博士 / 國立清華大學 / 生命科學系 / 95 / L-glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. Ionotropic glutamate receptors are divided into three subtypes, AMPA-, kainate- and NMDA-preferring receptors. AMPA receptors mediate fast excitatory neurotransmission in the vertebrate central nervous system. Oreochomis mossambicus (tilapia) expresses two duplicated Gria1 genes. All vertebrate AMPA receptor genes are alternatively spliced to create two isoforms, flip and flop. In general, AMPA receptors comprising the flip isoform are more active than those comprising the flop isoform. The agonist-activated activities of Gria1ai, i represents the flip isoform, were only detected in the presence of the desensitization attenuator, cyclothiazide. On the other hand, channel activities of Gria1αo, o represents the flop isoform, could be detected in the absence of cyclothiazide, implying that channel of Gria1αo was more active than that of Gria1ai. The Gria1αi and Gria1αo differed only in the 38-amino acid flip/flop region in front of the last transmembrane region. The flip sequence of tilapia Gria1a differed from that of mammalian Gria1 in four positions, which were individually mutated in this report to study their roles in controlling the tilapia Gria1a channel properties. Electrophysiological analyses showed that I740 residue is responsible for fast desensitization in the Gria1ai. Substitution of a serine at position 750 increased the binding affinity of kainate, and attenuated the desensitization evoked by glutamate in the presence of cyclothiazide. I740, A753 and R776 residues are responsible for the higher ratio of the glutamate-activated currents over the kainate-activated currents in the Gria1αi. The activated currents recorded from Xenopus oocytes coinjected with Gria1αi and Gria2βi were larger than that from oocytes injected with Gria1αi or Gria2βi only. Furthermore, the glutamate-activated currents in the Gria1αi and Gria2βi coexpressed oocytes displayed a linear current-voltage relationship. These results demonstrated that Gria1αi and Gria2βi can form functional heteromeric receptors.
Gria1β C-terminal region can interact with p22 in the yeast two-hybrid system and it coprecipitates with p22 in cells coexpressed both proteins. To study the physiological functions of the interaction between p22 and Gria1β C-terminal region, p22 and chimera 3αi/1βC21, which consisted of the Gria3α backbone with the Gria1β C-terminal region, were coexpressed in Xenopus oocytes. The ratio of glutamate-activated currents over the kainate-activated currents recorded in 3αi/1βC21 decreased by coexpressing p22. Addition of cyclothiazide rescued the p22 effects on 3αi/1βC21, suggesting that p22 was involved in increasing the desensitization of 3αi/1βC21 receptor.
| Identifer | oai:union.ndltd.org:TW/095NTHU5105004 |
| Date | January 2006 |
| Creators | Der-Wang Tzeng, 曾德旺 |
| Contributors | Wei-Yuan Chow, 周姽嫄 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 70 |
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