Kenta Kato

Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound

Canonical transient receptor potential (TRPC) channels control influxes of Ca2+ and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca2+ influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca2+ response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca2+-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.

Uric Acid Secretion from Adipose Tissue and Its Increase in Obesity

Background: Purine metabolism in adipose tissue is largely unknown. Results: Adipose tissue has abundant xanthine oxidoreductase activity. Uric acid is secreted from adipose tissues and cells, and the secretion is augmented in obese mice. Conclusion: Adipose tissue can secrete uric acid in mice. Significance: Dysfunction of obese adipose tissue could be related to overproduction of uric acid. Obesity is often accompanied by hyperuricemia. However, purine metabolism in various tissues, especially regarding uric acid production, has not been fully elucidated. Here we report, using mouse models, that adipose tissue could produce and secrete uric acid through xanthine oxidoreductase (XOR) and that the production was enhanced in obesity. Plasma uric acid was elevated in obese mice and attenuated by administration of the XOR inhibitor febuxostat. Adipose tissue was one of major organs that had abundant expression and activities of XOR, and adipose tissues in obese mice had higher XOR activities than those in control mice. 3T3-L1 and mouse primary mature adipocytes produced and secreted uric acid into culture medium. The secretion was inhibited by febuxostat in a dose-dependent manner or by gene knockdown of XOR. Surgical ischemia in adipose tissue increased local uric acid production and secretion via XOR, with a subsequent increase in circulating uric acid levels. Uric acid secretion from whole adipose tissue was increased in obese mice, and uric acid secretion from 3T3-L1 adipocytes was increased under hypoxia. Our results suggest that purine catabolism in adipose tissue could be enhanced in obesity.

Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain.

The Ca2+ release-activated Ca2+ channel is a principal regulator of intracellular Ca2+ rise, which conducts various biological functions, including immune responses. This channel, involved in store-operated Ca2+ influx, is believed to be composed of at least two major components. Orai1 has a putative channel pore and locates in the plasma membrane, and STIM1 is a sensor for luminal Ca2+ store depletion in the endoplasmic reticulum membrane. Here we have purified the FLAG-fused Orai1 protein, determined its tetrameric stoichiometry, and reconstructed its three-dimensional structure at 21-Å resolution from 3681 automatically selected particle images, taken with an electron microscope. This first structural depiction of a member of the Orai family shows an elongated teardrop-shape 150Å in height and 95Å in width. Antibody decoration and volume estimation from the amino acid sequence indicate that the widest transmembrane domain is located between the round extracellular domain and the tapered cytoplasmic domain. The cytoplasmic length of 100Å is sufficient for direct association with STIM1. Orifices close to the extracellular and intracellular membrane surfaces of Orai1 seem to connect outside the molecule to large internal cavities.

Three-dimensional Reconstruction Using Transmission Electron Microscopy Reveals a Swollen, Bell-shaped Structure of Transient Receptor Potential Melastatin Type 2 Cation Channel

Transient receptor potential melastatin type 2 (TRPM2) is a redox-sensitive, calcium-permeable cation channel activated by various signals, such as adenosine diphosphate ribose (ADPR) acting on the ADPR pyrophosphatase (ADPRase) domain, and cyclic ADPR. Here, we purified the FLAG-tagged tetrameric TRPM2 channel, analyzed it using negatively stained electron microscopy, and reconstructed the three-dimensional structure at 2.8-nm resolution. This multimodal sensor molecule has a bell-like shape of 18 nm in width and 25 nm in height. The overall structure is similar to another multimodal sensor channel, TRP canonical type 3 (TRPC3). In both structures, the small extracellular domain is a dense half-dome, whereas the large cytoplasmic domain has a sparse, double-layered structure with multiple internal cavities. However, a unique square prism protuberance was observed under the cytoplasmic domain of TRPM2. The FLAG epitope, fused at the C terminus of the ADPRase domain, was assigned by the antibody to a position close to the protuberance. This indicates that the agonist-binding ADPRase domain and the ion gate in the transmembrane region are separately located in the molecule.

