes, and this affinity decrease was modulatedby H+ and Ca2+. 2. Low concentrations of Tris, K+, Na+, Mg2+ and Ca2+ increased the association rate of [3H]MK-801 binding observed as in increased [3H]MK-801 binding under non-equilibriumconditions. High concentrations of these ions inhibited [3H]MK-801 binding. Furthermore, high concentrations of Mg2+, Na+ and Tris permitted glutamate and glycine to decrease [3H]MK-801 binding. In contrast, Ca2+ antagonized the glutamate- and glycine-induced decrease in [3H]MK-801 binding observed in the presence of Mg2+. 3. H+ decreased the association rate of [3H]MK-801 binding observed as a decreased [3H]MK-801 binding under non-equilibrium conditions. Also, H+ antagonized the glutamate- and glycine-induced decrease in [3H]MK-801 binding observed in the presence of Mg2+. In addition, H+ increases the desensitization of NMDA receptors expressed in Xenopus oocytes. 4. NMDA receptors in the spinal cord, as compared with those in the cerebral cortex, display low affinity for MK-801 and for inhibition by cations but high sensitivity to glycine and to glutamate and glycine antagonists. Taken together, these results suggest that glutamate and glycineregulate the affinity of the Mg2+-block and that thiseffect is modulated by cations. Furthermore, there arepharmacological differences between NMDA receptors in the cerebralcortex and spinal cord. These findings may be useful for developingnovel drugs that modulate NMDA receptor function in certainphysiological and pathological processes, such as synaptic plasticityand learning, ischaemia and epilepsy. Key words: NMDA receptor, Glutamate, Glycine,[3H]MK-801 binding, Magnesium, Calcium, Tris, Potassium,Sodium, Proton, Xenopus oocytes, Spinal cord, Cerebral cortex,Receptor binding, Electrophysiology. ISBN 91-628-2615-