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Study on the modulation mechanism of neuronal calcuim channels

神经钙通道调节机制的研究

基本信息

批准号：
09672222
负责人：
KANEKO Shuji
金额：
$ 1.79万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672222/
关键词：
Calcium channel Xenopus oocytes HEK cell opioid receptor spinal dorsal horn GTP-binding protein C fiber TRP channel セロトニン受容体 GABA受容体カルシウムチャネルリン酸化一次感覚神経神経終末

项目摘要

On the role of voltage-dependent calcium channels in the neurons, two lines of studies have been conducted : one was the experiment using Xenopus oocyte translation system in which modulation mechanism at the molecular level is to be studied ; the other was designed to investigate the modulation of calcium channel function at primary afferent nerve terminals in spinal cord slices.The outline of results are as follows :(1) Oocyte system : I have clarified that tonic inhibition of N-type channels by G-proteins is mediated by Gbetagamma subunit. The inhibition by Gbetagamma is competitive to that by protein kinase C, and washable by continuous intracellular perfusion with GTP-containing solution. Stimulation of coexpressed opioid receptors with agonist elicited a voltage-dependent inhibition of N-type channels, as shown in the tonic inhibition, however, the inhibition by opioids were voltage-resistant in the case of P/Q-type channels. The voltage-resistant inhibition could be isolated by applying GTPgammaS to N-type channels, and by intracellular perfusion with GTP-containing solution. I have also found two alternatively splicing variants of human N-type channels by RT-PCR.(2) Spinal slice : EPSPs were recorded from spinal cord slices with dorsal horn attached by blind patch clamp technique. The evoked responses were categorized into two groups : one is mediated by Adelta fibers ; the other is mediated by C fibers. Repetitive high-frequency stimulation of C fiber inputs induced a slow depolarization of spinal neurons. This slow depolarization was presynaptically inhibited either by agonists for m opioid, GABA_A, GABA_B, or 5-HT_<1A> receptors. I have also clarified that N-type channels are involved in the normal excitatory neurotransmission which is mediated by glutamic acid, and that P-type channels are involved in the slow depolarization of spinal neurons.

关于电压依赖性钙通道在神经元中的作用，已开展了两方面的研究：一是利用非洲爪哇卵母细胞翻译系统研究其分子水平的调控机制；二是研究脊髓脑片初级传入神经末梢钙通道功能的调节。结果如下：(1)卵母细胞系统：我已阐明G蛋白对N型通道的紧张性抑制是由Gbetagamma亚基介导的。Gbetagamma的抑制作用与蛋白激酶C竞争，并可通过含GTP的连续细胞内灌流进行清洗。用激动剂刺激共表达的阿片受体，可引起N型通道的电压依赖性抑制，表现为紧张性抑制，而在P/Q型通道中，阿片类药物的抑制是电压抵抗的。通过对N型通道施加GTP-GammaS，并用含GTP的细胞内灌流，可以分离出这种耐电压抑制。通过RT-PCR，我还发现了人类N型通道的两种选择性剪接变体。(2)脊髓切片：用盲膜片钳技术从背角附着的脊髓切片上记录到EPSP。诱发反应分为两组：一组由A-Delta纤维介导，另一组由C纤维介导。重复高频刺激C纤维传入引起脊髓神经元缓慢去极化。这种缓慢的去极化在突触前被M阿片、GABA_A、GABA_B受体激动剂或5-羟色胺受体激动剂抑制。我还阐明了N型通道参与谷氨酸介导的正常兴奋性神经传递，P型通道参与脊髓神经元的缓慢去极化。