An Optical Assay of the Transport Activity of CLC-7

Abstract

Osteoporosis, characterized by excessive osteoclast mediated bone resorbtion, affects millions of people. ClC-7 is a chloride-proton exchanger member of the CLC protein family localized in lysosomes and in the ruffled border of osteoclasts. Loss of function of ClC-7 leads to osteopetrosis, neurodegeneration and lysosomal storage disease. The osteopetrotic phenotype is explained by the fact that the ion transport activity of ClC-7 is essential for the osteoclast mediated bone resorption. Thus, blocking ClC-7 can be expected to provide an effective treatment of osteoporosis. Here, we describe a purely optical assay of ClC-7 function employing the E2GFP-dsRed Cl−/pH sensor1 fused to the C-terminus of ClC-7, carrying in addition a mutation of a leucine motif at the N-terminus resulting in a partial redirection of ClC-7 to the plasma membrane2-3. For functional activity, ClC-7 associates with the beta-subunit Ostm14. To ensure the simultaneous and equivalent expression of ClC-7 and Ostm1, we constructed a plasmid in which the two cDNAs are linked by a self-cleavable 2AP peptide4. In addition, we introduced a mutation of the gating glutamate (E245A) which renders the transporter voltage-independent and abolishes proton transport3. The assay consists in lowering [Cl]ext and monitoring the resulting decrease in ClC-7 mediated decrease of [Cl]int by measuring the E2GFP/DSRed fluorescence ratio. This simple assay can be applied in HTS and may lead to the design of specific drugs modulating ClC-7 activity.Supported by IIT, Seed project1. Arosio D et al (2010) Nature Methods7, 516.2. Stauber T, Jentsch TJ (2010). J Biol Chem 285: 34537.3. Leisle L et al. (2011 EMBO J. 30, 2140.4. Trishas G et al (2008). BMC Biol. Sep 15;6.