Molecular elucidation of CaMKII-mediated regulation of vascular receptor-operated Ca^<2+> entry channel TRPC6.

CaMKII介导的血管受体操纵的Ca ^ 2 进入通道TRPC6调节的分子阐明。

基本信息

批准号：
17590221
负责人：
INOUE Ryuji
金额：
$ 2.37万
依托单位：
Fukuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

TRPC6 is one of major TRP isoforms in vascular tissues and acts as a nonselective cation channel associated with vascular tone generation and remodeling. In our recent study, we have found that, in addition to a number of main mechanisms contributing the activation of this channel such as diacylyglycerol and IP3-receptor/calmodulin /phosphatidylinosides interaction, the phosphorylated state of the channel by calmodulin-dependent kinase II (CaMKII) greatly influence the process of activation both before and during receptor stimulation. To elucidate the molecular basis of this regulation, we have performed the following experiments. For this purpose, we performed a mutation analysis of CAMKII consensus motifs on wild-type TRPC6 and its chimera with N-terminal (NT) and transmembrane (TM) domains of TRPC7 (T776).Search for the CAMKII consensus motif `RXX(S/T)' identified 8 and 7 candidate sequences on the NT or TM regions of TRPC6 and T776, respectively, but not on the C-terminus. Alanine … More substitution in these sequences revealed that only the mutations T487A in wild type TRPC6 and T433A in T776 strongly attenuated Ba^<2+> influx evoked by carbachol (100μM) without affecting their cell-membrane localized expression. The critical importance of T487A for TRPC6 activation was also confirmed by patch clamp experiments, where carbachol-induced current (ITRPC6) was reduced from 9.4±2.3 to 1.2±0.4 (pA/pF, n=5) by alanine substitution. In addition, substitution of T487 with glutamine, which confers a permanently negative charge therefore, caused potentiation of the current and Ba2+ response to carbachol, with prolong deactivation after termination of receptor stimulation. On the other hand, the cell membrane localized expression of TRPC6 protein assessed by its immunofluorescence did not appreciably change by these mutations. Concidering the proposed membrane topology derived from the structural analysis of TRPC1, T487 in TRPC6 and T433 in T776 are likely located on a long intracellular stretch between the second and third TM domains (II- III loop). These results suggest, combined with the requisiteness of a putative calmodulin binding site (CIRB) for channel activation, that close spatial arrangement of CIRB and the II-III loop might allow effective phosphorylation of T487 by CAMKII. This might in turn prime and/or facilitate the TRPC6 channel gating toward 'opening', which might be greatly affected by the charged state of T487 and negatively charged phospholipids in its close vicinity.In the course of molecular elucidation of CaMKII-mediated regulation, we laso noticed that one of consensus phosphorylation motif T69 is common with that of protein kinase G (PKG), Since PKG is an important target of the ubiquitous physiological vasorelaxant nitric oxide (NO) and also since vasoconstrictor-induced Ca2+ entry is known to be strongly inhibited by activation of NO/cGMP/PKG pathway, we explored the potential role of this system in regulating TRPC6 channel activity. The magnitude of ITRPC6 was greatly inhibited by pretreatment with a NO donor, S-nitrosoacetyl penicillamine (SNAP) (by 70% at 100 μ M) in a voltage-independent manner, but cell-surface expression of TRPC6 protein was not appreciably reduced. Similar extent of inhibition was produced by a membrane-permeable analogue of cGMP 8-bromo cGMP (8-Br-cGMP; 100 g M). Both the inhibitory effects of SNAP and 8-Br-cGMP were nearly abolished by simultaneous administration of the PKG inhibitor KT5823 (10 μ M), PKG-specific inhibitory peptide DT-3 or alanine substitution for T69. These results suggest that PKG-mediated phosphorylation is another powerful mechanism to control TRPC6 channel activity, which may in situ operate as a tonic negative feedback via NO produced in adjacent endothelial cells or migrating inflammatory cells. Less

