Construction and validation of a homology model of the human voltage-gated proton channel hHV1

The topological similarity of voltage-gated proton channels (H_V1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether H_V1s have a similar structure. We present the construction and validation of a homology model of the human H_V1 (hH_V1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hH_V1 by exploiting the homology of hH_V1 with VSDs of K⁺ and Na⁺ channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hH_V1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp¹¹². Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hH_V1. Each Arg residue in the S4 helix of hH_V1 was replaced by His to test accessibility using Zn²⁺ as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg²¹¹, the third Arg residue in the S4 helix in hH_V1, remains accessible to the internal solution and is located near the charge transfer center, Phe¹⁵⁰.     

Peregrination of the selectivity filter delineates the pore of the human voltage-gated proton channel hHV1

Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hH_V1), because the proton concentration is >10⁶ times lower than that of other cations. Here we use “selectivity filter scanning” to elucidate the molecular requirements for proton-specific conduction in hH_V1. Asp¹¹², in the middle of the S1 transmembrane helix, is an essential part of the selectivity filter in wild-type (WT) channels. After neutralizing Asp¹¹² by mutating it to Ala (D112A), we introduced Asp at each position along S1 from 108 to 118, searching for “second site suppressor” activity. Surprisingly, most mutants lacked even the anion conduction exhibited by D112A. Proton-specific conduction was restored only with Asp or Glu at position 116. The D112V/V116D channel strikingly resembled WT in selectivity, kinetics, and ΔpH-dependent gating. The S4 segment of this mutant has similar accessibility to WT in open channels, because R211H/D112V/V116D was inhibited by internally applied Zn²⁺. Asp at position 109 allowed anion permeation in combination with D112A but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established externally to the constriction at F150. The three positions that permitted conduction all line the pore in our homology model, clearly delineating the conduction pathway. Evidently, a carboxyl group must face the pore directly to enable conduction. Molecular dynamics simulations indicate reorganization of hydrogen bond networks in the external vestibule in D112V/V116D. At both positions where it produces proton selectivity, Asp frequently engages in salt linkage with one or more Arg residues from S4. Surprisingly, mean hydration profiles were similar in proton-selective, anion-permeable, and nonconducting constructs. That the selectivity filter functions in a new location helps to define local environmental features required to produce proton-selective conduction.     

Oligomerization of the voltage-gated proton channel

The voltage-gated proton channel exists as a dimer, although each protomer has a separate conduction pathway, and when forced to exist as a monomer, most major functions are retained. However, the proton channel protomers appear to interact during gating. Proton channel dimerization is thought to result mainly from coiled-coil interaction of the intracellular C-termini. Several types of evidence are discussed that suggest that the dimer conformation may not be static, but is dynamic and can sample different orientations. Zn²⁺ appears to link the protomers in an orientation from which the channel(s) cannot open. A tandem WT-WT dimer exhibits signs of cooperative gating, indicating that despite the abnormal linkage, the correct orientation for opening can occur. We propose that C-terminal interaction functions mainly to tether the protomers together. Comparison of the properties of monomeric and dimeric proton channels speaks against the hypothesis that enhanced gating reflects monomer-dimer interconversion.Key words: voltage-gated proton channels, voltage gating, voltage-sensing domains, phagocytes, coiled-coil, oligomerization, proton currents, pH, dimerization, C-terminus     

The pore of the voltage-gated proton channel

In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containing?a VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 in?the transmembrane VSD to form the ion selectivity filter in the channel's open conformation.     

Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His²⁴⁴ or His²⁶⁶ residue. The binding of Zn²⁺ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co²⁺, Ca²⁺, Ni²⁺, Mg²⁺, and Mn²⁺. The strength of Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺ and Ni²⁺ binding to the site close to His²⁴⁴ is stronger than that of these divalent metal ions binding to the site close to His²⁶⁶.     

Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (I_ratio) were determined. The use of I_ratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q₁₀s for the proton currents were evaluated from the I_ratios. Q₁₀ exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q₁₀. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q₁₀ was decomposed into Q₁₀ of the channel and of the access resistances. Finally, the Q₁₀ for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.     

pH regulation and beyond: unanticipated functions for the voltage-gated proton channel, HVCN1

Electrophysiological studies have implicated voltage-gated proton channels in several specific cellular contexts. In neutrophils, they mediate charge compensation that is associated with the oxidative burst of phagocytosis. Molecular characterization of the hydrogen voltage-gated channel 1 (HVCN1) has enabled identification of unanticipated and diverse functions: HVCN1 not only modulates signaling from the B-cell receptor following B-cell activation and histamine release from basophils, but also mediates pH-dependent activation of spermatozoa, as well as acid secretion by tracheal epithelium. The importance of HVCN1 in pH regulation during phagocytosis was established by surprising evidence that indicated its first-responder role. In this review, we discuss recent findings from a functional perspective, and the potential of HVCN1 as a therapeutic target for autoimmune and other diseases.     

Clinicopathological and biological significance of human voltage-gated proton channel Hv1 protein overexpression in breast cancer

In our previous work, we showed for the first time that the voltage-gated proton channel Hv1 is specifically expressed in highly metastatic human breast tumor tissues and cell lines. However, the contribution of Hv1 to breast carcinogenesis is not well known. In this study, we showed that Hv1 expression was significantly correlated with the tumor size (p = 0.001), tumor classification (p = 0.000), lymph node status (p = 0.000), clinical stage (p = 0.000), and Her-2 status (p = 0.045). High Hv1 expression was associated significantly with shorter overall (p = 0.000) and recurrence-free survival (p = 0.000). In vitro, knockdown of Hv1 expression in the highly metastatic MDA-MB-231 cells decreased the cell proliferation and invasiveness, inhibited the cell proton secretion and intracellular pH recovery, and blocked the cell capacity of acidifying extracellular milieu. Furthermore, the gelatinase activity in MDA-MB-231 cells that suppressed Hv1 was reduced. In vivo, the breast tumor size of the implantation of the MDA-MB-231 xenografts in nude mice that were knocked down by Hv1 was dramatically smaller than that in the control groups. The results demonstrated that the inhibition of Hv1 function via knockdown of Hv1 expression can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Based on these results, we came to the conclusion that Hv1 is a potential biomarker for prognosis of breast cancer and a potential target for anticancer drugs in breast cancer therapy.     

Zn induces apoptosis in human highly metastatic SHG-44 glioma cells,through inhibiting activity of the voltage-gated proton channel Hv1

In contrast to the voltage-gated K⁺ channels, the voltage-gated proton channel Hv1 contains a voltage-sensor domain but lacks a pore domain. Here, we showed that Hv1 is expressed in the highly metastatic glioma cell SHG-44, but lowly in the poorly metastatic glioma cell U-251. Inhibition of Hv1 activity by 140 μM zinc chloride induces apoptosis in the human highly metastatic glioma cells. Zn²⁺ ions markedly inhibit proton secretion, and reduce the gelatinase activity in the highly metastatic glioma cells. In vivo, the glioma tumor sizes of the implantation of the SHG-44 xenografts in nude mice that were injected zinc chloride solution, were dramatically smaller than that in the controlled groups. The results demonstrated that the inhibition of Hv1 activity via Zn²⁺ ions can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Our results suggest that Zn²⁺ ions may be used as a potential anti-glioma drug for glioma therapy.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

The voltage-gated proton channel Hv1 has two pores, each controlled by one voltage sensor

In voltage-gated channels, ions flow through a single pore located at the interface between membrane-spanning pore domains from each of four subunits, and the gates of the pore are controlled by four peripheral voltage-sensing domains. In a striking exception, the newly discovered voltage-gated Hv1 proton channels lack a homologous pore domain, leaving the location of the pore unknown. Also unknown are the number of subunits and the mechanism of gating. We find that Hv1 is a dimer and that each subunit contains its own pore and gate, which is controlled by its own voltage sensor. Our experiments show that the cytosolic domain of the channel is necessary and sufficient for dimerization and that the transmembrane part of the channel is functional also when monomerized. The results suggest a mechanism of gating whereby the voltage sensor and gate are one and the same.     

