Kyohei Itsuki

PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels

Phosphatidylinositol 4,5-bisphosphate has a direct role in regulating receptor-operated TRPC channel activation and inactivation.

A self‐limiting regulation of vasoconstrictor‐activated TRPC3/C6/C7 channels coupled to PI(4,5)P2‐diacylglycerol signalling

•  From brain to digestive tract, electro‐chemical signals are broadly utilized to control the activity of the organs; however, the formation of such signals is very varied in each cell and still unknown in many cells. •  In this study, we found a novel mechanism for forming an electrical signal, produced by channels of the transient receptor potential canonical (TRPC) family of channels, which allow the permeation of ions such as sodium and calcium and are opened by the actions of hormones such as adrenaline and noradrenaline. •  Such hormones can activate an enzyme (phospholipase C) by which PI(4,5)P2, a member of the membrane lipid ‘phosphoinositide’, is degraded: the degradation of PI(4,5)P2 to produce an agonist (diacylglycerol) involved in the opening of TRPC channels, while the degradation itself is surprisingly critical to the closing of these channels. •  As a result of such a self‐limiting effect via membrane lipid degradation, TRPC channels can produce a unique electro‐chemical signal which is tightly bound to the arrangement of membrane lipid and hormones. •  Differential sensitivity for PIP2 of TRPC

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides

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)P2-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)P2 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.

Dynamics of receptor-operated Ca(2+) currents through TRPC channels controlled via the PI(4,5)P2-PLC signaling pathway.

Transient receptor potential canonical (TRPC) channels are Ca2+-permeable, nonselective cation channels that carry receptor-operated Ca2+ 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.

Quantitative measurement of Ca 2+-dependent calmodulin-target binding by Fura-2 and CFP and YFP FRET imaging in living cells

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 Ca(2+) concentration ([Ca(2+)](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 [Ca(2+)](i) values allowed for an effective separation of Fura-2 cross-talk signals and seCFP and seYFP cross-talk signals, permitting calculation of [Ca(2+)](i) and FRET with high fidelity. This correction approach was particularly effective at lower [Ca(2+)](i) levels, eliminating bleed-through signals that resulted in an artificial enhancement of FRET. By adopting this correction approach combined with stepwise [Ca(2+)](i) increases produced in living cells, we successfully elucidated steady-state relationships between [Ca(2+)](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 [Ca(2+)](i) versus FRET relationship for voltage-gated sodium, calcium, and TRPC6 channel CaM binding domains (IQ domain or CBD) revealed distinct sensitivities for [Ca(2+)](i). Moreover, the CaM binding strength at basal or subbasal [Ca(2+)](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 Ca(2+) and CaM changes rather than the local CaM-channel interface domain may be involved in Ca(2+)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 Ca(2+)-dependent functions.

Lipid-Mediated Mechanisms Involved in the Mechanical Activation of TRPC6 and TRPV4 Channels in the Vascular Tone Regulation

The transient receptor potential (TRP) proteins form a large Ca2+-permeable nonselective cation channel superfamily activated by physicochemical stimuli, and participate in a wide array of biological functions including sensory signal transduction. Recent investigations have disclosed that many of TRP channels expressed in the cardiovascular system (CVS) are activated by mechanical stresses operating therein such as membrane stretch, hypoosmolarity and shear stress. Although mechanisms proposed for mechanical signal transduction are diverse, accumulating evidence suggests that lipid mediators derived from phospholipase C (PLC)- and phospholipase A2 (PLA2)-dependent pathways may play central roles in the activation and regulation of these TRP channels. In this review, we focus on the lipid-mediated regulation of two TRP channels abundantly expressed in the CVS, i.e. TRPC6 and TRPV4, with particular interest in the synergistic interaction between receptor-mediated and mechanical stimulations, and discuss about their complex functional antagonism in vascular tone and blood pressure regulation.