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Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hH V 1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hH V 1, mutation of this motif did not affect cholesterol effects. The murine H V 1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the H V 1 protein. However, the data are fully explainable if H V 1 preferentially associates with cholesterol-dependent lipid domains, or "rafts." The rafts would be expected to concentrate in the membrane/glass interface and to be depleted from the electrically accessible patch membrane. This idea is supported by evidence that H V 1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hH V 1 greatly attenuated the cholesterol effect, but C truncation alone did not; this suggests that the N terminus is the region of attachment to lipid domains. Searching for abundant raft-associated proteins led to stomatin. Co-immunoprecipitation experiment results were consistent with hH V 1 binding to stomatin. The stomatin-mediated association of H V 1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct H V 1 to subcellular locations where it is needed, such as the phagosome in leukocytes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Recently, three proton channels (H V ) have been identified and characterized in Aplysia californica (AcH V 1-3). Focusing on AcH V 1 and AcH V 2, analysis of Transcriptome Shotgun Assembly and genomic databases of 91 molluscs identified H V homologous channels in other molluscs: channels homologous to AcH V 1 and to AcH V 2 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, H V 4. This new family has high homology to AcH V 1 and AcH V 2 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 H V 1 channels (~ 13 amino acids) and the much larger loops of molluscan H V 2 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, H V 4 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 (CgH V 4) and might be considered an archetypical minimal proton channel. Functional expression and electrophysiological characterization demonstrated that CgH V 4 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 H V 4 type channel, adding a new member to the recently expanded family of proton channels., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

With a single gene encoding H V 1 channel, proton channel diversity is particularly low in mammals compared to other members of the superfamily of voltage-gated ion channels. Nonetheless, mammalian H V 1 channels are expressed in many different tissues and cell types where they exert various functions. In the first part of this review, we regard novel aspects of the functional expression of H V 1 channels in mammals by differentially comparing their involvement in (1) close conjunction with the NADPH oxidase complex responsible for the respiratory burst of phagocytes, and (2) in respiratory burst independent functions such as pH homeostasis or acid extrusion. In the second part, we dissect expression of H V channels within the eukaryotic tree of life, revealing the immense diversity of the channel in other phylae, such as mollusks or dinoflagellates, where several genes encoding H V channels can be found within a single species. In the last part, a comprehensive overview of the biophysical properties of a set of twenty different H V channels characterized electrophysiologically, from Mammalia to unicellular protists, is given.

The voltage-gated proton channel, H V 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single-channel water permeability 2.3 × 10 -12 cm 3 s -1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the predicted value. Osmotic deflation of reconstituted lipid vesicles reveals negligible water permeability of the H V 1 wild-type channel and the D174A mutant open at 0 mV. The conductance of 1400 H + s -1 per wild-type channel agrees with the calculated diffusion limit for a ≈2 Å capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H + conductance by reducing the capture radius. At least one intervening amino acid contributes to H + conductance while interrupting the water wire across the membrane., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

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 (H V ) is tiny. To date, all species found to express H V 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 (AcH V 1, AcH V 2, and AcH V 3) are distributed throughout the whole animal. Patch-clamp analysis confirmed proton selectivity of these channels but they all differed markedly in gating. AcH V 1 gating resembled H V in mammalian cells where it is responsible for proton extrusion and charge compensation. AcH V 2 activates more negatively and conducts extensive inward proton current, properties likely to acidify the cytosol. AcH V 3, which differs from AcH V 1 and AcH V 2 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., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Gating of the voltage-gated proton channel H V 1 is strongly controlled by pH. There is evidence that this involves the sidechains of titratable amino acids that change their protonation state with changes of the pH. Despite experimental investigations to identify the amino acids involved in pH sensing only few progress has been made, including one histidine at the cytoplasmic side of the channel that is involved in sensing cellular pH. We have used constant pH molecular dynamics simulations in symmetrical and asymmetrical pH conditions across the membrane to investigate the pH- and ΔpH-dependent gating of the human H V 1 channel. Therefore, the p K a of every titratable amino acids has been assessed in single simulations. Our simulations captured initial conformational changes between a deactivated and an activated state of the channel induced solely by changes of the pH. The pH-dependent gating is accompanied by an outward displacement of the three S4 voltage sensing arginines that moves the second arginine past the hydrophobic gasket (HG) which separates the inner and outer pores of the channel. H V 1 activation, when outer pH increases, involves amino acids at the extracellular entrance of the channel that extend the network of interactions from the external solution down to the HG. Whereas, amino acids at the cytoplasmic entrance of the channel are involved in activation, when inner pH decreases, and in a network of interactions that extend from the cytoplasm up to the HG.

Voltage-gated proton channels (H V 1) are expressed in eukaryotes, including basal hexapods and polyneopteran insects. However, currently, there is little known about H V 1 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 H V 1 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 H V 1 in the phasmatodean stick insect Extatosoma tiaratum (Et). EtH V 1 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 H V 1 and might be instrumental in uncovering the physiological function of H V 1 in insects., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The respiratory burst of phagocytes is essential for human survival. Innate immune defence against pathogens relies strongly on reactive oxygen species (ROS) production by the NADPH oxidase (NOX2). ROS kill pathogens while the translocation of electrons across the plasma membrane via NOX2 depolarizes the cell. Simultaneously, protons are released into the cytosol. Here, we compare freshly isolated human polymorphonuclear leukocytes (PMN) to the granulocytes-like cell line PLB 985. We are recording ROS production while inhibiting the charge compensating and pH regulating voltage-gated proton channel (H V 1). The data suggests that human PMN and the PLB 985 generate ROS via a general mechanism, consistent of NOX2 and H V 1. Additionally, we advanced a mathematical model based on the biophysical properties of NOX2 and H V 1. Our results strongly suggest the essential interconnection of H V 1 and NOX2 during the respiratory burst of phagocytes. Zinc chelation during the time course of the experiments postulates that zinc leads to an irreversible termination of the respiratory burst over time. Flow cytometry shows cell death triggered by high zinc concentrations and PMA. Our data might help to elucidate the complex interaction of proteins during the respiratory burst and contribute to decipher its termination., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)