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，除了一些促进该通道激活的主要机制，如二酰基甘油和ip3受体/钙调素/磷脂酰亚苷相互作用外，该通道被钙调素依赖性激酶II （CaMKII）磷酸化的状态极大地影响了受体刺激之前和期间的激活过程。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）的n端（NT）和跨膜（TM）结构域嵌合体的CAMKII共识基序进行了突变分析。搜索CAMKII共识基序‘RXX（S/T）’，分别在TRPC6和T776的NT或TM区发现了8个和7个候选序列，但在c端没有发现。结果表明，只有野生型TRPC6中的T487A突变和T776中的T433A突变，在不影响其细胞膜局部表达的情况下，强烈减弱了100μM碳苯酚引起的Ba^<2+>内流。膜片钳实验也证实了T487A对TRPC6的激活至关重要，通过丙氨酸取代，碳甾醇诱导电流（ITRPC6）从9.4±2.3降低到1.2±0.4 （pA/pF, n=5）。此外，谷氨酰胺取代T487，因此赋予永久负电荷，导致电流和Ba2+对碳醇的反应增强，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蛋白的细胞膜定位表达并未因这些突变而发生明显变化。考虑到从TRPC1的结构分析得出的膜拓扑结构，TRPC6中的T487和T776中的T433可能位于第二和第三个TM结构域（II- III环）之间的细胞内延伸。这些结果表明，结合假定的钙调素结合位点（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有效磷酸化T487。这可能反过来启动和/或促进TRPC6通道的“打开”，这可能受到T487的带电状态和其附近带负电荷的磷脂的极大影响。在camkii介导调控的分子阐明过程中，我们还注意到共识的磷酸化基元之一T69与蛋白激酶G （PKG）的磷酸化基元相同，由于PKG是无处不在的生理血管松弛剂一氧化氮（NO）的重要靶点，并且由于已知血管收缩剂诱导的Ca2+进入被NO/cGMP/PKG通路的激活强烈抑制，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 -亚硝基乙酰青霉胺（SNAP）预处理后，ITRPC6的表达量在100 μ M下被抑制70%，且与电压无关，但细胞表面TRPC6蛋白的表达没有明显降低。cGMP 8-溴cGMP的膜渗透性类似物（8-Br-cGMP; 100 g M）也产生类似程度的抑制作用。同时给予PKG抑制剂KT5823 （10 μ M）、PKG特异性抑制肽DT-3或T69的丙氨酸替代，SNAP和8-Br-cGMP的抑制作用几乎被消除。这些结果表明pkg介导的磷酸化是控制TRPC6通道活性的另一个强有力的机制，它可能在原位通过邻近内皮细胞或迁移的炎症细胞产生的NO进行强直性负反馈。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