Increases in intracellular pH facilitate endocytosis and decrease availability of voltage-gated proton channels in osteoclasts and microglia

Key points • Voltage-gated H+ channels help to compensate for the pH and voltage disturbances generated by production of reactive oxygen species. • In this study, we investigated how changes in the intracellular pH levels control H+ channel activity in macrophage-lineage cells, osteoclasts and microglia. • An increase in intracellular pH decreased the numbers of H+ channels available at the plasma membrane through facilitation of dynamin-dependent endocytic internalization. • This inhibitory regulation mechanism for H+ channels is novel. • The results help us to understand better the significance of the intracellular pH levels in membrane dynamics and H+ channel availability, which, in turn, may modulate natural immunity. Abstract  Voltage-gated proton channels (H+ channels) are highly proton-selective transmembrane pathways. Although the primary determinants for activation are the pH and voltage gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pHi) in regulating the availability of H+ channels in osteoclasts and microglia. In whole-cell clamp recordings, the pHi was elevated after exposure to NH4Cl and returned to the control level after washout. However, the H+ channel conductance did not recover fully when the exposure was prolonged (>5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H+ channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage dependence for activation, were unchanged, the decreases in the H+ channel conductance were probably due to the decreases in H+ channels available at the plasma membrane. The decreases in the H+ channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH4Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H+ channels, pHi increases by these H+-transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H+ channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pHi increases in osteoclasts and microglia decrease the numbers of H+ channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.