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

TRPC6是血管组织中主要的Trp亚型之一，是一种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，除了二酰甘油和IP3受体/钙调蛋白/磷脂酰肌苷相互作用等多种主要机制外，钙调素依赖激酶II(CaMKII)的磷酸化状态对受体刺激前和受体刺激期间的激活过程都有很大影响。为了阐明这一调控的分子基础，我们进行了以下实验。为此，我们对野生型TRPC6及其与TRPC7(T776)N-末端(NT)和跨膜(TM)结构域的嵌合体进行了CaMKII共有基序的突变分析，寻找CaMKII共有基序‘RXX(S/T)’，分别在TRPC6和T776的NT和TM区域确定了8和7个候选序列，但在C末端没有发现。丙氨酸…这些序列的进一步替换表明，只有野生型TRPC6的T487A和T776的T433A突变强烈地减弱了卡巴胆碱(100μM)引起的Ba2+和Gt；内流，而不影响它们的细胞膜定位表达。膜片钳实验也证实了T487A对TRPC6激活的关键作用，丙氨酸替代使氨基甲胆碱诱发电流(ITRPC6)从9.4±2.3降至1.2±0.4(pA/pF，n=5)。此外，T487被谷氨酰胺取代，从而产生永久的负电荷，导致电流和Ba2+对氨基甲胆碱的反应增强，并在受体刺激终止后延长失活时间。另一方面，通过免疫荧光检测TRPC6蛋白在细胞膜上的定位表达并未因这些突变而发生明显变化。考虑到通过对TRPC1的结构分析得出的膜拓扑结构，TRPC6中的T487和T776中的T433很可能位于第二个和第三个TM结构域(II-III环)之间的较长的细胞内伸展上。这些结果表明，结合钙调素结合位点(CIRB)对通道激活的必要性，CIRB和II-III环的紧密空间排列可能允许CaMKII有效地磷酸化T487。这可能反过来启动和/或促进TRPC6通道通向‘开放’，这可能受到T487的带电状态及其附近带负电的磷脂的很大影响。在CaMKII介导的调节的分子阐明过程中，我们注意到一个共同的磷酸化基序T69与蛋白激酶G(PKG)是共同的，因为PKG是普遍存在的生理性血管松弛药一氧化氮(NO)的重要靶点，而且由于血管收缩药诱导的钙内流被已知强烈地被NO/cGMP/PKG通路激活所抑制，我们探索了这个系统在调节TRPC6通道活性中的潜在作用。NO供体S-亚硝乙酰青霉胺(SNAP)以电压不依赖的方式显著抑制ITRPC6的表达(在100μM时抑制70%)，但对细胞表面TRPC6蛋白的表达无明显影响。CGMP 8-bromo cGMP(8-Br-cGMP；100 g M)的膜通透性类似物也有类似程度的抑制作用。同时给予PKG抑制剂KT5823(10μM)、PKG特异性抑制肽DT-3或T69的丙氨酸替代，SNAP和8-BR-cGMP的抑制作用几乎消失。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有力的机制，它可能通过在邻近的内皮细胞或移行性炎症细胞中产生NO而在原位起到紧张性负反馈的作用。较少

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