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

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. Introduction: Hϩ is a key signaling ion in bone resorption. In addition to H ϩ pumps and exchangers, osteoclasts are equipped with H ϩ conductive pathways to compensate rapidly for pH imbalance. The H ϩ channel is distinct in its strong H ϩ extrusion ability and voltage-dependent gatings. Methods: To investigate how and when the H ϩ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H ϩ 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 ϩ current was enhanced by increasing the pH gradient across the plasma membrane (⌬pH), indicating that the H ϩ channel changed its activity by sensing ⌬pH. The reversal potential (V rev ) was a valuable tool for the real-time monitoring of ⌬pH in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 M) 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

A pH-stabilizing role of voltage-gated proton channels in IgE-mediated activation of human basophils

“…In phagocytes, they are thought to enable sustained NADPH oxidase activity by compensating for the electrogenic activity of the oxidase (3)(4)(5). Stimuli that activate NADPH oxidase in human eosinophils and neutrophils and in murine osteoclasts greatly enhance the opening of proton channels in these cells studied in perforated-patch configuration (6)(7)(8)(9)(10), largely via PKC phosphorylation (11). In contrast, phorbol myristate acetate (PMA) has no clear effect on proton currents in alveolar epithelial cells that lack NADPH oxidase (6).…”

Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells

“…A more profound difference was seen in response to stimulation by PMA. Agonists that activate PKC strongly amplify the proton conductance in many human and mammalian cells (8,9,(14)(15)(16)(17)(18), a phenomenon called "enhanced gating." Although both isoforms responded to PMA, the HVCN1 S response was significantly greater.…”

Early and late activation of the voltage-gated proton channel during lactic acidosis through pH-dependent and -independent mechanisms

“…The reversal potentials (V rev ) were obtained by either the tail-current method or the repolarization-pulse method [11,18,20]. In the former, the I-V relationships were obtained from instantaneous tail currents at different voltages following a constant voltage pulse (+100 mV, 1-2 s).…”

“…In most of recordings, 50 μM 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), a Cl − channel blocker, was added to the bath solution. The buffer molecules (Hepes) could not pass through the amphotericin B pores [20], and so the pH i was controlled by the intrinsic pH buffers. The membrane potential was measured under the current-clamp configuration in the standard Ringer solution: Cs-methanesulfonate in the pipette solution was replaced by K-gluconate.…”