Ca2+ signaling and HCO3- secretion by exocrine glands

Ca2 信号传导和外分泌腺的 HCO3- 分泌

• 批准号: 8743760
• 负责人: Shmuel Muallem
• 金额: $ 258.85万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8743760
• 关键词: 2-aminoethoxydiphenyl borate; Achlorhydria; Affect; Anions; Apical; Autophagocytosis; Bicarbonates; Binding Proteins; Biochemical; Biological; Biological Assay; Buffers; Cell membrane; Cell physiology; Cell surface; Cells; Charge; Complex; Coupled; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Disease; Duct (organ) structure; Ductal; Early Endosome; Enzymes; Epithelial; Epithelial Cells; Epithelium; Exocrine Glands; Family; Family member; Fluids and Secretions; Frequencies; Functional disorder; Ganglioside Sialidase Deficiency Disease; Gastric Acid; Gastric Parietal Cells; Gene Deletion; Genetic; Gland; Health; Histologic; Homeostasis; Hypertension; In Vitro; Inflammatory; Knockout Mice; Lateral; Lead; Liquid substance; Lysosomes; Mediating; Mediator of activation protein; Membrane; Membrane Protein Traffic; Minor salivary gland structure; Modeling; Molecular; Molecular Analysis; Mucins; Mucolipidoses; Mus; Mutation; NAADP; Organelles; Pancreas; Pancreatitis; Pathway interactions; Patients; Phenotype; Phosphotransferases; Physiological; Play; Process; Proteins; Receptor Activation; Recruitment Activity; Regulation; Role; STIM1 gene; Saliva; Salivary Glands; Secretory Cell; Signal Transduction; Solubility; Stress; Syndrome; TRP channel; TRPC3 ion channel; Time; Tissues; Toxic effect; Work; acute pancreatitis; cell injury; in vivo; late endosome; member; mouse model; mutant; pancreatic juice; premature; prevent; receptor; response; scaffold

Regulation of Ca2+ signaling by STIM1. Ca2+ influx by store-operated Ca2+ channels is a key component of the receptor-evoked Ca2+ signal. In all cells examined, transient receptor potential canonical (TRPC) channels mediate a significant portion of the receptor-stimulated Ca2+ influx. Recent studies have revealed how STIM1 activates TRPC1, but the role of STIM1 in TRPC channel activation by receptor stimulation is not fully understood. We established mutants of TRPC channels that could not be activated by STIM1 but were activated by the "charge-swap" mutant STIM1(K684E,K685E). WT but not mutant TRPC channels were inhibited by scavenging STIM1 while mutant TRPC channels were robustly activated by receptor stimulation. Moreover, STIM1 and STIM1(K684E,K685E) reciprocally affected receptor-activated WT and mutant TRPC channels. Together, these findings indicate that TRPC channels can function as STIM1-dependent and STIM1-independent channels, which increases the versatility of TRPC channel function and their role in receptor-stimulated Ca2+ influx. To understand the role of the native STIM1 and Orai1 in polarized Ca2+ signals in secretory epithelial cells we found that inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca2+ influx and frequency of Ca2+ oscillations. The native Orai1 localization is restricted to the apical pole of the lateral membrane and does not cluster in response to store depletion. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co-IP of STIM1/Orai1/IP3Rs/TRPCs is enhanced by cell stimulation and disrupted by 2APB. The polarized localization and recruitment of these proteins results in preferred Ca2+ entry that is initiated at the apical pole. The role of Ca2+ influx channels in disease was shown by demonstrating that genetic and pharmacological of the Ca2+ influx channel TRPC3 protect pancreas and salivary glands from the toxic effect of excessive Ca2+ influx. Another type of Ca2+ channels are the TRPMLs. Mutations in TRPML1, a lysosomal Ca2+-permeable TRP channel, lead to mucolipidosis type IV with achlorhydria. We produced Trpml1 null mice and demonstrated critical requirement for this channel in gastric acid secretion. Histologic and ultrastructural analyses revealed that Trpml1-/- parietal cells are damaged, although the intralysosomal Ca2+ content were unaffected in these cells. Cells express two organellar Ca2+ channels, TRPMLs and the NAADP-activated TPCs. A critical question is whether the two channels are related. We used molecular, biochemical and functional assays to show that the TRPMLs and TPCs are indepent channels. Mechanism and Regulation of Epithelial HCO3- secretion: This lab also study epithelial HCO3- transport in health and disease. Members of the SLC26 family of anion transporters play a critical role in epithelial HCO3- secretion. We characterize several features of the electrogenic members of the family to identify structural motifs that determine multiple functional modes of these transporters and explain how the transporters can function either as coupled or as uncoupled transporters. Fluid and HCO3- secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition. Secretion of ductal fluid and HCO3- in secretory glands is fueled by Na+/HCO3- cotransport mediated by basolateral NBCe1-B and by Cl-/HCO3- exchange mediated by luminal Slc26a6 and CFTR. To understand the mechanisms governing ductal secretion we showed that ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator IRBIT and by inhibition of PP1. In contrast, silencing the WNK and SPAK kinases increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO3- secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase-regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.