TRPC6 是血管组织中主要的 TRP 亚型之一，作为与血管张力生成和重塑相关的非选择性阳离子通道，在我们最近的研究中，我们发现，除了许多主要的促进机制外，该通道的激活还具有多种作用。作为二酰甘油和 IP3 受体/钙调蛋白/磷脂酰肌苷相互作用，钙调蛋白依赖性激酶 II 导致通道的磷酸化状态(CaMKII) 在受体刺激之前和期间极大地影响激活过程。为了阐明这种调节的分子基础，我们进行了以下实验，我们对野生型 TRPC6 上的 CAMKII 共有基序进行了突变分析。及其与 TRPC7 (T776) 的 N 端 (NT) 和跨膜 (TM) 结构域的嵌合体。搜索 CAMKII 共有基序“RXX(S/T)”，鉴定出 8和 7 个候选序列分别位于 TRPC6 和 T776 的 NT 或 TM 区域，但不在 C 末端。这些序列中的更多替换表明，只有野生型 TRPC6 中的突变 T487A 和 T776 中的 T433A 减弱了 Ba^<。 2+> 由卡巴胆碱 (100μM) 引起的流入，而不影响其细胞膜局部表达 T487A 的至关重要性。 TRPC6 激活也通过膜片钳实验得到证实，其中通过丙氨酸取代，卡巴胆碱诱导电流 (ITRPC6) 从 9.4±2.3 降低至 1.2±0.4 (pA/pF，n=5)。此外，用谷氨酰胺取代 T487。因此，它赋予永久负电荷，导致电流和 Ba2+ 对卡巴胆碱的反应增强，并在受体刺激终止后延长失活时间。另一方面，通过免疫荧光评估的 TRPC6 蛋白的细胞膜局部表达并没有因这些突变而明显改变，考虑到从 TRPC1、TRPC6 中的 T487 和 T776 中的 T433 的结构分析得出的拟议膜拓扑结构可能位于较长的细胞内延伸上。这些结果表明，结合通道的假定钙调蛋白结合位点（CIRB）的必要性。 CIRB 和 II-III 环的紧密空间排列可能允许 CAMKII 有效磷酸化 T487，这可能反过来引发和/或促进 TRPC6 通道门控“打开”，这可能会受到带电状态的极大影响。 T487 及其附近带负电的磷脂。在 CaMKII 介导的调节的分子阐明过程中，我们注意到共有磷酸化基序之一 T69 与由于 PKG 是普遍存在的生理性血管舒张剂一氧化氮 (NO) 的重要靶标，而且已知血管收缩剂诱导的 Ca2+ 进入会受到 NO/cGMP/PKG 途径激活的强烈抑制，因此我们探索了该系统在调节 TRPC6 通道活性中的潜在作用。NO 供体 S-亚硝基乙酰基预处理大大抑制了 ITRPC6 的强度。青霉胺 (SNAP)（100 μM 时减少 70%）以电压无关的方式进行，但 TRPC6 蛋白的细胞表面表达并未明显减少，cGMP 8-溴的膜渗透类似物也产生了类似的抑制程度。 cGMP（8-Br-cGMP；100 g M）的同时给药几乎消除了 SNAP 和 8-Br-cGMP 的抑制作用。 PKG 抑制剂 KT5823 (10 μM)、PKG 特异性抑制肽 DT-3 或 T69 的丙氨酸替代这些结果表明 PKG 介导的磷酸化是控制 TRPC6 通道活性的另一种强大机制，其可能在原位发挥补品作用。通过相邻内皮细胞或迁移炎症细胞产生的 NO 产生的负反馈较少。

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Transient receptor potential C3/6 channels are essential for angiotensin II-induced cardiac hypertrophy.

瞬时受体电位 C3/6 通道对于血管紧张素 II 诱导的心脏肥大至关重要。

DOI：
发表时间：
2006
期刊：
EMBO Journal 25(22)
影响因子：
0
作者：
Onohara N;Nishida M;Inoue R;他6名
通讯作者：
他6名

α_<12/13>-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts.

α_<12/13>介导的活性氧的产生对于心脏成纤维细胞中血管紧张素受体诱导的NFAT激活至关重要。

DOI：
发表时间：
2005
期刊：
Journal of Biological Chemistry 280(24)
影响因子：
0
作者：
Fujii;T.;Onohara;N.;Maruyama;Y;Tanabe;S.;Kobayashi;H.;Fukutomi;M.;Nagamatsu;Y;Nishihara;N.;Inoue;R;Sumimoto;H;Shibasaki;F;Nagao;T.;Nishida;M.;Kurose;H
通讯作者：
H

Cell membrane-derived lysophosphatidylcholine activates cardiac ryanodine receptor channels.

细胞膜来源的溶血磷脂酰胆碱激活心脏兰尼碱受体通道。