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

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Abstract
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. 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. 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). Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (delta(pH)), indicating that the H+ channel changed its activity by sensing delta(pH). The reversal potential (V(rev)) was a valuable tool for the real-time monitoring of delta(pH) in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 microM) 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 V(rev), 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 approximately 80% of cells, the PMA-induced augmentation in the current activity remained after compensating for the delta(pH) changes, implying that both delta(pH)-dependent and -independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward V(rev). 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.

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