Molecular Characterization of Flubendiamide Sensitivity in the Lepidopterous Ryanodine Receptor Ca2+ Release Channel

Flubendiamide is a benzenedicarboxamide derivative that shows selective insecticidal activity against lepidopterous insects. The specific modulatory effects of flubendiamide on ryanodine binding in insect muscle microsomal membranes suggest that the ryanodine receptor (RyR) Ca(2+) release channel is a primary target of flubendiamide. However, the molecular mechanisms underlying the species-specific action of flubendiamide are unclear. We have cloned cDNA encoding a novel RyR from the lepidopterous silkworm RyR (sRyR) and tested the sensitivity to flubendiamide of the recombinant sRyR in HEK293 cells. Confocal localization studies and Ca(2+) imaging techniques revealed that sRyRs form Ca(2+) release channels in the endoplasmic reticulum. Importantly, flubendiamide induced release of Ca(2+) through the sRyR, but not through the rabbit RyR isoforms. Photoaffinity labeling of sRyR deletion mutants using a photoreactive derivative revealed that flubendiamide is mainly incorporated into the transmembrane domain (amino acids 4111-5084) of the sRyR. The rabbit cardiac muscle isoform RyR2 (rRyR2) and the RyR mutant carrying a replacement of the transmembrane domain (residues 4084-5084) with its counterpart sequence from rRyR2 (residues 3936-4968) were not labeled by the photoreactive compound. This replacement in the sRyR significantly impaired the responses to flubendiamide but only marginally reduced the sensitivity to caffeine, a general RyR activator. Furthermore, deletion of the N-terminal sequence (residues 183-290) abolished the responses of the sRyR to flubendiamide but not the sensitivity to caffeine. Our results suggest that the transmembrane domain plays an important role in the formation of an action site for flubendiamide, while the N-terminus is a structural requirement for flubendiamide-induced activation of the sRyR.

Ca2+ influx and protein scaffolding via TRPC3 sustain PKCβ and ERK activation in B cells

Ca2+ signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca2+ entry activated by Ins(1,4,5)P3-induced Ca2+ release from endoplasmic reticulum, the importance of DAG-activated Ca2+ entry remains elusive. Here, we describe the physiological role of DAG-activated Ca2+ entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cβ (PKCβ) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCβ that maintained localization of PKCβ at the PM. Thus, TRPC3 functions as both a Ca2+-permeable channel and a protein scaffold at the PM for downstream PKCβ activation in B cells.

A Pathogenic C Terminus-truncated Polycystin-2 Mutant Enhances Receptor-activated Ca2+ Entry via Association with TRPC3 and TRPC7

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca2+ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca2+ influx that may lead to dysregulated cell growth in ADPKD.

Multiobjective Optimization Based on Expensive Robotic Experiments under Heteroscedastic Noise

In many engineering problems, including those related to robotics, optimization of the control policy for multiple conflicting criteria is required. However, this can be very challenging because of the existence of noise, which may be input dependent or heteroscedastic, and restrictions regarding the number of evaluations owing to the costliness of the experiments in terms of time and/or money. This paper presents a multiobjective optimization algorithm for expensive-to-evaluate noisy functions for robotics. We present a method for model selection between heteroscedastic and standard homoscedastic Gaussian process regression techniques to create suitable surrogate functions from noisy samples, and to find the point to be observed at the next step. This algorithm is compared against an existing multiobjective optimization algorithm, and then used to optimize the speed and head stability of the sidewinding gait of a snake robot.

Reply to Thinnes: Is There Competition in Trafficking of VDAC-cored VRAC and SOC in NE Differentiation of Cells?

This is a response to the letter by Friedrich Thinnes (1). As you point out, it is important to clarify the role of store-operated calcium (SOC) channels in VRAC (volume-regulated anion channel), because calcium entry through SOC channels seems to inhibit Cl− efflux through VRAC and further induces apoptosis of cancer cell line LNCaP cells (2–4). Some TRP (transient receptor potential) channels involved in SOC entry have been picked up as candidates for the SOC channel in LNCaP cells (5–7), but interaction between these channels and VRAC has not been sufficiently clarified. Furthermore, because Orai1 and STIM1 distribute ubiquitously in our body, they are expected to have a regulatory role over VRAC in various cells. Indeed, the SOC current in LNCaP cells exhibits some CRAC (Ca2+ release-activated Ca2+)-like properties (5). Their involvement in VRAC, however, has not yet been reported. Therefore, the present discussion should provide new insights as to how we can integrate ongoing research into VRAC and store-operated channels. In addition to the present three-dimensional structure of Orai1, we have described some TRP channel structures using EM (electron microscopy) image analysis (8, 9). The swollen structures of both store-operated Orai1 and TRPC3 channels should be able to accommodate and/or associate with various components, like the VRAC complex. Single particle reconstruction from EM images is very promising for the analysis of such “super” complexes because it does not require crystallization. Since volume control of cells is universal to physiological functions, including apoptosis in our body, single particle reconstruction of an SOC channel-VRAC “super complex” should enhance the analysis of cell-volume control machinery, which may be general to its related physiology.