Molecular elucidation of receptor-operated Ca^<2+> permeable cation channels with TRP as candidate proteins

以 TRP 作为候选蛋白对受体操纵的 Ca^2 > 渗透性阳离子通道进行分子阐明

基本信息

批准号：
12670088
负责人：
INOUE Ryuji
金额：
$ 2.3万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Stimulation of G-protein coupled or tyrosine kinase receptors often leads to activation of Ca2+-permeable cation channels (ROCCs) which seem to participate in a wide variety of living cells by evoking Ca2+ entry from the extracellular space in direct and indirect ways. Although the molecular identification of ROCCs remains entirely elusive, recent progress in understanding the Drosophila's visual signal transduction has revealed that the vertebrate homologues of transient receptor potential protein (TRPs) are promising candidates for these channels. Based on this knowledge, we have launched on correlating native ROCCs with distinct TRP isoforms at molecular level using the following approach; (1) detailed comparison of cation currents due to native ROCCs and recombinantly expressed TRPs in HEK293 cells with electrophysiological and Ca2+ imaging techniques; (2) detection of TRP mRNAs by RT-PCR and in situ hybridization techniques with TRP isoform specific probes; (3) effects of TRP isof … More orm specific antisense oligonucleotides on native ROCCs. As the results of these studies, we have found that TRP6 is likely to be the essential component of alpha-1 adrenoceptor-activated ROCCs which seem to serve as a membrane depolarizer activating voltage-dependent Ca2+ entry as well as a direct Ca2+ entry pathway that is activated in a store depletion-independent fashion. Thus, TRP6 could be an interesting molecular target of development of anti-hypertensive drugs having entirely new mechanisms (Circ Res. 88, 325-332, 2001). We have also obtained the evidence supporting that TRP6, together with its relative protein, LTRPC2, may form a heretrooligomeric complex which reproduces a number of hallmarks of muscarinic ROCCs widely distributed in the whole gut, some part of brain and adrenal chromaffin cells. In this heterooligomeric configuration, it appears that the signal transduction pathway involved switches from pertussiss toxin-insensitive to sensitive pathways. Such heteromultimeric association of different isoforms of TRP and TRP-related proteins might be responsible for enormous biodiversity of Ca2+ entry associated with cell receptor stimulation. Less

刺激G蛋白偶联或酪氨酸激酶受体通常会导致Ca2+可渗透阳离子通道（ROCC）的激活，这些通道（ROCC）似乎通过以直接和间接方式从细胞外空间中唤起Ca2+进入各种活细胞。尽管ROCC的分子鉴定仍然完全难以捉摸，但在理解果蝇的视觉信号转导方面的最新进展表明，瞬时受体势蛋白（TRP）的脊椎动物同源物（TRP）是这些通道的候选者。基于这些知识，我们使用以下方法在分子水平上纠正具有不同TRP同工型的天然ROCC。（1）通过电生理和Ca2+成像技术在HEK293细胞中详细比较阳离子电流以及在HEK293细胞中重组表达的TRP；（2）通过RT-PCR检测TRP mRNA和与TRP同工型特异性问题的原位杂交技术；（3）TRP ISOF…更多或特定的反义寡核苷酸对天然ROCC的影响。作为这些研究的结果，我们发现TRP6可能是Alpha-1 adrenoceptor激活的ROCC的重要组成部分，该ROCC似乎是一种膜去极化器激活电压依赖性CA2+进入以及直接的CA2+ CA2+进入途径，该途径在商店deppletion depletion-Intpertentententententententententententententententententent中被激活。这就是TRP6可能是具有全新机制的抗高血压药物发展的有趣分子靶标（Circ Res。88，325-332，2001）。我们还获得了支持TRP6及其相对蛋白LTRPC2的证据，可能会形成一种地球根源络合物，该复合体复制了整个肠道中广泛分布在整个肠道中的毒桃体ROCC的许多标志，这是脑和肾上腺脂肪蛋白细胞的某些部分。在这种杂气组合构型中，看来信号转导途径涉及从对毒素不敏感的敏感途径的开关。 TRP和与TRP相关的蛋白质不同同工型的异植物缔合可能负责与细胞受体刺激相关的Ca2+进入的巨大生物多样性。较少的