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The voltage-gated proton channel 1 (H(V)1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. H(V)1 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 H(V)1 channel. In the present study, we report the existence of an H(V)1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative H(V)1 orthologues in various polyneopteran insects. To confirm that these putative H(V)1 orthologues were functional channels, we studied the H(V)1 channel of Nicoletia phytophila (NpH(V)1), an insect of the Zygentoma order, in more detail. NpH(V)1 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, NpH(V)1 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. DatabaseNucleotide sequence data have been deposited in the GenBank database under accession number .

The voltage-gated proton channel 1 (H(V)1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. H(V)1 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 H(V)1 channel. In the present study, we report the existence of an H(V)1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative H(V)1 orthologues in various polyneopteran insects. To confirm that these putative H(V)1 orthologues were functional channels, we studied the H(V)1 channel of Nicoletia phytophila (NpH(V)1), an insect of the Zygentoma order, in more detail. NpH(V)1 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, NpH(V)1 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. DatabaseNucleotide sequence data have been deposited in the GenBank database under accession number .

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 P2Y(1), P2Y(2), P2Y(4), P2Y(11), and P2Y(13) receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca(2+) signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca(2+), which corresponded to internal Ca(2+) release followed by activation of store-operated Ca(2+) entry. The evoked Ca(2+) signals stimulated Ca(2+)-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, ATPgammaS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-alpha and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPgammaS) 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 G(s) protein-coupled P2Y(11) receptor and increased cAMP to negatively regulate TLR signaling., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

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 P2Y(1), P2Y(2), P2Y(4), P2Y(11), and P2Y(13) receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca(2+) signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca(2+), which corresponded to internal Ca(2+) release followed by activation of store-operated Ca(2+) entry. The evoked Ca(2+) signals stimulated Ca(2+)-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, ATPgammaS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-alpha and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPgammaS) 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 G(s) protein-coupled P2Y(11) receptor and increased cAMP to negatively regulate TLR signaling., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published