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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，目前进行了两方面的研究：一是利用非洲爪蟾卵母细胞翻译系统的实验，研究分子水平的调节机制；二是利用非洲爪蟾卵母细胞翻译系统进行实验，研究分子水平的调节机制。另一个旨在研究脊髓切片初级传入神经末梢钙通道功能的调节。结果概要如下：（1）卵母细胞系统：我已经阐明G蛋白对N型通道的强直抑制是由Gbetagamma亚基介导的。 Gbetagamma 的抑制作用与蛋白激酶 C 的抑制作用具有竞争性，并且可以通过用含 GTP 的溶液连续进行细胞内灌注来清洗。用激动剂刺激共表达的阿片受体会引起 N 型通道的电压依赖性抑制，如强直抑制所示，然而，在 P/Q 型通道的情况下，阿片类药物的抑制是电压抵抗的。通过将 GTPgammaS 应用于 N 型通道，并通过用含有 GTP 的溶液进行细胞内灌注，可以分离出耐电压抑制。我还通过RT-PCR发现了人类N型通道的两种选择性剪接变体。(2)脊髓切片：通过盲膜片钳技术从带有背角的脊髓切片记录EPSP。诱发反应分为两组：一组由Adelta纤维介导；一组由Adelta纤维介导。另一种是由 C 纤维介导的。 C 纤维输入的重复高频刺激引起脊髓神经元的缓慢去极化。这种缓慢的去极化在突触前被莫阿片、GABA_A、GABA_B或5-HT_ 1A 受体激动剂抑制。我还阐明了 N 型通道参与由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。