Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II
Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II
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DOI：
10.1113/jphysiol.2013.251249
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发表时间：
2013-06-01
影响因子：
5.5
通讯作者：
Inoue, Ryuji
中科院分区：
文献类型：
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作者：
Shi, Juan;Geshi, Naomi;Inoue, Ryuji
Ca 2 +/钙调蛋白（CaM）依赖性激酶II（CaMKII）通过调节多种Ca 2+渗透性通道（如非电压门控Ca 2+通道TRPC 6），在多种Ca 2+介导的细胞功能（包括心血管系统的生理学/病理生理学）中发挥关键作用。中心点在这项研究中，我们通过嵌合体、缺失和定点突变方法研究了CaMK II对其正调控的分子机制。中心点结果表明TRPC 6通道的两个空间上分离的位点，即C-末端肌醇-1，4，5-三磷酸受体/CaM结合结构域的远端部分和位于推定的第一胞内环上的Thr 487，对于CaMK II介导的TRPC 6通道调节是至关重要的。中心点该机制可能作为通过TRPC 6通道的Ca 2+内流的有效正反馈调节，与神经激素因子刺激磷脂酶C偶联受体后的细胞内和跨膜Ca 2+动员一致，从而微调心血管功能。这些功能的破坏可能导致病理状态，如心脏肥大和心律失常、高血压和动脉粥样硬化。摘要采用嵌合、缺失和定点突变等方法，探讨了钙调蛋白依赖性激酶II（CaMKII）调控小鼠瞬时受体电位通道TRPC 6的分子机制。由毒蕈碱激动剂卡巴胆碱（CCh; 100 μ m）诱导的TRPC 6表达HEK 293细胞中的电流（ICCh）被CaMK II特异性肽autocamtide-2相关抑制肽（AIP; 10 μ m）强烈减弱。TRPC 6/C7嵌合体实验表明，TRPC 6的C-末端序列是ICCh对AIP诱导的CaMKII抑制敏感所必需的。此外，TRPC 6的C-末端CaM/肌醇-1，4，5-三磷酸受体结合结构域（CIRB）的远端区域（Gln 855-Glu 877）的缺失足以消除ICCh。系统的丙氨酸扫描潜在的CaMKII磷酸化位点显示，Thr 487是唯一负责激活TRPC 6通道的受体刺激。用其他非极性氨基酸，即谷氨酰胺或天冬酰胺再现了Thr 487（T487 A）的丙氨酸突变的消除作用，而用谷氨酸或天冬氨酸的磷酸模拟突变部分地挽救了该作用。TRPC 6通道的细胞表达和分布没有随这些突变而显著改变。Myc标记的TRPC 6通道的电生理和免疫细胞化学数据表明，Thr 487最有可能位于细胞膜的胞内侧。过表达T487 A可显著降低A7 r5主动脉肌细胞Arg 8-加压素诱导的内源性TRPC 6样电流。基于这些结果，我们建议，一个C-末端结构域（大概是远端CIRB区域）周围的一个单一的CaMKII磷酸化位点Thr 487的最佳空间排列可能是必不可少的CaMKII介导的TRPC 6通道的调节。这种机制可能在天然环境中具有生理学意义，例如在血管平滑肌细胞中。
Key points center dot Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) plays pivotal roles in diverse Ca2+-mediated cellular functions including the physiology/pathophysiology of the cardiovascular system, through modulation of a variety of Ca2+-permeable channels such as a non-voltage-gated Ca2+ channel TRPC6. center dot In this study, we investigated the molecular mechanism underlying its positive regulation by CaMKII with chimera, deletion and site-directed mutagenesis approaches. center dot The results indicate that two spatially separated sites of TRPC6 channel, i.e. a distal part of the C-terminal inositol-1,4,5-trisphosphate receptor/CaM binding domain and Thr487 located on the putative first intracellular loop, are crucial for the CaMKII-mediated regulation of TRPC6 channels. center dot This mechanism may serve as an effective positive feedback regulation of Ca2+ influx through TRPC6 channels, in concert with intracellular and transmembrane Ca2+ mobilization upon phospholipase C-coupled receptor stimulation by neurohormonal factors, thereby fine-tuning the cardiovascular functions. center dot Disruption of these could lead to pathological states such as cardiac hypertrophy and arrhythmia, hypertension and atherosclerosis. Abstract The molecular mechanism underlying Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII)-mediated regulation of the mouse transient receptor potential channel TRPC6 was explored by chimera, deletion and site-directed mutagenesis approaches. Induction of currents (ICCh) in TRPC6-expressing HEK293 cells by a muscarinic agonist carbachol (CCh; 100 m) was strongly attenuated by a CaMKII-specific peptide, autocamtide-2-related inhibitory peptide (AIP; 10 m). TRPC6/C7 chimera experiments showed that the TRPC6 C-terminal sequence is indispensable for ICCh to be sensitive to AIP-induced CaMKII inhibition. Further, deletion of a distal region (Gln855-Glu877) of the C-terminal CaM/inositol-1,4,5-trisphosphate receptor binding domain (CIRB) of TRPC6 was sufficient to abolish ICCh. Systematic alanine scanning for potential CaMKII phosphorylation sites revealed that Thr487 was solely responsible for the activation of the TRPC6 channel by receptor stimulation. The abrogating effect of the alanine mutation of Thr487 (T487A) was reproduced with other non-polar amino acids, namely glutamine or asparagine, while being partially rescued by phosphomimetic mutations with glutamate or aspartate. The cellular expression and distribution of TRPC6 channels did not significantly change with these mutations. Electrophysiological and immunocytochemical data with the Myc-tagged TRPC6 channel indicated that Thr487 is most likely located at the intracellular side of the cell membrane. Overexpression of T487A caused significant reduction of endogenous TRPC6-like current induced by Arg8-vasopressin in A7r5 aortic myocytes. Based on these results, we propose that the optimal spatial arrangement of a C-terminal domain (presumably the distal CIRB region) around a single CaMKII phosphorylation site Thr487 may be essential for CaMKII-mediated regulation of TRPC6 channels. This mechanism may be of physiological significance in a native environment such as in vascular smooth muscle cells.