Molecular physiological characterization of Ca2+ signal-regulating ion channels that control vectorial substance transport

我们研究了质膜Ca~(2+)通透通道和内质网(ER)Ca~(2+)释放通道的分子同一性，以及它们的功能特性、细胞定位和在上皮细胞中的偶联，以建立上皮细胞钙信号矢量化的分子生理学基础。受体激活的TRPC通道在多种上皮细胞中广泛表达。TRPC3、C5、C6、C7在G蛋白偶联受体的刺激下被激活，而不依赖于钙离子储存/钙离子释放的耗竭。在内皮细胞质膜内陷小窝中，TRPC5与eNOS结合，并被NO激活，介导钙内流，控制受体诱导的NO产生级联反应的正反馈环路。NAD激活的TRPM2Ca~(2+)~(2+)通道也被证明与细胞在氧化应激下通过活性氧/氮物种或TNF而死亡有关。要了解分子机制…我们用鸡DT40 B淋巴细胞建立了一个缺失TRPC1的细胞系。细胞内钙离子进入减少，IP3；受体介导的钙释放减少，B细胞受体刺激引起的钙振荡，提示TRPC1在钙信号模式中起关键作用。此外，我们还证实了Ca^&lt；2+/IP_3信号的正反馈环路：由TRPC3-介导的Ca^&lt；2+&gt；进入引起磷脂酶C(PLC)γ的转位和随后的激活。为了了解连接结构的分子结构，我们表征了该结构所必需的分子。已经鉴定了三个连接素成员，它们有助于在可兴奋细胞中形成连接结构。结合蛋白Mitsugum29在内质网钙离子释放和SOC中起重要作用。肌浆网(SR)管腔内的Sarcalmenin对肌浆网的钙存储容量有重要作用。较少

We have studied molecular identity of plasma membrane Ca^<2+>-permeable channels and endoplasmic reticulum (ER) Ca^<2+> release channels, and their functional properties, cellular localization, and coupling in epithelial cells, to establish molecular physiological basis underlying vectorization of Ca^<2+> signals in epithelial transport.Abundant expression of receptor-activated TRPC channels was revealed in various epithelial cells. TRPC3, C5, C6, C7 were activated upon stimulation of G-protein-coupled receptors independently of depletion of Ca^<2+> stores/ Ca^<2+> release. TRPC5 was associated with eNOS in the plasma membrane invagination caveola in endothelial cells, and was activated by NO to mediate Ca^<2+> influx that controls a positive feedback loop for receptor-induced NO production cascade. The NAD-activated TRPM2 Ca^<2+> channel was also demonstrated to be linked to cell death under oxidative stress by reactive oxygen/nitrogen species or by TNF.To understand molecular mechani … More sms that determine spatio-temporal patterns of Ca^<2+> signaling, we used chicken DT40 B lymphocytes to create a cell line deficient of TRPC1. The cells showed reduction in store-operated Ca^<2+> (SOC) entry, IP3 ; receptor-mediated Ca^<2+> release, and Ca^<2+> oscillation upon B cell receptor stimulation, suggesting that TRPC1 is critical for patterning Ca^<2+> signals. Furthermore, we demonstrated a positive feedback loop for Ca^<2+>/ IP_3 signaling : translocation and subsequent activation of phospholipase C (PLC)γ elicited by TRPC3-mediating Ca^<2+> entry.For functional coupling between plasmamembrane and ER membrane in Ca^<2+> signaling, the junction structure formed by the two kinds of membrane is essential. To understand molecular architecture of the junction structure, we characterized molecules essential for the structure. Three Junctophilin members have been identified which contribute to formation of the junction structure in excitable cells. A junctional protein Mitsugumin29 plays an important role in ER Ca^<2+> release and SOC. Sarcalmenin in the lumen of sarcoplasmic reticulum (SR) contributes to Ca^<2+> storing capacity of SR. Less