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

我们研究了质膜Ca^<2+>-可渗透通道和内质网（ER） Ca^<2+>释放通道的分子特性，以及它们在上皮细胞中的功能特性、细胞定位和偶联，以建立上皮细胞运输中Ca^<2+>信号向量化的分子生理基础。受体激活的TRPC通道在多种上皮细胞中大量表达。TRPC3, C5, C6， C7在g蛋白偶联受体的刺激下被激活，而不依赖于Ca^<2+>储存的消耗/ Ca^<2+>释放。TRPC5与内皮细胞质膜内陷小窝中的eNOS相关，并被NO激活介导Ca^<2+>内流，从而控制受体诱导的NO级联生成的正反馈回路。nad激活的TRPM2 Ca^<2+>通道也被证明与活性氧/氮或TNF在氧化应激下的细胞死亡有关。为了进一步了解Ca^<2+>信号的时空模式，我们利用鸡DT40 B淋巴细胞建立了TRPC1缺失的细胞系。细胞的store- operation Ca^<2+> (SOC)入口IP3降低；受体介导的Ca^<2+>释放，以及B细胞受体刺激下Ca^<2+>振荡，表明TRPC1对Ca^<2+>信号的模式至关重要。此外，我们证明了Ca^<2+>/ IP_3信号的正反馈回路：trpc3介导的Ca^<2+>进入引发磷脂酶C (PLC)γ的易位和随后的激活。在Ca^<2+>信号传导过程中，质膜与内质膜之间的功能耦合，两种膜形成的结结构是必不可少的。为了了解结结构的分子结构，我们对结构所必需的分子进行了表征。已经确定了三种亲结蛋白成员，它们有助于可兴奋细胞中结结构的形成。连接蛋白Mitsugumin29在ER Ca^<2+>释放和SOC中起重要作用。肌浆网（SR）管腔内的肌钙蛋白对SR的Ca^<2+>储存能力的影响较小

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