MECHANISMS OF TRANSPORT AND REGULATION BY INTRA-AND EXTRACELLULAR LIGANDS OF CLC PROTEINS INVOLVED IN HUMAN GENETIC DISEASES

CLC proteins are a gene family with nine members in humans of which five are involved in monogenic hereditary diseases. Four CLC members are chloride selective ion channels that help to stabilize the membrane potential and are involved in whole body salt and water homeostasis. Among these is the ClC-Kb channel that is mutated in Bartter syndrome, a salt wasting nephropathy. The other five CLC proteins are found in the membranes of intracellular organelles. Only very recently has it been recognized that at least some (and probably all) of the intracellular CLC proteins are not chloride ion channels but secondary active chloride/proton antiporters. However, the implications of this surprising result and the precise mechanisms of functioning of these intracellular CLC transport proteins are only incompletely understood. In the present project, we intend to investigate in great detail the molecular and biophysical foundations of the transport mechanism of ClC-5, mutations of which cause Dent disease, a renal disorder that leads to kidney stones and eventual renal failure. This research will employ high resolution patch clamp recordings of heterologously expressed proteins combined with site-directed mutagenesis and innovative fluorescence imaging. In addition, we will explore the regulation of the ClC-5 transporter by intracellular nucleotide ligands, and the mechanism of regulation of CLC-K channels by extracellular inorganic (calcium ions, protons) and organic ligands. We hope to obtain insights into the functioning of the ClC-5 transporter, that will help to understand the mechanisms that lead to disease, and to develop rational strategies to treat Dent disease. The manipulation of CLC-K channel function by extracellular ligands may be helpful for the development of a therapeutic strategy for cases of Bartter syndrome with residual channel activity.