Functional role of intracellular chloride/proton exchangers ClC-3, ClC-4 and ClC-5 in neurosecretion

ClC-3、ClC-4和ClC-5是存在于多种细胞类型的内体区室中的氯离子/质子交换剂。它们对正常细胞功能的至关重要性通过与CLCN 4和CLCN 5中的突变相关的ClCN 3-/-小鼠和人类疾病的严重表型来说明。然而，这些转运蛋白如何防止这些特定的细胞功能障碍，以及它们如何参与不同的细胞功能，在很大程度上仍然不清楚。我们的目的是描述的作用，ClC-3，ClC-4，和ClC-5在神经分泌使用肾上腺嗜铬细胞。我们将在培养的嗜铬细胞中研究不同氯离子质子交换剂的亚细胞定位。我们将研究氯/H+交换剂在儿茶酚胺积累和分泌中的作用，结合使用高分辨率电容测量，笼状Ca 2+和电流法的闪光光解。我们将利用几种工程小鼠模型来修饰ClC-3、ClC-4和ClC-5的表达和功能，或者切除ClC-3和/或ClC-4并敲低ClC-5，或者表达具有修饰的功能的ClC-3（Clcn 3E 281 Q/E281 Q），或者仅表达某些ClC-3剪接变体。我们希望我们的实验将提供新的见解不同的剪接变体的作用，异二聚体组装的重要性和主要的运输功能的ClC-3，ClC-4，和ClC-5的积累中的儿茶酚胺和释放LDCV。

ClC-3, ClC-4, and ClC-5 are chloride/proton exchangers present in endosomal compartments of multiple cell types. Their crucial importance for normal cell function is illustrated by a severe phenotype of Clcn3-/- mice and human diseases associated with mutations in CLCN4 and CLCN5. However, how these transporters prevent these particular cell dysfunctions and how they are involved in different cellular functions has remained largely ununderstood. We aim to describe the role of ClC-3, ClC-4, and ClC-5 in neurosecretion using adrenal chromaffin cells. We will study the subcellular localization of the different chloride proton/exchangers in cultured chromaffin cells. We will study the role of Cl-/H+ exchangers in catecholamine accumulation and secretion, using a combination of high-resolution capacitance measurements, flash photolysis of caged Ca2+ and amperometry. We will modify ClC-3, ClC-4 and ClC-5 expression and function utilizing several engineered mouse models, either ablating ClC-3 and/or ClC-4 and knock-down ClC-5, or expressing ClC-3 with modified function (Clcn3E281Q/E281Q), or exclusively expressing certain ClC-3 splice variants. We expect that our experiments will provide novel insights into the role of distinct splice variants, the importance of heterodimeric assemblies and the main transport functions of ClC-3, ClC-4, and ClC-5 in the accumulation of catecholamine and release from LDCV.