Transcendent Aspects of Proton Channels.

A handful of biological proton-selective ion channels exist. Some open at positive or negative membrane potentials, others open at low or high pH, and some are light activated. This review focuses on common features that result from the unique properties of protons. Proton conduction through water or proteins differs qualitatively from that of all other ions. Extraordinary proton selectivity is needed to ensure that protons permeate and other ions do not. Proton selectivity arises from a proton pathway comprising a hydrogen-bonded chain that typically includes at least one titratable amino acid side chain. The enormously diverse functions of proton channels in disparate regions of the phylogenetic tree can be summarized by considering the chemical and electrical consequences of proton flux across membranes. This review discusses examples of cells in which proton efflux serves to increase pHi, decrease pHo, control the membrane potential, generate action potentials, or compensate transmembrane movement of electrical charge.

Interior pH Sensing Residue of Human Voltage-Gated Proton Channel Hv1 is Histidine 168.

The molecular mechanisms governing the human voltage-gated proton channel hHv1 remain elusive. Here we used membrane-enabled hybrid-solvent continuous constant pH molecular dynamics (CpHMD) simulations with pH replica exchange to further evaluate the recently obtained structural models of hHv1 in the closed (hyperpolarized) and open (depolarized) states (Geragotelis, Tobias et al., Proc. Natl. Acad. Sci. USA 2020) and explore potential pH-sensing residues. The CpHMD titration at a set of symmetric pH conditions revealed three residues that can gain or lose protons upon channel depolarization. Among them residue H168 at the intracellular end of the S3 helix switches from the deprotonated to the protonated state and its protonation is correlated with the increased tilting of the S3 helix during the transition from the closed- to the open state. Thus, the simulation data suggest H168 as an interior pH sensor, in support of a recent finding based on electrophysiological experiments of Hv1 mutants (Cherny, DeCoursey et al., J. Gen. Physiol. 2018). We propose that protonation of H168 acts as a key that unlocks the closed channel configuration by increasing the flexibility of the S2-S3 linker, which increases the tilt angle of S3 and enhances the mobility of the S4 helix, thus promoting channel opening. Our work represents an important step towards deciphering the pH-dependent gating mechanism of hHv1.

Interaction with stomatin directs human proton channels into cholesterol-dependent membrane domains.

Many membrane proteins are modulated by cholesterol. Here we report profound effects of cholesterol depletion and restoration on the human voltage-gated proton channel, hHV1, in excised patches but negligible effects in the whole-cell configuration. Despite the presence of a putative cholesterol-binding site, a CARC motif in hHV1, mutation of this motif did not affect cholesterol effects. The murine HV1 lacks a CARC sequence but displays similar cholesterol effects. These results argue against a direct effect of cholesterol on the HV1 protein. However, the data are fully explainable if HV1 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 HV1 channels can diffuse between seal and patch membranes when suction is applied. Simultaneous truncation of the large intracellular N and C termini of hHV1 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 hHV1 binding to stomatin. The stomatin-mediated association of HV1 with cholesterol-dependent lipid domains provides a mechanism for cells to direct HV1 to subcellular locations where it is needed, such as the phagosome in leukocytes.

The HVCN1 voltage-gated proton channel contributes to pH regulation in canine ventricular myocytes.

Regulation of intracellular pH (pHi ) in cardiomyocytes is crucial for cardiac function; however, currently known mechanisms for direct or indirect extrusion of acid from cardiomyocytes seem insufficient for energetically efficient extrusion of the massive H+ loads generated under in vivo conditions. In cardiomyocytes, voltage-sensitive H+ channel activity mediated by the HVCN1 proton channel would be a highly efficient means of disposing of H+ , while avoiding Na+ loading, as occurs during direct acid extrusion via Na+ /H+ exchange or indirect acid extrusion via Na+ -HCO3- cotransport. PCR and immunoblotting demonstrated expression of HVCN1 mRNA and protein in canine heart. Patch clamp analysis of canine ventricular myocytes revealed a voltage-gated H+ current that was highly H+ -selective. The current was blocked by external Zn2+ and the HVCN1 blocker 5-chloro-2-guanidinobenzimidazole. Both the gating and Zn2+ blockade of the current were strongly influenced by the pH gradient across the membrane. All characteristics of the observed current were consistent with the known hallmarks of HVCN1-mediated H+ current. Inhibition of HVCN1 and the NHE1 Na+ /H+ exchanger, singly and in combination, showed that either mechanism is largely sufficient to maintain pHi in beating cardiomyocytes, but that inhibition of both activities causes rapid acidification. These results show that HVCN1 is expressed in canine ventricular myocytes and provides a major H+ extrusion activity, with a capacity similar to that of NHE1. In the beating heart in vivo, this activity would allow Na+ -independent extrusion of H+ during each action potential and, when functionally coupled with anion transport mechanisms, could facilitate transport-mediated CO2 disposal. KEY POINTS: Intracellular pH (pHi ) regulation is crucial for cardiac function, as acidification depresses contractility and causes arrhythmias. H+ ions are generated in cardiomyocytes from metabolic processes and particularly from CO2 hydration, which has been shown to facilitate CO2 venting from mitochondria. Currently, the NHE1 Na+ /H+ exchanger is viewed as the dominant H+ extrusion mechanism in cardiac muscle. We show that the HVCN1 voltage-gated proton channel is present and functional in canine ventricular myocytes, and that HVCN1 and NHE1 both contribute to pHi regulation. HVCN1 provides an energetically efficient mechanism of H+ extrusion that would not cause Na+ loading, which can cause pathology, and that could contribute to transport-mediated CO2 disposal. These results provide a major advance in our understanding of pHi regulation in cardiac muscle.

HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species.

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels.

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this "gating pore" when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open-closed gating, but strikingly, at negative voltages where "normal" gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 µM Zn2+ Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family.

Voltage-gated proton channels (H(V)) are unique, in part because the ion they conduct is unique. H(V) channels are perfectly selective for protons and have a very small unitary conductance, both arguably manifestations of the extremely low H(+) concentration in physiological solutions. They open with membrane depolarization, but their voltage dependence is strongly regulated by the pH gradient across the membrane (ΔpH), with the result that in most species they normally conduct only outward current. The H(V) channel protein is strikingly similar to the voltage-sensing domain (VSD, the first four membrane-spanning segments) of voltage-gated K(+) and Na(+) channels. In higher species, H(V) channels exist as dimers in which each protomer has its own conduction pathway, yet gating is cooperative. H(V) channels are phylogenetically diverse, distributed from humans to unicellular marine life, and perhaps even plants. Correspondingly, H(V) functions vary widely as well, from promoting calcification in coccolithophores and triggering bioluminescent flashes in dinoflagellates to facilitating killing bacteria, airway pH regulation, basophil histamine release, sperm maturation, and B lymphocyte responses in humans. Recent evidence that hH(V)1 may exacerbate breast cancer metastasis and cerebral damage from ischemic stroke highlights the rapidly expanding recognition of the clinical importance of hH(V)1.

The intimate and controversial relationship between voltage-gated proton channels and the phagocyte NADPH oxidase.

One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase complex and voltage-gated proton channels (HV 1). By the time I entered this field, there had already been substantial progress toward understanding NADPH oxidase, but HV 1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then-recent discoveries of Henderson, Chappell, and colleagues in 1987-1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane: NADPH oxidase moves electrons and HV 1 moves protons. The consequences of electrogenic NADPH oxidase activity on both membrane potential and pH strongly self-limit this enzyme. Fortunately, both consequences specifically activate HV 1, and HV 1 activity counteracts both consequences, a kind of yin-yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship between NADPH oxidase and HV 1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyte NADPH oxidase - an industrial strength producer of reactive oxygen species (ROS) - to myriad other cells that produce orders of magnitude less ROS for signaling purposes. These cells with their seven NADPH oxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come.

Aspartate 112 is the selectivity filter of the human voltage-gated proton channel.

The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel, H(V)1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes, histamine secretion by basophils, sperm capacitation, and airway pH. The most selective ion channel known, H(V)1 shows no detectable permeability to other ions. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions. Here we identify aspartate 112 as a crucial component of the selectivity filter of H(V)1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H(V)1 requires an acidic group at the selectivity filter.

Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells.

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr(29). Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.

The Voltage-Gated Proton Channel: A Riddle, Wrapped in a Mystery, inside an Enigma.

The main properties of the voltage-gated proton channel (HV1) are described in this review, along with what is known about how the channel protein structure accomplishes its functions. Just as protons are unique among ions, proton channels are unique among ion channels. Their four transmembrane helices sense voltage and the pH gradient and conduct protons exclusively. Selectivity is achieved by the unique ability of H3O(+) to protonate an Asp-Arg salt bridge. Pathognomonic sensitivity of gating to the pH gradient ensures HV1 channel opening only when acid extrusion will result, which is crucial to most of its biological functions. An exception occurs in dinoflagellates in which influx of H(+) through HV1 triggers the bioluminescent flash. Pharmacological interventions that promise to ameliorate cancer, asthma, brain damage in ischemic stroke, Alzheimer's disease, autoimmune diseases, and numerous other conditions await future progress.

Voltage-gated proton channel in a dinoflagellate.

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kH(V)1: it is proton-specific and activated by depolarization, its g(H)-V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp(51)) results in loss of proton-specific conduction. Indirect evidence suggests that kH(V)1 is monomeric, unlike other proton channels. Furthermore, kH(V)1 differs from all known proton channels in activating well negative to the Nernst potential for protons, E(H). This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability.

NOX5 in human spermatozoa: expression, function, and regulation.

Physiological and pathological processes in spermatozoa involve the production of reactive oxygen species (ROS), but the identity of the ROS-producing enzyme system(s) remains a matter of speculation. We provide the first evidence that NOX5 NADPH oxidase is expressed and functions in human spermatozoa. Immunofluorescence microscopy detected NOX5 protein in both the flagella/neck region and the acrosome. Functionally, spermatozoa exposed to calcium ionophore, phorbol ester, or H(2)O(2) exhibited superoxide anion production, which was blocked by addition of superoxide dismutase, a Ca(2+) chelator, or inhibitors of either flavoprotein oxidases (diphenylene iododonium) or NOX enzymes (GKT136901). Consistent with our previous overexpression studies, we found that H(2)O(2)-induced superoxide production by primary sperm cells was mediated by the non-receptor tyrosine kinase c-Abl. Moreover, the H(V)1 proton channel, which was recently implicated in spermatozoa motility, was required for optimal superoxide production by spermatozoa. Immunoprecipitation experiments suggested an interaction among NOX5, c-Abl, and H(V)1. H(2)O(2) treatment increased the proportion of motile sperm in a NOX5-dependent manner. Statistical analyses showed a pH-dependent correlation between superoxide production and enhanced sperm motility. Collectively, our findings show that NOX5 is a major source of ROS in human spermatozoa and indicate a role for NOX5-dependent ROS generation in human spermatozoa motility.