The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn(2+). Here we present a crystal structure of mouse Hv1 in the resting state at 3.45-Å resolution. The structure showed a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. Two out of three arginines in S4 were located below the phenylalanine constituting the gating charge-transfer center. The extracellular region of each protomer coordinated a Zn(2+), thus suggesting that Zn(2+) stabilizes the resting state of Hv1 by competing for acidic residues that otherwise form salt bridges with voltage-sensing positive charges on S4. These findings provide a platform for understanding the general principles of voltage sensing and proton permeation.

The voltage-gated proton channel, Hv1 (VSOP) has a voltage-sensor domain (VSD) but lacks an authentic pore domain, and the VSD of Hv1 plays dual roles of voltage sensing and proton permeation. Hv1 is required for high-level superoxide production by phagocytes through its tight functional coupling with NADPH oxidase to eliminate pathogens. Hv1 is also expressed in human sperm and has been suggested to regulate motility through activating pH-sensitive calcium channels. The activities of Hv1 also have pathological implications, such as exacerbation of ischemic brain damage and progression of cancer. In this study, our crystal structure of mouse Hv1 (mHv1) showed a "closed umbrella" shape with a long helix consisting of the cytoplasmic coiled-coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. We also found a Zn2+ion at the extracellular region of mHv1 protomer. The binding of Zn2+strongly suggested that the crystal structure of mHv1 represents the resting state, since Zn2+specifically inhibits activities of voltage-gated proton channels. Actually, two out of three arginines as sensor residues (R204 and R207) were located lower than the conserved phenylalanine, F146, on the S2 in a charge transfer center. This makes contrast with previous structures of other VSDs in the activated state where many positive residues of S4 were located upper than the conserved phenylalanine. Additionally, the crystal structure of mHv1 highlighted two hydrophobic barriers. Aspartic acid (D108), which is critical for proton selective permeation, was located facing intracellular vestibule below the inner hydrophobic barrier, thereby being accessible to water from the cytoplasm. Another hydrophobic layer of extracellular side probably ensures interruption of the proton pathway of mHv1 in resting state. These findings provide a novel platform for understanding the general principles of voltage sensing and proton permeation.

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5′ position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] and phosphatidylinositol 4,5-bisphosphate [PI (4,5)P 2 ] upon voltage depolarization. However, it is unclear whether VSPs also have 3′ phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P 3 . TLC assay showed that the 3′ phosphate of PI(3,4,5)P 3 was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ] was removed by VSPs. Monitoring of PI(3,4)P 2 levels with the pleckstrin homology (PH) domain from tandem PH domaincontaining protein (TAPP1) fused with GFP (PH TAPP1 -GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5′ phosphatase activity of VSP toward PI(3,4,5)P 3 . However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P 3 is dephosphorylated at the 5′ position, PI(3,4)P 2 is then dephosphorylated at the 3′ position. These results suggest that substrate specificity of the VSP changes with membrane potential.phosphoinositide | ascidian P hosphoinositides serve as not only components of biological membranes, but also as coordinators of diverse cellular events including proliferation, cell migration, vesicle turnover, and ion transport. Numerous phosphatases and kinases that regulate phosphoinositide availability have been identified (1, 2), and defects or enhancements of these enzymes lead to tumorigenesis, metabolic disorders, and degeneration. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a wellcharacterized phosphatase that dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] (3). Defect or loss of PTEN leads to generation or progression of tumors (4, 5), and enhancement of PTEN underlies diabetes (6). We have shown that a sea squirt ortholog of one PTEN-related phosphatase, transmembrane phosphatase with tensin homology (TPTE)/ TPTE and PTEN homologous inositol lipid phosphatase (TPIP), designated as Ciona intestinalis VSP (Ci-VSP), dephosphorylates phosphoinositides that depend on membrane potential (7, 8). Ci-VSP has a voltage-sensor domain (VSD) consisting of four transmembrane segments and a PTEN-like region. Despite its sequence similarity to PTEN in its active center, Ci-VSP exhibits 5′ phosphatase activity toward PI(3,4,5)P 3 and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], unlike PTEN. Such distinct substrate specificity from PTEN partly d...

Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wildtype (248 -576), wild-type (236 -576), and G365A mutant (248 -576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.The voltage-sensing phosphatase (VSP) 3 was discovered through the survey of the genome of the ascidian, Ciona intestinalis, as a hybrid protein that has a voltage sensor domain (VSD) consisting of four ␣-helices (S1-S4) for voltage sensing and a cytoplasmic region encoding a phosphatidylinositol phosphatase domain (1). The VSP gene is conserved from sea urchins to humans where it is expressed in the testis (2). In the recent report, C. intestinalis VSP (Ci-VSP) is expressed in the cells of the stomach, intestine, and blood of juveniles detected by whole mount in situ hybridization (3). The VSD of VSP bears homology to the VSD of voltage-gated ion channels and a recently identified voltage-gated proton channel protein that contains only the VSD without pore domain, VSOP or Hv1 (4, 5). The Ci-VSP cytoplasmic region, which consists of a phosphatase domain (PD) and a C2 domain, shares high sequence similarity with the phosphatase and tensin homolog (PTEN) (1, 6). The amino acid sequence of the Ci-VSP cytoplasmic region has similarity to that of PTEN with 36% identity. A unique feature of both invertebrate and vertebrate VSPs is that depolarization induces phosphoinositide phosphatase activity through coupling of VSD to the phosphatase region (7-9). The enzymatic activity of Ci-VSP increases in the range of membrane potentials, from Ϫ80 to 100 mV, correlating with the extent of voltage sensor movement (7-8). Ci-VSP most likely operates as a monomer as shown by a study of single molecule imaging in heterologous expression system (10). The linker region between the VSD and ...

The voltage sensor domain (VSD) is the key module for voltage sensing in voltage-gated ion channels and voltage-sensing phosphatases. Structurally, both the VSD and the recently discovered voltage-gated proton channels (Hv channels) voltage sensor only protein (VSOP) and Hv1 contain four transmembrane segments. The fourth transmembrane segment (S4) of Hv channels contains three periodically aligned arginines (R1, R2, R3). It remains unknown where protons permeate or how voltage sensing is coupled to ion permeation in Hv channels. Here we report that Hv channels truncated just downstream of R2 in the S4 segment retain most channel properties. Two assays, site-directed cysteinescanning using accessibility of maleimide-reagent as detected by Western blotting and insertion into dog pancreas microsomes, both showed that S4 inserts into the membrane, even if it is truncated between the R2 and R3 positions. These findings provide important clues to the molecular mechanism underlying voltage sensing and proton permeation in Hv channels.ion conduction | membrane topology | membrane insertion | voltage sensor V oltage sensor domain (VSD) is a protein module that senses transmembrane voltage and regulates ion permeation through the pore (1-3) and phosphoinositide turnover by the phosphatase (4). The fourth transmembrane segment (S4) of VSD contains periodically aligned positively charged residues (arginine and lysine), which play a critical role in voltage sensing (1, 2). Recent analyses of the crystal structures of voltage-gated potassium channels (5, 6) showed that basic residues in S4 form salt bridges with negatively charged residues from other helices, and these salt bridges are formed at both sides of a hydrophobic residue such as phenylalanine. This suggests a focused electric field within a crevice surrounded by transmembrane helices (6), which would enable extracellular and intracellular aqueous environments to enter deeply into the membrane through a narrow crevice. One line of functional evidence for this narrow crevice is that mutation within the VSD creates an ion permeation pathway that is activated upon hyperpolarization (7−9). Such ion conduction is called an "omegacurrent" (9) or "gating pore current" (10). Similar conductances through the VSD are elicited in a mutated form of human voltagegated sodium channels (SCN4a) found in patients with periodic paralysis (11, 12) and in flatworm potassium channels (13).Recently, the protein, which consists solely of a VSD, was identified in tunicate, mouse (14) and human (15) and called voltage sensor only protein (VSOP) or Hv1. Mouse VSOP (mVSOP) is found within the phagosomes of white blood cells, and is essential for robust production of superoxide anions (16, 17) and maintenance of intracellular pH (18) in phagocytes. Despite the lack of a pore domain, these proteins function as voltage-gated proton channels (Hv channels) in heterologous expression systems (14,15) and when reconstituted into artificial membranes (19). S4 of both mVSOP and Hv1 contain three argini...