Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice

Acid transport is required for bone synthesis by osteoblasts. The osteoblast basolateral surface extrudes acid by Na⁺/H⁺ exchange, but apical proton uptake is undefined. We found high expression of the Cl⁻/H⁺ exchanger ClC3 at the bone apical surface. In mammals ClC3 functions in intracellular vesicular chloride transport, but when we found Cl⁻ dependency of H⁺ transport in osteoblast membranes, we queried whether ClC3 Cl⁻/H⁺ exchange functions in bone formation. We used ClC3 knockout animals, and closely-related ClC5 knockout animals: In vitro studies suggested that both ClC3 and ClC5 might support bone formation. Genotypes were confirmed by total exon sequences. Expression of ClC3, and to a lesser extent of ClC5, at osteoblast apical membranes was demonstrated by fluorescent antibody labeling and electron microscopy with nanometer gold labeling. Animals with ClC3 or ClC5 knockouts were viable. In ClC3 or ClC5 knockouts, bone formation decreased ~40 % by calcein and xylenol orange labeling in vivo. In very sensitive micro-computed tomography, ClC5 knockout reduced bone relative to wild type, consistent with effects of ClC3 knockout, but varied with specific histological parameters. Regrettably, ClC5-ClC3 double knockouts are not viable, suggesting that ClC3 or ClC5 activity are essential to life. We conclude that ClC3 has a direct role in bone formation with overlapping but probably slightly smaller effects of ClC5. The mechanism in mineral formation might include ClC H⁺ uptake, in contrast to ClC3 and ClC5 function in cell vesicles or other organs.