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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

TRPC 6是血管组织中主要的TRP亚型之一，并且作为与血管张力产生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，除了一些主要的机制，如二酰基甘油和IP 3-受体/钙调蛋白/磷脂酰肌醇的相互作用，磷酸化状态的通道钙调蛋白依赖性激酶II（CaMKII）极大地影响激活的过程之前和期间的受体刺激。为了阐明这种调节的分子基础，我们进行了以下实验。为此，我们对野生型TRPC 6及其与TRPC 7（T776）的N-末端（NT）和跨膜（TM）结构域的嵌合体上的CAMKII共有基序进行了突变分析。搜索CAMKII共有基序“RXX（S/T）”，分别在TRPC 6和T776的NT或TM区域上鉴定出8个和7个候选序列，但不在C-末端。丙氨酸 ...更多信息这些序列中的替换显示，只有野生型TRPC 6中的T487 A突变和T776中的T433 A突变强烈减弱了卡巴胆碱（100μM）诱发的Ba^2+内流，而不影响它们的细胞膜定位表达。T487 A对TRPC 6激活的关键重要性也通过膜片钳实验证实，其中氨甲酰胆碱诱导的电流（ITRPC 6）通过丙氨酸取代从9.4±2.3降低至1.2±0.4（pA/pF，n=5）。此外，取代T487与谷氨酰胺，这赋予一个永久的负电荷，因此，导致增强的电流和Ba 2+对卡巴胆碱的反应，延长失活后终止受体刺激。另一方面，通过其免疫荧光评估的TRPC 6蛋白的细胞膜定位表达没有因这些突变而明显改变。考虑到源自TRPC 1的结构分析的所提出的膜拓扑结构，TRPC 6中的T487和T776中的T433可能位于第二和第三TM结构域（II-III环）之间的长细胞内伸展上。这些结果表明，结合一个假定的钙调蛋白结合位点（CIRB）的通道激活的requisiteness，CIRB和II-III环的紧密空间排列可能允许有效的磷酸化T487的CAMK II。在对CaMKII介导的调控进行分子生物学研究的过程中，我们发现T69的磷酸化基序与蛋白激酶G（PKG）的磷酸化基序有共同点，而T487的磷酸化基序与PKG的磷酸化基序有共同点，因此，T487的磷酸化基序与PKG的磷酸化基序有共同点，而PKG的磷酸化基序与T487的磷酸化基序有共同点。由于PKG是无处不在的生理性血管舒张剂一氧化氮（NO）的重要靶点，并且已知NO/cGMP/PKG通路的激活强烈抑制血管收缩剂诱导的Ca 2+内流，因此我们探讨了该系统在调节TRPC 6通道活性中的潜在作用。用NO供体S-亚硝基乙酰青霉胺（SNAP）预处理（100 μ M时抑制70%），以电压非依赖性方式极大地抑制了ITRPC 6的幅度，但TRPC 6蛋白的细胞表面表达没有明显减少。类似程度的抑制产生的膜渗透类似物的cGMP 8-溴cGMP（8-Br-cGMP; 100克M）。同时给予PKG抑制剂KT 5823（10 μ M）、PKG特异性抑制肽DT-3或丙氨酸替代T69几乎消除了SNAP和8-Br-cGMP的抑制作用。这些结果表明PKG介导的磷酸化是控制TRPC 6通道活性的另一种强有力的机制，其可以通过邻近内皮细胞或迁移的炎症细胞中产生的NO原位作为紧张性负反馈起作用。少