STIM1 对 Ca2+ 信号传导的调节。钙池操纵的 Ca2+ 通道的 Ca2+ 流入是受体诱发的 Ca2+ 信号的关键组成部分。在所有检查的细胞中，瞬时受体电位规范 (TRPC) 通道介导受体刺激的 Ca2+ 流入的很大一部分。最近的研究揭示了 STIM1 如何激活 TRPC1，但 STIM1 在受体刺激激活 TRPC 通道中的作用尚不完全清楚。我们建立了不能被 STIM1 激活但被“电荷交换”突变体 STIM1（K684E，K685E）激活的 TRPC 通道突变体。 WT而非突变TRPC通道被清除STIM1抑制，而突变TRPC通道被受体刺激强烈激活。此外，STIM1和STIM1(K684E，K685E)相互影响受体激活的WT和突变TRPC通道。总之，这些发现表明 TRPC 通道可以作为 STIM1 依赖性和 STIM1 独立通道发挥作用，这增加了 TRPC 通道功能的多功能性及其在受体刺激的 Ca2+ 流入中的作用。为了了解天然 STIM1 和 Orai1 在分泌上皮细胞极化 Ca2+ 信号中的作用，我们发现抑制 Orai1、STIM1 或删除 TRPC1 可减少 Ca2+ 流入和 Ca2+ 振荡频率。天然的 Orai1 定位仅限于侧膜的顶极，并且不会响应存储耗尽而聚集。出乎意料的是，细胞刺激导致天然 STIM1 极化募集到顶端和侧面区域，从而募集到有或没有 Orai1 的区域。 TRPC1 在质膜的顶端和基底外侧区域表达。 STIM1/Orai1/IP3Rs/TRPC 的 Co-IP 通过细胞刺激而增强，并被 2APB 破坏。这些蛋白质的极化定位和募集导致从顶极开始的首选 Ca2+ 进入。 Ca2+ 内流通道在疾病中的作用通过证明 Ca2+ 内流通道 TRPC3 的遗传和药理学作用来保护胰腺和唾液腺免受过量 Ca2+ 内流的毒性作用而得到证实。 另一种类型的 Ca2+ 通道是 TRPML。 TRPML1（一种溶酶体 Ca2+ 渗透性 TRP 通道）的突变会导致 IV 型粘脂沉积症并伴有胃酸缺乏。我们培育了 Trpml1 缺失小鼠，并证明了胃酸分泌对该通道的关键需求。组织学和超微结构分析表明，Trpml1-/- 壁细胞受损，但这些细胞中溶酶体内的 Ca2+ 含量不受影响。细胞表达两种细胞器 Ca2+ 通道：TRPML 和 NAADP 激活的 TPC。一个关键问题是这两个渠道是否相关。我们使用分子、生化和功能测定来证明 TRPML 和 TPC 是独立的通道。 上皮 HCO3- 分泌的机制和调节：该实验室还研究健康和疾病中上皮 HCO3- 的转运。阴离子转运蛋白 SLC26 家族的成员在上皮 HCO3- 分泌中发挥着关键作用。我们表征了该家族的产电成员的几个特征，以确定决定这些转运蛋白多种功能模式的结构基序，并解释转运蛋白如何作为偶联或非偶联转运蛋白发挥作用。 体液和 HCO3- 分泌是上皮细胞的基本功能，决定体液体积和离子组成。分泌腺中导管液和 HCO3- 的分泌是由基底外侧 NBCe1-B 介导的 Na+/HCO3- 共转运和管腔 Slc26a6 和 CFTR 介导的 Cl-/HCO3- 交换促进的。为了了解控制导管分泌的机制，我们发现小鼠导管分泌通过 NBCe1-B/CFTR 激活剂 IRBIT 的沉默和 PP1 的抑制而受到抑制。相反，沉默 WNK 和 SPAK 激酶会增加分泌。分子分析表明，WNK 激酶充当招募 SPAK 的支架，SPAK 磷酸化 CFTR 和 NBCe1-B，从而减少它们的细胞表面表达。 IRBIT 通过将 PP1 募集到复合物中，使 CFTR 和 NBCe1-B 去磷酸化，除了刺激它们的活性外，还可以恢复它们的细胞表面表达，从而对抗 WNK 和 SPAK 的作用。这些发现强调了 IRBIT 在上皮液和 HCO3 分泌中的关键作用，并提供了 IRBIT 协调这些过程的分子机制。它们还对涉及全身液体稳态、高血压和囊性纤维化的 WNK/SPAK 激酶调节过程有影响。