The G215R mutation in the Cl-/H+-antiporter CLC-7 found in ADO II osteopetrosis does not abolish function but causes a severe trafficking defect

Abstract

Background: ClC-7 is a ubiquitous transporter which is broadly expressed in mammalian tissues. It is implied in the pathogenesis of lysosomal storage disease and osteopetrosis. Because of its endosomal/lysosomal localization it is still poorly characterized. Methodology/Principal Findings: An electrophysiological characterization of rat ClC-7 using solid-supported membranebased electrophysiology is presented. The measured currents show the characteristics of ClC-7 and confirm its function as a Cl-/H+-antiporter. We have used rat ClC-7 in CHO cells as a model system to investigate the functionality and cellular localization of the wt transporter and its variant G213R ClC-7 which is the analogue of human G215R ClC-7 responsible for autosomal dominant osteopetrosis type II. Our study shows that rat G213R ClC-7 is functional but has a localization defect in CHO cells which prevents it from being correctly targeted to the lysosomal membrane. The electrophysiological assay is tested as a tool for drug discovery. The assay is validated with a number of drug candidates. It is shown that ClC-7 is inhibited by DIDS, NPPB and NS5818 at micromolar concentrations. Conclusions/Significance: It is suggested that the scenario found in the CHO model system also applies to the human transporter and that mislocalization rather than impaired functionality of G215R ClC-7 is the primary cause of the related autosomal dominant osteopetrosis type II. Furthermore, the robust solid-supported membrane-based electrophysiological assay is proposed for rapid screening for potential ClC-7 inhibitors which are discussed for treatment of osteoporosis. © 2010 Schulz et al.