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Voltage gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Although these molecules are distinct entities, with no proven physical interaction, their presence and activity in many cells appears to be coordinated. We describe these interactions and discuss several types of mechanisms that might explain them.

The voltage-gated proton channel 1 ( HV1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV1 channel. In the present study, we report the existence of an HV1-type proton channel in insects. We searched insect transcriptome shotgun assembly ( TSA) sequence databases and found putative HV1 orthologues in various polyneopteran insects. To confirm that these putative HV1 orthologues were functional channels, we studied the HV1 channel of Nicoletia phytophila (Np HV1), an insect of the Zygentoma order, in more detail. Np HV1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, Np HV1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels. Database Nucleotide sequence data have been deposited in the GenBank database under accession number . [ABSTRACT FROM AUTHOR]

Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pHi regulation. The "enhanced gating mode" appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-δ phosphorylation sites in the human voltage-gated proton channel, Hv1 (Hvcn1), Thr29 and Ser97, both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-δ, Enhanced gating was evaluated by expressing wild-type (WT) or mutant Hv1 channels in LK35.2 cells, a B cell hybridoma, Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect seas reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT Hv1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr29 is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes. [ABSTRACT FROM AUTHOR]

The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable of clearing invading pathogens but can also inflict damage to host tissues. It is therefore important to prevent an overshoot of the TLR-induced response where necessary, and here we show that extracellular ATP is capable of doing this in human monocytes. Using reverse transcription-PCR, we showed that monocytes express P2Y1, P2Y2, P2Y4, P2Y11, and P2Y13 receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca2+ signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca2+, which corresponded to internal Ca2+ release followed by activation of store-operated Ca2+ entry. The evoked Ca2+ signals stimulated Ca2+-activated K+ channels, producing transient membrane hyperpolarization. In addition, ATP promoted cytoskeleton reorganization and cell migration; however, unlike chemoattractants, the migration was non-directional and further analysis showed that ATP did not activate Akt, essential for sensing gradients. When TLR2, TLR4, or TLR2/6 were stimulated with their respective ligands, ATPγS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-α and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPγS) strongly increased cAMP levels, and, moreover, the TLR-response was inhibited by forskolin, whereas UTP neither increased cAMP nor inhibited the TLR-response. Thus, our data suggest that ATP promotes non-directional migration and, importantly, acts as a ‘host tissue damage’ signal via the Gs protein-coupled P2Y11 receptor and increased cAMP to negatively regulate TLR signaling. [ABSTRACT FROM AUTHOR]