Functional and structural studies of human CLC chloride proteins involved in genetic diseases

The most abundant anion in the human body is chloride, which is necessary for metabolism, for maintenance of the membrane potential, and for keeping acid-basic balance in cellular compartments. Members of the CLC family of chloride transporting proteins are essential mediators of the Cl- movement across membranes. The human genome encodes 9 CLC genes; of these 5 are Cl-/H+ antiporters and 4 are Cl- channels. Four of them form protein complexes where the CLC pore forming proteins associate with specific β-subunits (CLC-Ka and –Kb with Barttin; CLC-7 with Ostm1 CLC-2 with GlialCAM). In general, association of the CLCs with the auxiliary β-subunits ensures proper targeting and stability of the complex. Defects in genes encoding CLC proteins or their auxiliary subunits are associated with hereditary diseases. Loss of function mutations in the renal CLC-Ks and their subunit Barttin are associated with type III and IV Bartter syndrome, while mutations in the lysosomal CLC-7/Ostm1 complex lead to osteopetrosis and lysosomal storage diseases. Mutations in the CLC-2/GlialCAM complex cause brain oedema and megalencephalic leukoencephalopathy (MLC). However, the knowledge of the basic structural and mechanistic properties underlying CLC complex assembly is still poorly investigated. Through a combination of electrophysiological techniques, fluorescence and biochemical assays this project aims at providing the first description of the molecular interactions between CLC proteins and their corresponding subunits and to determine the structure of the associated complexes. Our hope is to gain the necessary structural and functional information to develop new drugs and therapeutic strategies.