Tatsuki Kurokawa

Multimeric nature of voltage-gated proton channels

Voltage-gated potassium channels are comprised of four subunits, and each subunit has a pore domain and a voltage-sensing domain (VSD). The four pore domains assemble to form one single central pore, and the four individual VSDs control the gate of the pore. Recently, a family of voltage-gated proton channels, such as HV or voltage sensor only protein (VSOP), was discovered that contain a single VSD but no pore domain. It has been assumed that VSOP channels are monomeric and contain a single VSD that functions as both the VSD and the pore domain. It remains unclear, however, how a protein that contains only a VSD and no pore domain can conduct ions. Using fluorescence measurements and immunoprecipitation techniques, we show here that VSOP channels are expressed as multimeric channels. Further, FRET experiments on constructs with covalently linked subunits show that VSOP channels are dimers. Truncation of the cytoplasmic regions of VSOP reduced the dimerization, suggesting that the dimerization is caused mainly by cytoplasmic protein–protein interactions. However, these N terminus- and C terminus-deleted channels displayed large proton currents. Therefore, we conclude that even though VSOP channels are expressed mainly as dimers in the cell membrane, single VSOP subunits could function independently as proton channels.

The cytoplasmic coiled-coil mediates cooperative gating temperature sensitivity in the voltage-gated H+ channel Hv1

Hv1/VSOP is a dimeric voltage-gated H(+) channel in which the gating of one subunit is reportedly coupled to that of the other subunit within the dimer. The molecular basis for dimer formation and intersubunit coupling, however, remains unknown. Here we show that the carboxy terminus ends downstream of the S4 voltage-sensor helix twist in a dimer coiled-coil architecture, which mediates cooperative gating. We also show that the temperature-dependent activation of H(+) current through Hv1/VSOP is regulated by thermostability of the coiled-coil domain, and that this regulation is altered by mutation of the linker between S4 and the coiled-coil. Cooperative gating within the dimer is also dependent on the linker structure, which circular dichroism spectrum analysis suggests is α-helical. Our results indicate that the cytoplasmic coiled-coil strands form continuous α-helices with S4 and mediate cooperative gating to adjust the range of temperatures over which Hv1/VSOP operates.

Functionality of the voltage-gated proton channel truncated in S4

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 cysteine-scanning 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.

3′ Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP)

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)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] 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)P3. TLC assay showed that the 3′ phosphate of PI(3,4,5)P3 was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] was removed by VSPs. Monitoring of PI(3,4)P2 levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PHTAPP1-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)P3. However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P3 is dephosphorylated at the 5′ position, PI(3,4)P2 is then dephosphorylated at the 3′ position. These results suggest that substrate specificity of the VSP changes with membrane potential.

Crystal Structure of the Cytoplasmic Phosphatase and Tensin Homolog (PTEN)-like Region of Ciona intestinalis Voltage-sensing Phosphatase Provides Insight into Substrate Specificity and Redox Regulation of the Phosphoinositide Phosphatase Activity

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, wild-type (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.

Gating of the designed trimeric/tetrameric voltage-gated H+ channel

The voltage‐gated H+ channel assembles as a dimer by the cytoplasmic coiled‐coil domain. This study focuses on understanding the structural characteristics and functional significance of dimerization. Monomeric, trimeric and tetrameric channels can be engineered by changing the assembly state of the coiled coil by mutation, and interestingly, they show functional currents. However, only the native dimeric form shows successful cooperative gating, which is of physiological importance in the phagosomal production of reactive oxygen species. These results help us to understand better why the native form of the channel is a dimer from a standpoint of molecular structure and physiological function.

Long α helices projecting from the membrane as the dimer interface in the voltage-gated H+ channel

Continuous helices extending from the transmembrane region to the cytoplasmic region form a dimeric interface to regulate activation of the voltage-gated H+ channel.

Potential Role of Voltage‐Sensing Phosphatases in Regulation of Cell Structure Through the Production of PI(3,4)P2

Voltage‐sensing phosphatase, VSP, consists of the transmembrane domain, operating as the voltage sensor, and the cytoplasmic domain with phosphoinositide‐phosphatase activities. The voltage sensor tightly couples with the cytoplasmic phosphatase and membrane depolarization induces dephosphorylation of several species of phosphoinositides. VSP gene is conserved from urochordate to human. There are some diversities among VSP ortholog proteins; range of voltage of voltage sensor motions as well as substrate selectivity. In contrast with recent understandings of biophysical mechanisms of VSPs, little is known about its physiological roles. Here we report that chick ortholog of VSP (designated as Gg‐VSP) induces morphological feature of cell process outgrowths with round cell body in DF‐1 fibroblasts upon its forced expression. Expression of the voltage sensor mutant, Gg‐VSPR153Q with shifted voltage dependence to a lower voltage led to more frequent changes of cell morphology than the wild‐type protein. Coexpression of PTEN that dephosphorylates PI(3,4)P2 suppressed this effect by Gg‐VSP, indicating that the increase of PI(3,4)P2 leads to changes of cell shape. In addition, visualization of PI(3,4)P2 with the fluorescent protein fused with the TAPP1‐derived pleckstrin homology (PH) domain suggested that Gg‐VSP influenced the distribution of PI(3,4)P2. These findings raise a possibility that one of the VSPs functions could be to regulate cell morphology through voltage‐sensitive tuning of phosphoinositide profile. J. Cell. Physiol. 229: 422–433, 2014.

Comparison between mouse and sea urchin orthologs of voltage-gated proton channel suggests role of S3 segment in activation gating.

The voltage-gated proton channel, Hv1, is expressed in blood cells, airway epithelium, sperm and microglia, playing important roles in diverse biological contexts including phagocytosis or sperm maturation through its regulation of membrane potential and pH. The gene encoding Hv1, HVCN1, is widely found across many species and is also conserved in unicellular organisms such as algae or dinoflagellates where Hv1 plays role in calcification or bioluminescence. Voltage-gated proton channels exhibit a large variation of activation rate among different species. Here we identify an Hv1 ortholog from sea urchin, Strongylocentrotus purpuratus, SpHv1. SpHv1 retains most of key properties of Hv1 but exhibits 20-60 times more rapid activation kinetics than mammalian orthologs upon heterologous expression in HEK293T cells. Comparison between SpHv1 and mHv1 highlights novel roles of the third transmembrane segment S3 in activation gating of Hv1.

The cytochrome bcc-aa3-type respiratory chain of Rhodococcus rhodochrous.

Rhodococcus rhodochrous is an active soil bacterium belonging to the Nocardia group of high GC gram-positive bacteria. It is rich in various enzymes and thus important in the industrial production of chemicals and bioremediation. In this work, the respiratory chain of this aerobic organism was investigated and characterized. Grown under highly aerobic conditions, the membrane fraction of R. rhodochrous cells only contained a-, b- and c-type cytochromes, suggesting that it is the cytochrome bcc-aa(3)-type pathway that mainly operates under these conditions. In contrast, the d-type cytochrome was also present under microaerobic conditions, indicating that the alternative pathway of the bd-type oxidase works in these circumstances. In addition, the results of H(+)/O ratio measurements indicate that these two pathways have different energy efficiencies.