Intracellular ClC-3 chloride channels promote bone resorption in vitro through organelle acidification in mouse osteoclasts

ClC-7 [Cl.sup.-] channels expressed in osteoclasts are important for bone resorption since it has been shown that disruption of the ClCN7 gene in mice leads to severe osteopetrosis. We have previously reported that [Cl.sup.-] currents recorded from mouse osteoclasts resemble those of ClC-3 [Cl.sup.-] channels. The aim of the present study was to determine the expression of ClC-3 channels in mouse osteoclasts and their functional role during bone resorption. We detected transcripts for both CIC-7 and CIC-3 channels in mouse osteoclasts by RT-PCR. The expression of ClC-3 was confirmed by immunocytochemical staining. Mouse osteoclasts lacking ClC-3 [Cl.sup.-] channels (Cl[C-3.sup.-/-] osteoclasts) derived from ClCN3 gene-deficient mice (Cl[C-3.sup.-/-]) showed lower bone resorption activity compared with Cl[C-3.sup.+/+] osteoclasts derived from wild-type mice (Cl[C-3.sup.+/+]). Treatment of Cl[C-3.sup.+/+] osteoclasts with small interfering RNA (siRNA) against ClC-3 also significantly reduced bone resorption activity. Electrophysiological properties of basal and hypotonicity-induced [Cl.sup.-] currents in Cl[C-3.sup.-/-] osteoclasts did not differ significantly from those in Cl[C-3.sup.+/+] osteoclasts. Using immunocytochemistry, ClC-3 was colocalized with lysosome-associated membrane protein 2. Using pH-sensitive dyes, organelle acidification activity in Cl[C-3.sup.-/-] osteoclasts was weaker than in Cl[C-3.sup.+/+] osteoclasts. Treatment of Cl[C-3.sup.+/+] osteoclasts with siRNA against ClC-3 also reduced the organelle acidification activity. In conclusion, ClC-3 [Cl.sup.-] channels are expressed in intracellular organelles of mouse osteoclasts and contribute to osteoclastic bone resorption in vitro through organelle acidification. knockout mice; [Cl.sup.-] current; lysosome-associated membrane protein; pH-sensitive dye staining