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 Hv1 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 mouse and human VPC proteins are >90% identical. Hv1 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+. Hv1 is present in phagocytic leukocytes and is required for the oxidative burst that underlies microbial killing via the gp91phox phagocyte NADPH oxidase associated cytochrome b558 (CytB) family (TC #1.A.20). This argues against the contested claim that gp91phox 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.

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).

 

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

H+ (in) → H+ (out)



This family belongs to the .

 

References:

and DeCoursey TE. (2015). The Voltage-Gated Proton Channel: A Riddle, Wrapped in a Mystery, inside an Enigma. Biochemistry. 54(21):3250-68.

Bennett, A.L. and I.S. Ramsey. (2017). CrossTalk opposing view: proton transfer in Hv1 utilizes a water wire, and does not require transient protonation of a conserved aspartate in the S1 transmembrane helix. J. Physiol. 595: 6797-6799.

Boonamnaj, P. and P. Sompornpisut. (2018). Insight into the Role of the Hv1 C-Terminal Domain in Dimer Stabilization. J Phys Chem B. [Epub: Ahead of Print]

Castillo K., Pupo A., Baez-Nieto D., Contreras GF., Morera FJ., Neely A., Latorre R. and Gonzalez C. (2015). Voltage-gated proton (Hv1) channels, a singular voltage sensing domain. FEBS Lett. 589(22):3471-8.

Chamberlin A., Qiu F., Rebolledo S., Wang Y., Noskov SY. and Larsson HP. (2014). Hydrophobic plug functions as a gate in voltage-gated proton channels. Proc Natl Acad Sci U S A. 111(2):E273-82.

Chanda, B., and F. Bezanilla (2008). A common pathway for charge transport through voltage-sensing domains. Neuron 57: 345-51.

Chaves, G., C. Derst, A. Franzen, Y. Mashimo, R. Machida, and B. Musset. (2016). Identification of an HV 1 Voltage-Gated Proton Channel in Insects. FEBS J. [Epub: Ahead of Print]

Cherny, V.V., D. Morgan, B. Musset, G. Chaves, S.M. Smith, and T.E. DeCoursey. (2015). Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel, hHV1. J Gen Physiol 146: 343-356.

Cherny, V.V., D. Morgan, S. Thomas, S.M.E. Smith, and T.E. DeCoursey. (2018). Histidine is crucial for ΔpH-dependent gating of the human voltage-gated proton channel, hH1. J Gen Physiol. [Epub: Ahead of Print]

De La Rosa, V. and I.S. Ramsey. (2018). Gating Currents in the Hv1 Proton Channel. Biophys. J. 114: 2844-2854.

DeCoursey, T.E. (2008). Voltage-gated proton channels: what's next? J. Physiol. 586: 5305-5324.

Demaurex, N. and A. El Chemaly. (2010). Physiological roles of voltage-gated proton channels in leukocytes. J. Physiol. 588: 4659-4665.

Fujiwara, Y., T. Kurokawa, and Y. Okamura. (2014). Long α helices projecting from the membrane as the dimer interface in the voltage-gated H+ channel. J Gen Physiol 143: 377-386.

Gianti, E., L. Delemotte, M.L. Klein, and V. Carnevale. (2016). On the role of water density fluctuations in the inhibition of a proton channel. Proc. Natl. Acad. Sci. USA 113: E8359-E8368.

Gonzalez, C., H.P. Koch, B.M. Drum, and H.P. Larsson. (2010). Strong cooperativity between subunits in voltage-gated proton channels. Nat Struct Mol Biol 17: 51-56.

Gonzalez, C., S. Rebolledo, M.E. Perez, and H.P. Larsson. (2013). Molecular mechanism of voltage sensing in voltage-gated proton channels. J Gen Physiol 141: 275-285.

Hong, L., V. Singh, H. Wulff, and F. Tombola. (2015). Interrogation of the intersubunit interface of the open Hv1 proton channel with a probe of allosteric coupling. Sci Rep 5: 14077.

Koch, H.P., T. Kurokawa, Y. Okochi, M. Sasaki, Y. Okamura, and H.P. Larsson. (2008). Multimeric nature of voltage-gated proton channels. Proc. Natl. Acad. Sci. USA 105: 9111-9116.

Lee, S.Y., J.A. Letts, and R. Mackinnon. (2008). Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1. Proc. Natl. Acad. Sci. USA 105: 7692-7695.

Lee, S.Y., J.A. Letts, and R. MacKinnon. (2009). Functional reconstitution of purified human Hv1 H+ channels. J. Mol. Biol. 387: 1055-1060.

Li, Q., R. Shen, J.S. Treger, S.S. Wanderling, W. Milewski, K. Siwowska, F. Bezanilla, and E. Perozo. (2015). Resting state of the human proton channel dimer in a lipid bilayer. Proc. Natl. Acad. Sci. USA 112: E5926-5935.

Lishko, P.V., I.L. Botchkina, A. Fedorenko, and Y. Kirichok. (2010). Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel. Cell 140: 327-337.

