Kyohei Itsuki

Kyushu Dental University, Kitakyūshū, Fukuoka, Japan

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Publications (10)38.82 Total impact

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    ABSTRACT: Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that carry receptor-operated Ca(2+) currents (ROCs) triggered by receptor-induced, phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction, and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4,5)P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4,5)P2 and the possible role of PI(4,5)P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4,5)P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4,5)P2 using a model simulation that could explain the complicated effects of PI(4,5)P2 on different ROCs.
    Frontiers in Pharmacology 02/2015; 6:22. DOI:10.3389/fphar.2015.00022 · 3.80 Impact Factor

  • Biophysical Journal 01/2015; 108(2):282a. DOI:10.1016/j.bpj.2014.11.1542 · 3.97 Impact Factor
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    ABSTRACT: Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C-insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.
    The Journal of General Physiology 02/2014; 143(2):183-201. DOI:10.1085/jgp.201311033 · 4.79 Impact Factor
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    Biophysical Journal 01/2014; 106(2):334a. DOI:10.1016/j.bpj.2013.11.1916 · 3.97 Impact Factor
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    Kyohei Itsuki · Yuko Imai · Yasushi Okamura · Ryuji Inoue · Masayuki X. Mori ·

    Biophysical Journal 01/2013; 104(2):454-. DOI:10.1016/j.bpj.2012.11.2514 · 3.97 Impact Factor
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    ABSTRACT: TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P(2)-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P(2) regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.
    Channels (Austin, Tex.) 05/2012; 6(3):206-9. DOI:10.4161/chan.20883 · 2.32 Impact Factor
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    Yuko Imai · Kyohei Itsuki · Yasushi Okamura · Ryuji Inoue · Mori X. Masayuki ·

    Biophysical Journal 01/2012; 102(3):534-. DOI:10.1016/j.bpj.2011.11.2921 · 3.97 Impact Factor
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    Yuko Imai · Kyohei Itsuki · Yasushi Okamura · Ryuji Inoue · Masayuki X Mori ·
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    ABSTRACT: Activation of transient receptor potential (TRP) canonical TRPC3/C6/C7 channels by diacylglycerol (DAG) upon stimulation of phospholipase C (PLC)-coupled receptors results in the breakdown of phosphoinositides (PIPs). The critical importance of PIPs to various ion-transporting molecules is well documented, but their function in relation to TRPC3/C6/C7 channels remains controversial. By using an ectopic voltage-sensing PIP phosphatase (DrVSP), we found that dephosphorylation of PIPs robustly inhibits currents induced by carbachol (CCh), 1-oleolyl-2-acetyl-sn-glycerol (OAG) or RHC80267 in TRPC3, TRPC6 and TRPC7 channels, though the strength of the DrVSP-mediated inhibition (VMI) varied among the channels with a rank order of C7>C6>C3. Pharmacological and molecular interventions suggest that depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is most likely the critical event for VMI in all three channels.When the PLC catalytic signal was vigorously activated through overexpression of the muscarinic type-I receptor (M1R), the inactivation of macroscopic TRPC currents was greatly accelerated in the same rank order as the VMI, and VMI of these currents was attenuated or lost. VMI was also rarely detected in vasopressin-induced TRPC6-like currents inA7r5 vascular smooth muscle cells, indicating that the inactivation by PI(4,5)P₂ depletion underlies the physiological condition. Simultaneous fluorescence resonance energy transfer (FRET)-based measurement of PI(4,5)P₂ levels and TRPC6 currents confirmed that VMI magnitude reflects the degree of PI(4,5)P₂ depletion. These results demonstrate that TRPC3/C6/C7 channels are differentially regulated by depletion of PI(4,5)P₂, and that the bimodal signal produced by PLC activation controls these channels in a self-limiting manner.
    The Journal of Physiology 12/2011; 590(Pt 5):1101-19. DOI:10.1113/jphysiol.2011.221358 · 5.04 Impact Factor
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    Masayuki X Mori · Yuko Imai · Kyohei Itsuki · Ryuji Inoue ·
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    ABSTRACT: Calcium dynamics and its linked molecular interactions cause a variety of biological responses; thus, exploiting techniques for detecting both concurrently is essential. Here we describe a method for measuring the cytosolic Ca2+ concentration ([Ca2+](i)) and protein - protein interactions within the same cell, using Fura-2 and superenhanced cyan and yellow fluorescence protein (seCFP and seYFP, respectively) FRET imaging techniques. Concentration-independent corrections for bleed-through of Fura-2 into FRET cubes across different time points and [Ca2+](i) values allowed for an effective separation of Fura-2 cross-talk signals and seCFP and seYFP cross-talk signals, permitting calculation of [Ca2+](i) and FRET with high fidelity. This correction approach was particularly effective at lower [Ca2+](i) levels, eliminating bleed-through signals that resulted in an artificial enhancement of FRET. By adopting this correction approach combined with stepwise [Ca2+](i) increases produced in living cells, we successfully elucidated steady-state relationships between [Ca2+](i) and FRET derived from the interaction of seCFP-tagged calmodulin (CaM) and the seYFP-fused CaM binding domain of myosin light chain kinase. The [Ca2+](i) versus FRET relationship for voltage-gated sodium, calcium, and TRPC6 channel CaM binding domains (IQ domain or CBD) revealed distinct sensitivities for [Ca2+](i). Moreover, the CaM binding strength at basal or subbasal [Ca2+](i) levels provided evidence of CaM tethering or apoCaM binding in living cells. Of the ion channel studies, apoCaM binding was weakest for the TRPC6 channel, suggesting that more global Ca2+ and CaM changes rather than the local CaM-channel interface domain may be involved in Ca2+CaM-mediated regulation of this channel. This simultaneous Fura-2 and CFP- and YFP-based FRET imaging system will thus serve as a simple but powerful means of quantitatively elucidating cellular events associated with Ca2+-dependent functions.
    Biochemistry 05/2011; 50(21):4685-96. DOI:10.1021/bi200287x · 3.02 Impact Factor
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    Mori X. Masayuki · Yuko Imai · Kyohei Itsuki · Ryuji Inoue ·

    Biophysical Journal 02/2011; 100(3):99-. DOI:10.1016/j.bpj.2010.12.745 · 3.97 Impact Factor

Publication Stats

37 Citations
38.82 Total Impact Points


  • 2015
    • Kyushu Dental University
      Kitakyūshū, Fukuoka, Japan
  • 2014-2015
    • Kyushu University
      • • Department of Dental Science
      • • Faculty of Dental Science
      Hukuoka, Fukuoka, Japan
  • 2011-2013
    • Fukuoka University
      • Department of Physiology
      Hukuoka, Fukuoka, Japan