Characterization of acid flux in osteoclasts from patients harboring a G215R mutation in ClC-7 |
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| Authors: | Kim Henriksen Jeppe Gram Anita Vibsig Neutzsky-Wulff Vicki Kaiser Jensen Morten H Dziegiel Jens Bollerslev Morten A Karsdal |
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| Institution: | 1. Computational Biochemistry and Biophysics Laboratory (CBBL), U_Science Tech (UST), Universitat de Vic — Universitat Central de Catalunya (UVic-UCC), 08500 Vic, Barcelona, Spain;2. Servei de Neurologia–Neuroimmunologia, Centre d''Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d''Hebron (VHIR), Hospital Universitari Vall d''Hebron, 08035 Barcelona, Spain;1. College of Medicine, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA;2. Department of Cell Biology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA;3. Neural and Behavioral Science Graduate Program, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA;4. The Robert Furchgott Center for Neural and Behavioral Science, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA;5. School of Graduate Studies, SUNY Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA;1. Metabolic Bone Disease and Genetic Research Unit, Department of Osteoporosis and Bone Diseases, Shanghai Jiao Tong University affiliated with the Sixth People''s Hospital, 600 Yi-Shan Rd., Shanghai 200233, PR China;2. Department of Orthopedic Surgery, Shanghai Jiao Tong University affiliated with the Sixth People''s Hospital, 600 Yi-Shan Rd., Shanghai 200233, PR China |
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| Abstract: | The chloride-proton antiporter ClC-7 has been speculated to be involved in acidification of the lysosomes and the resorption lacunae in osteoclasts; however, neither direct measurements of chloride transport nor acidification have been performed.Human osteoclasts harboring a dominant negative mutation in ClC-7 (G215R) were isolated, and used these to investigate bone resorption measured by CTX-I, calcium release and pit scoring. The actin cytoskeleton of the osteoclasts was also investigated. ClC-7 enriched membranes from the osteoclasts were isolated, and used to test acidification rates in the presence of a V-ATPase and a chloride channel inhibitor, using a H+ and Cl? driven approach. Finally, acidification rates in ClC-7 enriched membranes from ADOII osteoclasts and their corresponding controls were compared.Resorption by the G215R osteoclasts was reduced by 60% when measured by both CTX-I, calcium release, and pit area when comparing to age and sex matched controls. In addition, the ADOII osteoclasts showed no differences in actin ring formation. Finally, V-ATPase and chloride channel inhibitors completely abrogated the H+ and Cl? driven acidification. Finally, the acid influx was reduced by maximally 50% in the ClC-7 deficient membrane fractions when comparing to controls.These data demonstrate that ClC-7 is essential for bone resorption, via its role in acidification of the lysosomes and resorption lacunae in osteoclasts. |
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