Mishra, A.K., A. Kumar, S. Yadav, M. Anand, B. Yadav, R. Nigam, S.K. Garg, and D.K. Swain. (2019). Functional insights into voltage gated proton channel (Hv1) in bull spermatozoa. Theriogenology 136: 118-130.

Morgan, D., B. Musset, K. Kulleperuma, S.M. Smith, S. Rajan, V.V. Cherny, R. Pomès, and T.E. Decoursey. (2013). Peregrination of the selectivity filter delineates the pore of the human voltage-gated proton channel hHV1. J Gen Physiol 142: 625-640.

Musset, B., M. Capasso, V.V. Cherny, D. Morgan, M. Bhamrah, M.J. Dyer, and T.E. DeCoursey. (2010). Identification of Thr29 as a critical phosphorylation site that activates the human proton channel Hvcn1 in leukocytes. J. Biol. Chem. 285: 5117-5121.

Nelson, R.D., G. Kuan, M.H. Saier, Jr., and M. Montal. (1999). Modular assembly of voltage-gated channel proteins: a sequence analysis and phylogenetic study. J. Mol. Microbiol. Biotechnol. 2: 281-287.

Okamura, Y., Y. Fujiwara, and S. Sakata. (2015). Gating mechanisms of voltage-gated proton channels. Annu. Rev. Biochem. 84: 685-709.

Okuda, H., Y. Yonezawa, Y. Takano, Y. Okamura, and Y. Fujiwara. (2016). Direct Interaction between the Voltage Sensors Produces Cooperative Sustained Deactivation in Voltage-gated H+ Channel Dimers. J. Biol. Chem. 291: 5935-5947.

Pathak, M.M., T. Tran, L. Hong, B. Joós, C.E. Morris, and F. Tombola. (2016). The Hv1 proton channel responds to mechanical stimuli. J Gen Physiol 148: 405-418.

Ramsey, I.S., M.M. Moran, J.A. Chong, and D.E. Clapham. (2006). A voltage-gated proton-selective channel lacking the pore domain. Nature 440: 1213-1216.

Ramsey, I.S., Y. Mokrab, I. Carvacho, Z.A. Sands, M.S. Sansom, and D.E. Clapham. (2010). An aqueous H+ permeation pathway in the voltage-gated proton channel Hv1. Nat Struct Mol Biol 17: 869-875.

Sakata, S. and Y. Okamura. (2018). Dynamic structural rearrangements and functional regulation of voltage-sensing phosphatase. J. Physiol. [Epub: Ahead of Print]

Sakata, S., M. Matsuda, A. Kawanabe, and Y. Okamura. (2017). Domain-to-domain coupling in voltage-sensing phosphatase. Biophys Physicobiol 14: 85-97.

Sakata, S., N. Miyawaki, T.J. McCormack, H. Arima, A. Kawanabe, N. Özkucur, T. Kurokawa, Y. Jinno, Y. Fujiwara, and Y. Okamura. (2016). Comparison between mouse and sea urchin orthologs of voltage-gated proton channel suggests role of S3 segment in activation gating. Biochim. Biophys. Acta. 1858: 2972-2983. [Epub: Ahead of Print]

Sanders, C.R. and J.M. Hutchison. (2018). Membrane properties that shape the evolution of membrane enzymes. Curr. Opin. Struct. Biol. 51: 80-91. [Epub: Ahead of Print]

Sasaki, M., M. Takagi, and Y. Okamura. (2006). A voltage sensor-domain protein is a voltage-gated proton channel. Science 312: 589-592.

Smith, S.M., D. Morgan, B. Musset, V.V. Cherny, A.R. Place, J.W. Hastings, and T.E. Decoursey. (2011). Voltage-gated proton channel in a dinoflagellate. Proc. Natl. Acad. Sci. USA 108: 18162-18167.

Tombola, F., M.H. Ulbrich, and E.Y. Isacoff. (2008). The voltage-gated proton channel Hv1 has two pores, each controlled by one voltage sensor. Neuron. 58: 546-556.

Tombola, F., M.H. Ulbrich, and E.Y. Isacoff. (2009). Architecture and gating of Hv1 proton channels. J. Physiol. 587: 5325-5329.

Villalba-Galea, C.A. (2014). Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition. Biophys. J. 107: 1564-1572.

Zhang, J., X. Chen, Y. Xue, N. Gamper, and X. Zhang. (2018). Beyond voltage-gated ion channels: Voltage-operated membrane proteins and cellular processes. J Cell Physiol. [Epub: Ahead of Print]

Examples:

TC#NameOrganismal TypeExample
1.A.51.1.1

The voltage-gated proton channel, mVSOP (269 aas and 2 TMSs) (Sasaki et al., 2006).  A hydrophobic plug functions as the gate (Chamberlin et al. 2013). Gating currents reveal that voltage-sensor (VS) activation and proton-selective aqueous conductance opening are thermodynamically distinct steps in the Hv1 activation pathway and show that pH changes directly alter VS activation. Gating cooperativity, pH-dependent modulation, and a high degree of H+ selectivity have been demonstrated (De La Rosa and Ramsey 2018).

