Antibodies against ClC7 inhibit extracellular acidification-induced Cl− currents and bone resorption activity in mouse osteoclasts

Abstract

The Cl⁻ channel/transporter ClC7 is crucial for osteoclastic bone resorption and might become a therapeutic target for osteoporosis. In this study, we raised anti-ClC7 polyclonal antibodies against three different peptide sequences, including G215, P249, and R286, which are the mutation regions found in autosomal dominant osteopetrosis type II patients and examined the effects of these antibodies on the ClC7 Cl⁻ current induced by extracellular acidification (acid-activated Cl⁻ current) using the whole-cell patch clamp technique and bone resorption activity in mouse osteoclasts. Intracellular dialysis of osteoclasts with antibodies to intracellular G215 (Ab-G215) and extracellular application of antibodies to extracellular P249 (Ab-P249) or R286 (Ab-R286) inhibited the acid-activated Cl⁻ current. These antibodies also suppressed the acid-activated Cl⁻ current in ClC7 overexpressing Raw264.7 cells; however, Cl⁻ currents evoked by hypotonic stimulation and the inherent inwardly rectifying K+ currents in mouse osteoclasts were unaffected by these antibodies. Furthermore, extracellularly applied Ab-P249 and Ab-R286 also reduced bone resorption activity. Our results demonstrate that these antibodies specifically block ClC7 in mouse osteoclasts. Thus, anti-ClC7 antibodies have potential promise for treatment of osteoporosis.

The ClC-7 Chloride Channel Is Downregulated by Hypoosmotic Stress in Human Chondrocytes

“…ClC-7 currents are known to be activated by extracellular acidic pH (Diewald et al, 2002;Kajiya et al, 2009;Ohgi et al, 2011). In our experiments, the Cl 2 currents were not affected by pH 6.0 (2.8 6 0.4 pA/pF at 1100 mV, n 5 4, P .…”

Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties

“…Since extracellular acidification was observed to activate anionic currents conducted via a number of ClC channel/transporter family members, including ClC-0 (Chen and Chen, 2001), ClC-2 (Jordt and Jentsch, 1997;Stroffekova et al, 1998), ClC-3 (Matsuda et al, 2010a, and ClC-7 (Diewald et al, 2002;Kajiya et al, 2009), it is possible that some ClC members are associated with the molecular entity of ASOR. ClC-3 (Matsuda et al, 2010a;Wang et al, 2012) and ClC-7 (Kajiya et al, 2009;Ohgi et al, 2011) were suggested to be responsible for the ASOR molecule. However, this possibility was recently ruled out in light of the following observations.…”

Acid- and Volume-Sensitive Chloride Currents in Microglial Cells

“…The biophysical and pharmacological profile of this current is remarkably similar in different cell types. Its activation threshold is around pH 5.0, it is strongly outwardly rectifying, shows time-dependent activation at constant positive membrane potentials (V mem ), maintains an ion permeability sequence of SCN − > I − > NO 3 − > Br − > Cl − , and it is blocked by a wide range of unspecific Cl − channel blockers such as DIDS, NBBP, niflumic acid, or phloretin [1,2,3,4,5,6,7,8,9,10,11,12,13,14].…”

“…While the role of VSOR channels in cell volume homeostasis as efflux pathway for anions and osmolytes is increasingly understood, functions of the ASOR current are less obvious, given that massive acidification is required to activate the conductance. E.g., an involvement in acidotoxic necrotic cell death, bone reabsorption, and spermatogenesis has been suggested in epithelial HeLa cells and mouse cortical neurons [9,10], osteoclasts [7,8], and Sertoli cells [1], respectively.…”