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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

G蛋白偶联受体或酪氨酸激酶受体的刺激通常导致Ca 2+渗透性阳离子通道（ROCC）的激活，其似乎通过以直接和间接方式引起Ca 2+从细胞外空间进入而参与多种活细胞。虽然ROCCs的分子鉴定仍然完全难以捉摸，最近的进展，在了解果蝇的视觉信号转导已经揭示，脊椎动物的同源物的瞬时受体电位蛋白（TRP）是有希望的候选人，这些通道。基于这些知识，我们开始使用以下方法在分子水平上将天然ROCC与不同的TRP亚型相关联：（1）通过电生理和Ca 2+成像技术详细比较HEK 293细胞中天然ROCC和重组表达的TRP引起的阳离子电流;（2）通过RT-PCR和TRP亚型特异性探针原位杂交技术检测TRP mRNA;（3）TRP的作用 ...更多信息在天然ROCC上的反义寡核苷酸。作为这些研究的结果，我们已经发现TRP 6可能是α-1肾上腺素受体激活的ROCC的基本组分，其似乎充当膜去极化剂激活电压依赖性Ca 2+进入以及直接Ca 2+进入途径，其以不依赖于储存耗尽的方式被激活。因此，TRP 6可能是开发具有全新机制的抗高血压药物的有趣分子靶点（Circ Res. 88，325-332，2001）。我们还获得了支持TRP 6及其相关蛋白LTRPC 2可能形成异源寡聚复合物的证据，该复合物复制了广泛分布于整个肠道、脑的某些部分和肾上腺嗜铬细胞的毒蕈碱ROCC的许多特征。在这种异源寡聚体构型中，似乎涉及从百日咳毒素不敏感到敏感途径的信号转导途径的切换。TRP和TRP相关蛋白的不同亚型的这种异源多聚体关联可能是负责与细胞受体刺激相关的Ca 2+进入的巨大生物多样性。少