Search

Recently, three proton channels (HV) have been identified and characterized in Aplysia californica (AcHV1‐3). Focusing on AcHV1 and AcHV2, analysis of Transcriptome Shotgun Assembly and genomic databases of 91 molluscs identified HV homologous channels in other molluscs: channels homologous to AcHV1 and to AcHV2 were found in 90 species (56 full‐length sequences) and in 33 species (18 full‐length sequences), respectively. Here, we report the discovery of a fourth distinct proton channel family, HV4. This new family has high homology to AcHV1 and AcHV2 and was identified only in bivalvian molluscs (13 species, 12 full‐length sequences). Typically, these channels possess an extracellular S1–S2 loop of intermediate size (~ 20 amino acids) compared to the shorter loops of molluscan HV1 channels (~ 13 amino acids) and the much larger loops of molluscan HV2 channels (> 65 amino acids). The characteristic voltage‐sensor motif in S4 possesses only two arginine residues with the common third arginine being replaced by a lysine. Moreover, HV4 channels are much smaller with only around 200 amino acids in total length. The smallest functional channel found so far in nature (189 amino acids) is expressed in the pacific oyster Crassostrea gigas (CgHV4) and might be considered an archetypical minimal proton channel. Functional expression and electrophysiological characterization demonstrated that CgHV4 shares distinctive hallmarks of other investigated proton channels as high proton selectivity, slow activation, and pH‐ and voltage‐regulated gating. This work is the first description of a HV4 type channel, adding a new member to the recently expanded family of proton channels. [ABSTRACT FROM AUTHOR]

Voltage‐gated ion channels, whose first identified function was to generate action potentials, are divided into subfamilies with numerous members. The family of voltage‐gated proton channels (HV) is tiny. To date, all species found to express HV have exclusively one gene that codes for this unique ion channel. Here we report the discovery and characterization of three proton channel genes in the classical model system of neural plasticity, Aplysia californica. The three channels (AcHV1, AcHV2, and AcHV3) are distributed throughout the whole animal. Patch‐clamp analysis confirmed proton selectivity of these channels but they all differed markedly in gating. AcHV1 gating resembled HV in mammalian cells where it is responsible for proton extrusion and charge compensation. AcHV2 activates more negatively and conducts extensive inward proton current, properties likely to acidify the cytosol. AcHV3, which differs from AcHV1 and AcHV2 in lacking the first arginine in the S4 helix, exhibits proton selective leak currents and weak voltage dependence. We report the expansion of the proton channel family, demonstrating for the first time the expression of three functionally distinct proton channels in a single species. [ABSTRACT FROM AUTHOR]

Voltage‐gated proton channels (HV1) are expressed in eukaryotes, including basal hexapods and polyneopteran insects. However, currently, there is little known about HV1 channels in insects. A characteristic aspartate (Asp) that functions as the proton selectivity filter (SF) and the RxWRxxR voltage‐sensor motif are conserved structural elements in HV1 channels. By analysing Transcriptome Shotgun Assembly (TSA) databases, we found 33 polyneopteran species meeting these structural requirements. Unexpectedly, an unusual natural variation Asp to glutamate (Glu) at SF was found in Phasmatodea and Mantophasmatodea. Additionally, we analysed the expression and function of HV1 in the phasmatodean stick insect Extatosoma tiaratum (Et). EtHV1 is strongly expressed in nervous tissue and shows pronounced inward proton conduction. This is the first study of a natural occurring Glu within the SF of a functional HV1 and might be instrumental in uncovering the physiological function of HV1 in insects. [ABSTRACT FROM AUTHOR]

The HV1 voltage‐gated proton (HV1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best‐described physiological inhibitor of HV1 is Zn2+. Externally applied ZnCl2 drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn2+ and Cd2+ on HV1 from Nicoletia phytophila, NpHV1. We introduced mutations at potential Zn2+ coordination sites and measured Zn2+ inhibition in different extracellular pH, with Zn2+ concentrations up to 1000 μm. Zn2+ inhibition in NpHV1 was quantified by the slowing of the activation time constant and a positive shift of the conductance–voltage curve. Replacing aspartate in the S3‐S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage–conductance curve, such that Zn2+ inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn2+ in the S3‐S4 linker. A simple Hodgkin Huxley model of NpHV1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high‐resolution clear native gel electrophoresis (hrCNE) confirmed that NpHV1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development. [ABSTRACT FROM AUTHOR]

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 are strongly inhibited by Zn2+, which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn2+. We hypothesize that high-affinity Zn2+ binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn2+ effects were weaker in monomeric channels. Mutation of His193 and His140 in various combinations and in tandem dimers revealed that channel opening was slowed by Zn2+ only when at least one His was present in each monomer, suggesting that in wild-type (WT) HV1, Zn2+ binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn2+ appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer–dimer interconversion occurs during activation of phagocytes appears to be ruled out. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

The potassium channels TASK-1 and TASK-3 show high sequence homology but differ in their sensitivity to extracellular divalent cations. Heterologous expression in HEK293 cells showed that the single-channel conductance of TASK-3 increased approximately four-fold after removal of external divalent cations, whereas the conductance of TASK-1 was unaffected. Replacing the glutamate at position 70 of TASK-3 by a lysine or arginine residue abolished the sensitivity to divalent cations. The reverse mutation in TASK-1 (K70E) induced sensitivity to divalent cations. The organic polycations spermine and ruthenium red modulated the conductance of TASK-3 in a similar way as Ca2+ or Mg2+. Our data suggest that these effects were mediated by shielding of the negative charges in the extracellular loops of TASK-3. Whole-cell currents carried by TASK-3 channels were inhibited by spermine and ruthenium red even in the presence of external divalent cations. These data suggest that, in addition to their effect on single-channel conductance, spermine and ruthenium red decreased the open probability of TASK-3 channels, probably by binding to residue E70. The standing outward current in thalamocortical relay neurons, which is largely carried by TASK channels, was also inhibited by divalent cations and spermine. Using the differential sensitivity of TASK-1 and TASK-3 to divalent cations and spermine we found that about 20% of the standing outward current in thalamocortical relay neurons flows through TASK-3 channels. We conclude from our results that inhibition of TASK-3 channels may contribute to the neuromodulatory effect of spermine released from neurons during repetitive activity or during hypoxia. [ABSTRACT FROM AUTHOR]