The gp91phox component of NADPH oxidase is not the voltage-gated proton channel in phagocytes, but it helps

During the "respiratory burst," the NADPH oxidase complex of phagocytes produces reactive oxygen species that kill bacteria and other invaders (Babior, B. M. (1999) Blood 93, 1464-1476). Electron efflux through NADPH oxidase is electrogenic (Henderson, L. M., Chappell, J. B., and Jones, O. T. G. (1987) Biochem. J. 246, 325-329) and is compensated by H(+) efflux through proton channels that reportedly are contained within the gp91(phox) subunit of NADPH oxidase. To test whether gp91(phox) functions as a proton channel, we studied H(+) currents in granulocytes from X-linked chronic granulomatous disease patients lacking gp91(phox) (X-CGD), the human myelocytic PLB-985 cell line, PLB-985 cells in which gp91(phox) was knocked out by gene targeting (PLB(KO)), and PLB-985 knockout cells re-transfected with gp91(phox) (PLB(91)). H(+) currents in unstimulated PLB(KO) cells had amplitude and gating kinetics similar to PLB(91) cells. Furthermore, stimulation with the phorbol ester phorbol 12-myristate 13-acetate increased H(+) currents to a similar extent in X-CGD, PLB(KO), and PLB(91) cells. Thus, gp91(phox) is not the proton channel in unstimulated phagocytes and does not directly mediate the increase of proton conductance during the respiratory burst. Changes in H(+) channel gating kinetics during NADPH oxidase activity are likely crucial to the activation of H(+) flux during the respiratory burst.     

The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel

Kv voltage-gated potassium channels share a cytoplasmic assembly domain, T1. Recent mutagenesis of two T1 C-terminal loop residues implicates T1 in channel gating. However, structural alterations of these mutants leave open the question concerning direct involvement of T1 in gating. We find in mammalian Kv1.2 that gating depends critically on residues at complementary T1 surfaces in an unusually polar interface. An isosteric mutation in this interface causes surprisingly little structural alteration while stabilizing the closed channel and increasing the stability of T1 tetramers. Replacing T1 with a tetrameric coiled-coil destabilizes the closed channel. Together, these data suggest that structural changes involving the buried polar T1 surfaces play a key role in the conformational changes leading to channel opening.     

Tryptophan 67 in the human VPAC(1) receptor: crucial role for VIP binding

The human receptor subtype for VIP and PACAP, referred to as VPAC(1) receptor, has a large N-terminal extracellular domain which is critical for VIP binding. We further investigated this domain by mutating 12 amino acid residues which could participate in the formation of a tight bend (W67) or a coiled coil motif. They were changed to alanine (A) and the cDNAs were transiently transfected into Cos cells. All mutants but W67A exhibited K(d) values similar to that of the wild-type receptor. For the W67A mutant, no specific (125)I-VIP binding could be observed. Mutants at the W67 site were further characterized after stable transfection of epitope-tagged VPAC(1) receptor-GFP fusion proteins into CHO cells. W67A, W67E, W67H, and W67K mutants neither bound VIP nor mediated adenylyl cyclase activation by VIP. The W67F mutant mediated stimulation of adenylyl cyclase only at high VIP concentrations. Microscopic analysis and antibody binding experiments showed that all mutants were similarly expressed at the cell surface of CHO cells. Therefore tryptophan 67 in the human VPAC(1) receptor plays a crucial role in VIP binding due, in part, to its aromatic moiety.     