Animals

mVSOP of Mus musculus (Q9DCE4)

 
1.A.51.1.2

The voltage-gated proton channel, Hv1, Hv1 or HVCN1 (273 aas) (Ramsey et al., 2006). Thr29 is a phosphorylation site that activates the HVCN1 channel in leukocytes (Musset et al., 2010). The condctivity pore has been delineated and depends of a carboxyl group (Asp or Glu) in the channel (Morgan et al. 2013). The 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 its biological functions (DeCoursey 2015). An exception occurs in dinoflagellates (see 1.A.51.1.4) in which H+ influx through HV1 triggers a bioluminescent flash. The gating mechanism of Hv1, cooperativity within dimers and the sensitivity to metal ions have been reviewed (Okamura et al. 2015). How this channel is activated by cytoplasmic [H+] and depolarization of the membrane potential has been proposed by Castillo et al. 2015. The extracellular ends of the first transmembrane segments form the intersubunit interface that mediates coupling between binding sites, while the coiled-coil domain does not directly participate in the process (Hong et al. 2015). Deep water penetration through hHv1 has been observed, suggesting a highly focused electric field, comprising two helical turns along the fourth TMS. This region likely contains the H+ selectivity filter and the conduction pore. A 3D model offers an explicit mechanism for voltage activation based on a one-click sliding helix conformational rearrangement (Li et al. 2015).  Trp-207 enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating (Cherny et al. 2015).  The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively (Okuda et al. 2016).  Segment S3 plays a role in activating gating (Sakata et al. 2016).  Two sites have been identified: one is the binding pocket of 2GBI (accessible to ligands from the intracellular side); the other is located at the exit site of the proton permeation pathway (Gianti et al. 2016).   Crystal structures of Hv1 dimeric channels revealed that the primary contacts between the two monomers are in the C-terminal domain (CTD), which forms a coiled-coil structure. Molecular dynamics (MD) simulations of full-length and truncated CTD models revealed a strong contribution of the CTD to the packing of the TMSs (Boonamnaj and Sompornpisut 2018).  Histidine-168 is essential for the ΔpH-dependent gating (Cherny et al. 2018). Proton transfer in Hv1 utilizes a water wire, and does not require transient protonation of a conserved aspartate in the S1 transmembrane helix (Bennett and Ramsey 2017).  Hv1 channels are present in bull spermatozoa, and these regulate sperm functions like hypermotility, capacitation and acrosome reaction through a complex interplay between different pathways involving cAMP, PKC, and Catsper (Mishra et al. 2019).

Animals

Hv1 of Homo sapiens (Q96D96)

 
1.A.51.1.3

Voltage-gated proton channel, HvCN1; VSOP; VSX1 (Sasaki et al., 2006).  Exhibits voltage and pH-dependent gating as well as Zn2+-reactivity. In the dimeric strcuture, each subumit has a proton channel. TMS4 appears to be the voltage sensor.  Subunit cooperativity has been demonstrated (Gonzalez et al. 2010).

 

 

Animals

HvCN1 of Ciona intestinalis (Q1JV40)

 
1.A.51.1.4

Voltage-gated proton-specific monomeric channel, kHv1. Activated by depolarization; functions in signaling and excitability to trigger bioluminescence (Smith et al., 2011).  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).

Dinoflagellates

kHv1 of Karlodinium veneficum (G5CPN9)

 
1.A.51.1.5

Proton channel protein, NpHv1 of 239 aas and 4 TMSs.  Proton selectivity, and pH- and voltage-dependent gating have been demoonstrated. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, led to a loss of proton-selective conduction (Chaves et al. 2016).

NpHv1 of Nicoletia phytophila

 
Examples:

TC#NameOrganismal TypeExample
1.A.51.2.1

The voltage-sensor containing phosphatase, VSP, of 576 aas and 4 TMSs N-terminal to the phosphatase domain. The enzyme region of VSP contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization (Zhang et al. 2018). The coupling between the two domains has been studied (Sakata et al. 2017). Membrane depolarization activates the phosphatase activity of the enzyme, presumably via electroconformational coupling between the sensor domain and the enzyme (Sanders and Hutchison 2018). Both the phosphatase domain and the C2 domain move with similar timing upon membrane depolarization (Sakata and Okamura 2018).

 

VSP of Ciona intestinalis (Transparent sea squirt) (Ascidia intestinalis)

 
1.A.51.2.2

Voltage-sensing phosphatase-2, VSP2, isoform X1, of 509 aas with 4 N-terminal TMSs that comprise the voltage sensor. 

VSP2 of Xenopus laevis (African clawed frog)

 
1.A.51.2.3

Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase, TPTE2, isoform gamma of522 aas and 4 TMSs.

TPTE2 of Homo sapiens