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，已经进行了两条研究：一种是使用爪蟾卵母细胞翻译系统的实验，其中可以研究分子水平的调节机制；另一个旨在研究脊髓切片中原发性传入神经末端对钙通道功能的调节。结果的轮廓如下：（1）卵母细胞系统：我已经澄清了Gbetagagamma subunit介导的G蛋白对N型通道的滋补抑制作用。 Gbetagamma的抑制作用与蛋白激酶C的竞争能力，可以通过含GTP溶液的连续细胞内灌注清洗。用激动剂刺激共表达的阿片类受体会引起电压依赖性抑制N型通道的抑制作用，如补品抑制所示，在P/Q-Type通道的情况下，阿片类药物的抑制是抗电压的。可以通过将GTPGAMMAS施加到N型通道，并使用含GTP的溶液来分离抗电压抑制。我还发现了通过RT-PCR的两个人类N型通道的剪接变体。（2）脊柱切片：EPSP是从脊髓切片中记录的，并通过盲贴夹技术连接背侧角。诱发的反应分为两组：一组是由阿德尔塔纤维介导的；另一个是由C纤维介导的。 C纤维输入的重复高频刺激会引起脊柱神经元的缓慢去极化。这种缓慢的去极化被激动剂前突触前抑制了M opioid，GABA_A，GABA_B或5-HT_ <1A>受体。我还阐明了N型通道参与正常兴奋性神经传递，该神经传递是由谷氨酸介导的，并且P型通道参与脊柱神经元缓慢的去极化。