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

Sammanfattning: NMDA receptors are glutamate-regulated ion channels that are permeable to Ca2+, Na+ and K+ and are sensitive to voltage-dependent Mg2+-block. To investigate the modulatory effects of amino acids and cations on NMDA receptors and the possible regional differences in the pharmacology of 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-801 in membrane preparations from the rat cerebral cortex and spinal cord. [3H]MK-801 binds selectively to the inside of the channel 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 in NMDA receptors expressed in Xenopus oocytes, and this affinity decrease was modulated by 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-equilibrium conditions. 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 glycine regulate the affinity of the Mg2+-block and that this effect is modulated by cations. Furthermore, there are pharmacological differences between NMDA receptors in the cerebral cortex and spinal cord. These findings may be useful for developing novel drugs that modulate NMDA receptor function in certain physiological and pathological processes, such as synaptic plasticity and learning, ischaemia and epilepsy.