Autosomal dominant osteopetrosis II (ADOII) is a relatively benign disorder caused by a missense mutation in the ClCN7 gene. In this study, we characterize the osteoclasts from patients with ADOII, caused by a G215R mutation, and investigate the effect on osteoclast function in vitro. Osteoclasts from ADOII patients and healthy age-and sex-matched controls, were used to evaluate osteoclastogenesis, cell fusion, acidification, and resorptive activity. ADOII osteoclasts in vivo have increased number and size. However, in vitro we observed no significant changes in the osteoclast formation rate, the morphology, and the expression of markers, such as cathepsin K and tartrate-resistant acid phosphatase. When mature ADOII osteoclasts were investigated on mineralized bone, they degraded the bone material, however only to 10 to 20% of the level in controls. We show by acridine orange, that the reduced chloride transport leads to reduced acidification. We show that the residual activity is sensitive to inhibitors of cathepsins and chloride channels, confirming that resorption is reduced but present. In conclusion, this is the first functional in vitro study of human ADOII osteoclasts. We show normal osteoclastogenesis in ADOII osteoclasts. However, the residual activity of the ClC-7 channel in ADOII osteoclasts does not allow sufficient acidification and thereby resorption. The osteoclasts ability to dissolve the inorganic phase of bone is essential for the degradation of the organic bone matrix, and thereby for the maintenance of the skeleton. The inorganic phase of bone consists of hydroxyapatite crystals, and the dissolution of this matrix requires a decrease in pH, which is facilitated by active transport of protons into the resorption lacunae.

The endogenous volume-regulated anion channel (VRAC) from HEK293 cells was pharmacologically characterized using the whole-cell patch-clamp technique. Under isotonic conditions a small (1.3 nS), Ca(2+)-independent Cl conductance was measured. However, swelling at 75% tonicity activated a VRAC identified as an outward-rectifying anion current ( P(l) > P(Cl) > P(gluconate)), which was ATP-dependent and showed inactivation at positive potentials. Activation of this current followed a sigmoid time course, reaching a plateau conductance of 42.6 nS after 12-15 min ( t(1/2) = 7 min). The pharmacology of this VRAC was investigated using standard Cl(-)-channel blockers (NPPB, DIDS, and tamoxifen) as well as a new group (acidic di-aryl ureas) of Cl(-)-channel blockers (NS1652, NS3623, NS3749, and NS3728). The acidic di-aryl ureas were originally synthezised for inhibition of the human erythrocyte Cl(-) conductance in vivo. NS3728 was the most potent VRAC blocker in this series ( IC(50) = 0.40 micro M) and even more potent than tamoxifen (2.2 micro M). NS3728 accelerated channel inactivation at positive potentials. These results show that acidic di-aryl ureas constitute a promising starting point for the synthesis of potent inhibitors of VRAC.

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