Regulatory Mechanisms and Physiological Relevance of a Voltage‐Gated H+ Channel in Murine Osteoclasts: Phorbol Myristate Acetate Induces Cell Acidosis and the Channel Activation

• Published in: Journal of bone and mineral research Vol. 18; no. 11; pp. 2069 - 2076
• Main Authors: Mori, Hiroyuki, Sakai, Hiromu, Morihata, Hirokazu, Kawawaki, Junko, Amano, Hitoshi, Yamano, Tsunekazu, Kuno, Miyuki
• Format: Journal Article
• Language: English
• Published: Washington, DC John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR) 01.11.2003; American Society for Bone and Mineral Research
• Subjects: Acidosis - chemically induced; Acidosis - metabolism; Animals; Biological and medical sciences; cell acidosis; Electric Conductivity; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Ion Channel Gating - drug effects; Ion Channels - metabolism; Male; Membrane Potentials - drug effects; Mice; osteoclast; Osteoclasts - drug effects; Osteoclasts - metabolism; Patch-Clamp Techniques; pH homeostasis; phorbol ester; proton channel; Protons; Skeleton and joints; Tetradecanoylphorbol Acetate - pharmacology; Vertebrates: osteoarticular system, musculoskeletal system; phorbol ester; Rodentia; Electrophysiology; Homeostasis; Acidity; Activation; Ionic channel; Osteoclast; cell acidosis; Acetate; Osteoarticular system; proton channel; Ester; Vertebrata; Mammalia; Mouse; Animal; pH homeostasis; Proton; pH; Bone
• ISSN: 0884-0431; 1523-4681
• DOI: 10.1359/jbmr.2003.18.11.2069

The voltage‐gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis. Introduction: H+ is a key signaling ion in bone resorption. In addition to H+ pumps and exchangers, osteoclasts are equipped with H+ conductive pathways to compensate rapidly for pH imbalance. The H+ channel is distinct in its strong H+ extrusion ability and voltage‐dependent gatings. Methods: To investigate how and when the H+ channel is available in functional osteoclasts, the effects of phorbol 12‐myristate 13‐acetate (PMA), an activator for protein kinase C, on the H+ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage‐colony stimulating factor (M‐CSF). Results and Conclusions: Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (ΔpH), indicating that the H+ channel changed its activity by sensing ΔpH. The reversal potential (Vrev) was a valuable tool for the real‐time monitoring of ΔpH in clamped cells. In the permeabilized patch, PMA (10 nM‐1.6 μM) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of Vrev, suggesting that intracellular acidification occurred. The PMA‐induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K+‐rich alkaline solution, probably through the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine. In ∼80% of cells, the PMA‐induced augmentation in the current activity remained after compensating for the ΔpH changes, implying that both ΔpH‐dependent and ‐independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward Vrev. These data suggest that the H+ channel may contribute to regulation of the pH environments and the membrane potential in osteoclasts activated by protein kinase C.