Bowie, Derek; (1992) Functional properties of vertebrate non-nmda excitatory amino acid receptors expressed in Xenopus laevis oocytes. Doctoral thesis (Ph.D.), University College London.

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Abstract

The Xenopus oocyte expression system was used to express non-N-methyl-D-aspartate (non-NMDA) receptors from mammalian (calf and rat) and avian (chick) brains. In each case, the properties of the expressed receptors were examined using two-electrode current and voltage clamp techniques and compared by examining their pharmacology using dose-response curve analysis (D/R) and constructing current-voltage (l-V) relationships using non-NMDA agonists. Fully-grown immature oocytes (stages V and VI) removed from female Xenopus laevis were microinjected with exogenous messenger ribonucleic acid (mRNA) and after a period of incubation (2-3 days), became responsive to a variety of agonists including central nervous system (CNS) transmitters. Responses evoked by bath application of non-NMDA receptor agonists including kainic acid (KA), domoic acid (Dom) and 5-bromowillardiine (BrW) all produced large, non-desensitizing membrane currents, whereas the responses to amino-3-hydroxy-5-methyl-5-isoxazolepropionic acid (AMPA), quisqualic acid (QA) and L-glutamic acid (L-Glu) were much smaller and showed signs of desensitization. The rank order of agonist-potency determined from D/R curves was QA > AM PA > BrW > KA in all mRNA-injected oocytes. Non-NMDA receptors expressed from each mRNA preparation had a similar sensitivity to KA and conventional non-NMDA receptor antagonists did not differentiate between agonist-evoked responses in oocytes injected with each different mRNA. l-V relationships determined for each agonist were dependent on the mRNA preparation. In oocytes injected with calf or chick brain mRNA, the membrane current evoked by KA (Dom and BrW) exhibited inward rectification at positive (>-i-20mV) membrane potentials. At positive potentials, the degree of rectification observed was dose dependent but agonist-independent. In contrast, in oocytes injected with rat brain mRNA, the response to KA was relatively voltage-independent at negative potentials and exhibited outward rectification at positive potentials. The l-V relationship determined for AMPA (QA and L-Glu) was the same in all mRNA-injected oocytes exhibiting a similar voltage-sensitivity to the KA-response observed in oocytes injected with rat brain mRNA. The estimated reversal potentials (VREV -6mV ± 2mV) for all non-NMDA receptor agonists were quite similar. In all mRNA-injected oocytes, the response to KA (Dom and BrW) was antagonised by other excitatory amino acid (EAA) receptor agonists with the following rank order of inhibition; QA > AMPA > L-Glu > NMDA. In oocytes injected with rat brain mRNA, AMPA antagonised the KA-response in a competitive manner. Moreover, a comparison between the amplitude of KA and AMPA responses in the same oocyte exhibited a direct correlation consistent with both agonists acting on the same receptor-ionophore complex. Despite this, the responses evoked by KA and AMPA could still be pharmacologically separated with thiocyanate (SCN ) ions which selectively antagonized only AMPA responses. In oocytes injected with calf or chick brain mRNA, the response to KA was antagonised by AMPA, QA and L-Glu in an apparently non-competitive manner. In some cases, the response to KA was still antagonised even in the absence of any measurable response to QA. A comparison between the amplitude of KA-responses with AMPA, QA or L-Glu in the same oocyte did not show a direct correlation. It is concluded that different types of non-NMDA receptor, equi-sensitive to KA, were successfully expressed in oocytes from mammalian and avian brain mRNA. In each case, the receptor exhibited similar pharmacological properties to nascent receptors in mammalian and avian neurones: however, novel aspects of receptor function were revealed from l-V relationships and antagonism studies with SCN' ions. The likelihood of non-NMDA receptor heterogeneity is considered.

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