Characterization of acid flux in osteoclasts from patients harboring a G215R mutation in ClC-7.
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.
Henriksen
Kim
K
Nordic Bioscience A/S, Herlev, Denmark. kh@nordicbioscience.com
Gram
Jeppe
J
Neutzsky-Wulff
Anita Vibsig
AV
Jensen
Vicki Kaiser
VK
Dziegiel
Morten H
MH
Bollerslev
Jens
J
Karsdal
Morten A
MA
D000143:Acids / Q000378:metabolism; D001120:Arginine / Q000235:genetics; D001862:Bone Resorption / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D005799:Genes, Dominant; D005998:Glycine / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism*; D009154:Mutation; D010010:Osteoclasts / Q000378:metabolism* / Q000648:ultrastructure; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000143:Acids; C097540:CLCN7 protein, human; D018118:Chloride Channels; D001120:Arginine; D025262:Vacuolar Proton-Translocating ATPases; D002118:Calcium; D005998:Glycine