The selectivity filter of the voltage-gated sodium channel is involved in channel activation

Amino acids located in the outer vestibule of the voltage-gated Na+ channel determine the permeation properties of the channel. Recently, residues lining the outer pore have also been implicated in channel gating. The domain (D) IV P-loop residue alanine 1529 forms a part of the putative selectivity filter of the adult rat skeletal muscle (mu1) Na+ channel. Here we report that replacement of alanine 1529 by aspartic acid enhances entry to an ultra-slow inactivated state. Ultra-slow inactivation is characterized by recovery time constants on the order of approximately 100 s from prolonged depolarizations and by the fact that entry to this state can be reduced by binding to the pore of a mutant mu-conotoxin GIIIA, suggesting that ultra-slow inactivation may reflect a structural rearrangement of the outer vestibule. The voltage dependence of ultra-slow inactivation in DIV-A1529D is U-shaped, with a local maximum near -60 mV, whereas activation is maximal only above -20 mV. Furthermore, a train of brief depolarizations produces more ultra-slow inactivation than a single maintained depolarization of the same duration. These data suggest that ultra-slow inactivation emanates from "partially activated" closed states and that the P-loop in DIV may undergo a conformational change during channel activation, which is accentuated by DIV-A1529D.     

Interaction of human immunodeficiency virus gp120 with the voltage-gated potassium channel BEC1

Retrovirus replication critically depends on a dynamic interplay between retroviral and host proteins. We report on the binding of the surface subunit (glycoprotein 120 (gp120)) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) to the cytoplasmic C-terminus of the voltage-gated potassium channel BEC1 (brain-specific ether-a-go-go-like channel 1), an interaction that can result in the repression of BEC’s activity and the inhibition of HIV-1 particle-release. BEC1 protein was found to be expressed in T cells and macrophages, the major target cells of HIV-1. Thus, gp120/BEC1 interaction may be involved in HIV-1 life cycle and/or pathogenesis.

Structured summary

MINT-7968695: BEC1 (uniprotkb:Q9ULD8) physically interacts (MI:0915) with gp160 (uniprotkb:P04578) by anti bait coimmunoprecipitation (MI:0006)MINT-7968714: BEC1 (uniprotkb:Q9ULD8) physically interacts (MI:0915) with gp160 (uniprotkb:P04578) by anti tag coimmunoprecipitation (MI:0007)MINT-7968675: BEC1 (uniprotkb:Q9ULD8) physically interacts (MI:0915) with gp160 (uniprotkb:P04578) by pull down (MI:0096)     

Calmodulin regulation of the calcium-leak channel Sec61 is unique to vertebrates

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, such as those formed by Sec61 complexes in the ER membrane, would interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism of intracellular signaling. We identified a calmodulin (CaM) binding motif in the cytosolic N-terminus of Sec61α from Canis familiaris that binds CaM, but not Ca²⁺-free apo-CaM, with nanomolar affinity and sequence specificity. In single channel lipid bilayer measurements, CaM potently mediated Sec61-channel closure in a Ca²⁺-dependent manner. No functional CaM binding motif was identified in the corresponding region of Sec61p from Saccharomyces cerevisiae, and no channel closure occurred in the presence of CaM and Ca²⁺. Therefore, CaM binding to the cytosolic N-terminus of Sec61α is involved in limiting Ca²⁺-leakage from the ER in C. familiaris but not S. cerevisiae.     

Calmodulin regulation of the calcium-leak channel Sec61 is unique to vertebrates

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, such as those formed by Sec61 complexes in the ER membrane, would interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism of intracellular signaling. We identified a calmodulin (CaM) binding motif in the cytosolic N-terminus of Sec61α from Canis familiaris that binds CaM, but not Ca(2+)-free apo-CaM, with nanomolar affinity and sequence specificity. In single channel lipid bilayer measurements, CaM potently mediated Sec61-channel closure in a Ca(2+)-dependent manner. No functional CaM binding motif was identified in the corresponding region of Sec61p from Saccharomyces cerevisiae, and no channel closure occurred in the presence of CaM and Ca(2+). Therefore, CaM binding to the cytosolic N-terminus of Sec61α is involved in limiting Ca(2+)-leakage from the ER in C. familiaris but not S. cerevisiae.