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腔室中的氯离子/质子交换体。Clcn3-/-小鼠和与CLCN4和CLCN5突变相关的人类疾病的严重表型表明，它们对正常细胞功能至关重要。然而，这些转运蛋白如何预防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。我们的目的是利用肾上腺染色质细胞来描述ClC-3、ClC-4和ClC-5在神经分泌中的作用。我们将研究不同氯离子质子交换剂在培养的嗜铬细胞中的亚细胞定位。我们将研究Cl-/H+交换剂在儿茶酚胺积累和分泌中的作用，使用高分辨率电容测量，笼状Ca2+的闪光光解和安培法的组合。我们将利用几种工程小鼠模型修改ClC-3、ClC-4和ClC-5的表达和功能，要么切除ClC-3和/或ClC-4并敲除ClC-5，要么表达具有修改功能的ClC-3 (Clcn3E281Q/E281Q)，要么只表达某些ClC-3剪接变体。我们期望我们的实验将为不同剪接变异体的作用、异二聚体组装的重要性以及ClC-3、ClC-4和ClC-5在儿茶酚胺积累和LDCV释放中的主要运输功能提供新的见解。

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.