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

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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.