1.A.51 The Voltage-gated Proton Channel (VPC) Family

Voltage-gated ion channels of the VIC family (1.A.1) consist of four 6 TMS domains. TMSs 1-4 in the 4 subunits or domains constitute the voltage sensor while TMSs 5 and 6 comprise the ion channel (Nelson et al., 1999). Proteins consisting only of the sensor domain (TMSs 1-4) have been identified. Sasaki et al. (2006) identified the mouse RIKEN cDNA 0610039P13 in the GenBank database as such a 4 TMS protein and named it mouse voltage sensor domain-only protein (mVSOP). They showed it is a voltage-gated proton channel. Similarly, Ramsey et al. (2006) identified a human gene called H_v1 as a voltage-gated proton channel. The latter group had previously identified an ascidian voltage-sensing domain homologous to those in VIC family members, that gates (regulates) the associated phosphatase activity rather than a channel activity.  Purified Hv channels, free of all other proteins, by themselves, catalyze H⁺ fluxes (Lee et al., 2009).  Hv1 most likely forms an internal water wire for selective proton transfer, and interactions between water molecules and S4 arginines may underlie coupling between voltage- and pH-gradient sensing (Ramsey et al. 2010).  It also responds to mechanical stress (Pathak et al. 2016).  The S4 segment of these channels transits between three major conformational states, and only the transitions between the inward and outward conformations are highly dependent on voltage and pH (Han et al. 2022). Arachidonic acid reverses cholesterol and zinc inhibition of human voltage-gated proton channels (Han et al. 2023).

The mouse and human VPC proteins are >90% identical. H_v1 is activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H⁺-selective, and Zn²⁺-sensitive. Three arg residues in S4 regulate channel gating, and two his residues are required for extracellular inhibition by Zn²⁺. H_v1 is present in phagocytic leukocytes and is required for the oxidative burst that underlies microbial killing via the gp91^phox phagocyte NADPH oxidase associated cytochrome b₅₅₈ (CytB) family (TC #1.A.20). This argues against the contested claim that gp91^phox is itself (or contains) H⁺ channel activity. The human voltage gated Hv1 is a homodimer (Lee et al., 2008) with two pores and gates (Tombola et al., 2008). The dimer interface corresponds to the interface between the voltage sensor and pore in Kv channels (Lee et al., 2008; DeCoursey, 2008). The monomer is probably also functional (Koch et al., 2008; Tombola et al., 2008).

Homologues of VPCs are found in ascidians, zebrafish, Xenopus and mammals (Sasaki et al., 2006). While TMSs 2 and 3 contain well-conserved negatively charged residues, TMS4 contains positively charged residues. These charged residues presumably confer upon these transmembrane proteins both their H⁺ channel activities and their voltage sensor capacities. Of the VIC family members, these proteins show greatest sequence similarity with the voltage sensors of voltage-gated Na⁺ channels. Because they lack the channel domain (TMSs 5 and 6) of VIC family members, the VPC family is considered distinct from the VIC family. However, it can be considered to be in the VIC superfamily.

Voltage-gated ion channels derive their voltage sensitivity from the movement of specific charged residues in response to a change in transmembrane potential. Several studies on mechanisms of voltage sensing in ion channels support the idea that these gating charges move through a well-defined permeation pathway. This gating pathway in a voltage-gated ion channel can also be mutated to transport free cations, including protons (Chanda and Bezanilla, 2008). The discovery of proton channels homologous to voltage-sensing domains suggests that the same gating pathway is used by voltage-dependent proton transporters. Several biological proton-selective ion channels exist (DeCoursey 2023). Some open at positive or negative membrane potentials, others open at low or high pH, and some are light activated. The review by DeCoursey 2023 focuses on common features that result from the unique properties of protons.

The Hv1 channel of humans (1.A.51.1.2) contains a voltage-sensing domain (VSD), similar to those of voltage-gated sodium, potassium and calcium channels. The pore domain of these other channels, which forms a central pore at the interface of the four subunits, is missing in Hv1. Tombola et al. 2009 review efforts to understand the structural organization of Hv1 channels. They discuss the relationship between the gating of Hv1 and the gating of ion-conducting pores recently discovered in the VSDs of mutant voltage-gated potassium and sodium channels.  Flagellar Hv1-dependent proton conductance in human sperm is activated by membrane depolarization, an alkaline extracellular environment, endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa (Lishko et al. 2010).

