Yoshifumi Okochi

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.

Voltage-gated proton channel is expressed on phagosomes

Voltage-gated proton channel has been suggested to help NADPH oxidase activity during respiratory burst of phagocytes through its activities of compensating charge imbalance and regulation of pH. In phagocytes, robust production of reactive oxygen species occurs in closed membrane compartments, which are called phagosomes. However, direct evidence for the presence of voltage-gated proton channels in phagosome has been lacking. In this study, the expression of voltage-gated proton channels was studied by Western blot with the antibody specific to the voltage-sensor domain protein, VSOP/Hv1, that has recently been identified as the molecular correlate for the voltage-gated proton channel. Phagosomal membranes of neutrophils contain VSOP/Hv1 in accordance with subunits of NADPH oxidases, gp91, p22, p47 and p67. Superoxide anion production upon PMA activation was significantly reduced in neutrophils from VSOP/Hv1 knockout mice. These are consistent with the idea that voltage-gated proton channels help NADPH oxidase in phagocytes to produce reactive oxygen species.

Enzyme Domain Affects the Movement of the Voltage Sensor in Ascidian and Zebrafish Voltage-sensing Phosphatases

The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN).The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.

Diverse regulation of sensory signaling by C. elegans nPKC-epsilon/eta TTX-4

Molecular and pharmacological studies in vitro suggest that protein kinase C (PKC) family members play important roles in intracellular signal transduction. Nevertheless, the in vivo roles of PKC are poorly understood. We show here that nPKC‐epsilon/eta TTX‐4 in the nematode Caenorhabditis elegans is required for the regulation of signal transduction in various sensory neurons for temperature, odor, taste, and high osmolality. Interestingly, the requirement for TTX‐4 differs in different sensory neurons. In AFD thermosensory neurons, gain or loss of TTX‐4 function inactivates or hyperactivates the neural activity, respectively, suggesting negative regulation of temperature sensation by TTX‐4. In contrast, TTX‐4 positively regulates the signal sensation of ASH nociceptive neurons. Moreover, in AWA and AWC olfactory neurons, TTX‐4 plays a partially redundant role with another nPKC, TPA‐1, to regulate olfactory signaling. These results suggest that C. elegans nPKCs regulate different sensory signaling in various sensory neurons. Thus, C. elegans provides an ideal model to reveal genetically novel components of nPKC‐mediated molecular pathways in sensory signaling.

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.

Bidirectional regulation of thermotaxis by glutamate transmissions in Caenorhabditis elegans

In complex neural circuits of the brain, massive information is processed with neuronal communication through synaptic transmissions. It is thus fundamental to delineate information flows encoded by various kinds of transmissions. Here, we show that glutamate signals from two distinct sensory neurons bidirectionally affect the same postsynaptic interneuron, thereby producing the opposite behaviours. EAT‐4/VGLUT (vesicular glutamate transporter)‐dependent glutamate signals from AFD thermosensory neurons inhibit the postsynaptic AIY interneurons through activation of GLC‐3/GluCl inhibitory glutamate receptor and behaviourally drive migration towards colder temperature. By contrast, EAT‐4‐dependent glutamate signals from AWC thermosensory neurons stimulate the AIY neurons to induce migration towards warmer temperature. Alteration of the strength of AFD and AWC signals led to significant changes of AIY activity, resulting in drastic modulation of behaviour. We thus provide an important insight on information processing, in which two glutamate transmissions encoding opposite information flows regulate neural activities to produce a large spectrum of behavioural outputs.

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.

Reversal of Salt Preference Is Directed by the Insulin/PI3K and Gq/PKC Signaling in Caenorhabditis elegans

Animals search for foods and decide their behaviors according to previous experience. Caenorhabditis elegans detects chemicals with a limited number of sensory neurons, allowing us to dissect roles of each neuron for innate and learned behaviors. C. elegans is attracted to salt after exposure to the salt (NaCl) with food. In contrast, it learns to avoid the salt after exposure to the salt without food. In salt-attraction behavior, it is known that the ASE taste sensory neurons (ASEL and ASER) play a major role. However, little is known about mechanisms for learned salt avoidance. Here, through dissecting contributions of ASE neurons for salt chemotaxis, we show that both ASEL and ASER generate salt chemotaxis plasticity. In ASER, we have previously shown that the insulin/PI 3-kinase signaling acts for starvation-induced salt chemotaxis plasticity. This study shows that the PI 3-kinase signaling promotes aversive drive of ASER but not of ASEL. Furthermore, the Gq signaling pathway composed of Gqα EGL-30, diacylglycerol, and nPKC (novel protein kinase C) TTX-4 promotes attractive drive of ASER but not of ASEL. A putative salt receptor GCY-22 guanylyl cyclase is required in ASER for both salt attraction and avoidance. Our results suggest that ASEL and ASER use distinct molecular mechanisms to regulate salt chemotaxis plasticity.

Autoimmune disorder phenotypes in Hvcn1-deficient mice.

H(v) channels (voltage-gated proton channels) are expressed in blood cells, microglia and some types of epithelial cells. In neutrophils H(v) channels regulate the production of reactive oxygen species through regulation of membrane potential and intracellular pH. H(v) channels have also been suggested to play a role in sperm physiology in the human. However, the functions of the Hv channel at the whole-body level are not fully understood. In the present paper we show that Hvcn1 (voltage-gated hydrogen channel 1)-knockout mice show splenomegaly, autoantibodies and nephritis, that are reminiscent of human autoimmune diseases phenotypes. The number of activated T-cells was larger in Hvcn1-deficient mice than in the wild-type mice. Upon viral infection this was remarkably enhanced in Hvcn1-deficient mice. The production of superoxide anion in T-cells upon stimulation with PMA was significantly attenuated in the Hvcn1-deficient mice. These results suggest that H(v) channels regulate T-cell homoeostasis in vivo.

The voltage-gated proton channel Hv1/VSOP inhibits neutrophil granule release

Neutrophil granule exocytosis is crucial for host defense and inflammation. Neutrophils contain 4 types of granules, the exocytotic release of which is differentially regulated. This exocytosis is known to be driven by diverse mediators, including calcium and nucleotides, but the precise molecular mechanism remains largely unknown. We show in the present study that voltage‐gated proton (Hv) channels are necessary for the proper release of azurophilic granules in neutrophils. On activation of NADPH oxidase by PMA and IgG, neutrophils derived from Hvcn1 gene knockout mouse exhibited greater secretion of MPO and elastase than WT cells. In contrast, release of LTF enriched in specific granules was not enhanced in these cells. The excess release of azurophilic granules in Hv1/VSOP‐deficient neutrophils was suppressed by inhibiting NADPH oxidase activity and, in part, by valinomycin, a potassium ionophore. In addition, Hv1/VSOP‐deficient mice exhibited more severe lung inflammation after intranasal Candida albicans infection than WT mice. These findings suggest that the Hv channel acts to specifically dampen the release of azurophilic granules through, in part, the suppression of increased positive charges at the plasma membrane accompanied by the activation of NADPH oxidase in neutrophils.