Structural and functional investigation of the human CLC-7/OSTM1 complex

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Members of the chloride channel (CLC) family occur in all phyla, with nine members present in mammals. The chloride-proton antiporter CLC-7 belongs to the CLC family and is the major CLC transporter in lysosomes, late endosomes and in the ruffled border, the acid-secreting plasma membrane domain of osteoclasts. Mutations of CLC-7 in humans or mice cause osteopetrosis and lysosomal storage diseases. The Cl- transporter CLC-7 reveals several features typical for the CLC family including its dimeric topology and the architecture of the ion translocation pathway. Also, the transporter obeys the classical CLC transporter ratio of two Cl- to one H+. However, CLC-7 has some very intriguing features which distinguish it from the rest of the CLC family. For instance, the voltage-dependent activation and deactivation of CLC-7 is much slower than other CLC transporters. Furthermore, CLC-7 is the only member that requires an accessory protein, osteopetrosis-associated membrane protein 1 (OSTM1), for both stability and function. CLC-7 and OSTM1 are co-localized in lysosomes and in the ruffled border of osteoclasts. OSTM1 is a type 1 transmembrane protein containing a large, highly glycosylated N-terminus and single membrane-spanning helix and short C-terminus. CLC-7-, OSTM1- and CLC-7/OSTM1-deficient mice have short life spans and develop severe osteopetrosis, retinal degeneration, lysosomal storage diseases, and neurodegeneration. So far, the mechanism of CLC-7 regulation by its accessory protein OSTM1 remains an open question. I propose a multidisciplinary approach combining structural, electrophysiological and biochemical tools for investigation of CLC-7/OSTM1. During my first months in the host laboratory, I was able to establish the protocol for purification of CLC-7 from Gallus gallus. I then analyzed the purified sample by cryo-electron microscopy (cryo-EM), which revealed over 3,000,000 well-distributed single particles. Next, I aim to investigate the function and structure of human CLC-7 alone (Aim 1) and CLC-7 in complex with OSTM1 (Aim 2). Comparison of the cryo-EM structure of the complex with the isolated form of CLC-7 will facilitate identification of important regulatory regions, and will help unravel the unique regulatory nature of the CLC-7 protein. I will then further investigate the regulatory mechanism of CLC-7 by fluorescence-based flux assays and electrophysiological measurements, using lipid-reconstituted samples of CLC-7 and CLC-7/OSTM1. These investigations will ultimately determine the mechanism of CLC-7/OSTM1 interaction and regulation. Beyond the molecular characterization of CLC-7/OSTM1, these studies will promote our understanding of the physiological and pathological roles of the CLC-7/OSTM1 complex.