Voltage-gated proton channels are designed to extrude large quantities of cytosolic acid in response to depolarising voltages. The discovery of the Hvcn1 gene and the generation of mice lacking the channel molecule have confirmed several postulated functions of proton channels in leukocytes. In neutrophils and macrophages, proton channels are required for high-level production of superoxide anions by the phagocytic NADPH oxidase, a bactericidal enzyme essential for host defence against infections. In B lymphocytes, proton channels are required for low-level production of superoxide that boosts the production of antibodies. Proton channels sustain the activity of immune cells in several ways. By extruding excess cytosolic acid, proton channels prevent deleterious acidification of the cytosol and at the same time deliver protons required for chemical conversion of the superoxide secreted by membrane oxidases. By moving positive charges across membranes, proton channels limit the depolarisation of the plasma membrane, promoting the electrogenic activity of NADPH oxidases and the entry of calcium ions into cells. Acid extrusion by proton channels is not restricted to leukocytes but also mediates the intracellular alkalinisation required for the activation of spermatozoids. Proton channels are therefore multitalented channels that control male fertility as well as our innate and adaptive immunity (Demaurex & El Chemaly et al., 2010).

Voltage-gated proton (Hv) channels play an essential role in phagocytic cells by generating a hyperpolarizing proton current that electrically compensates for the depolarizing current generated by the NADPH oxidase during the respiratory burst, thereby ensuring a sustained production of reactive oxygen species by the NADPH oxidase in phagocytes to neutralize engulfed bacteria. Neutralizations of three charged residues in the fourth transmembrane domain, S4, reduce the voltage dependence of activation (Gonzalez et al. 2013). The middle S4 charged residue moves from a position accessible from the cytosolic solution to a position accessible from the extracellular solution, suggesting that this residue moves across most of the membrane electric field during voltage activation. The charge movement of these three S4 charges accounts for almost all of the measured gating charge in Hv channels (Gonzalez et al. 2013). 

Hv assembles as a dimeric channel, and the two transmembrane channel domains function cooperatively, mediated by the coiled-coil assembly domain in the cytoplasmic C terminus. A picture of the dimer configuration based on the analyses of interactions among the two voltage sensor domains (VSDs) and a coiled-coil domain has been presented (Fujiwara et al. 2014). The two S4 helices are probably situated closely in the dimeric channel. Continuous helices stretching from the transmembrane to the cytoplasmic region in the dimeric interface may regulate channel activation in the Hv dimer.  The voltage sensing domain (VSD) of the voltage-gated proton channel, Hv1, mediates a H⁺-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH) (Villalba-Galea 2014). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To understand the mechanism for V-ΔpH coupling in Hv1, H⁺ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pHI) (Villalba-Galea 2014). Opening of the Hv1 channel is preceded by a voltage-independent transition. For Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. 

The voltage-gated proton channel Hv1 plays a critical role in the fast proton translocation that underlies a wide range of physiological functions, including the phagocytic respiratory burst, sperm motility, apoptosis, and metastatic cancer. Both voltage activation and proton conduction are carried out by a voltage-sensing domain (VSD) with strong similarity to canonical VSDs in voltage- dependent cation channels and enzymes (Li et al. 2015).

Hv1 is a dimer of two voltage-sensing domains (VSDs), each containing a pore pathway, a voltage sensor (S4), and a gate (S1), forming its own ion channel (Mony et al. 2020). Opening of the two channels in the dimer is cooperative. Part of the cooperativity is due to association between coiled-coil domains that extend intracellularly from the S4s. Interactions between the transmembrane portions of the subunits may also contribute. Using functional analysis of a mutagenesis scan, biochemistry, and modeling, Mony et al. 2020 found that the subunits form a dimer interface along the entire length of S1, and also have intersubunit contacts between S1 and S4. These interactions exert a strong effect on gating, in particular on the stability of the open state. These results suggest that gating in Hv1 is tuned by extensive VSD-VSD interactions between the gates and voltage sensors of the dimeric channel.

The generalized transport reaction catalyzed by members of the VPC family is:

H⁺ (in) → H⁺ (out)