Document Detail

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides.
Jump to Full Text
MedLine Citation:
PMID:  22760061     Owner:  NLM     Status:  MEDLINE    
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.
Kyohei Itsuki; Yuko Imai; Yasushi Okamura; Kihachiro Abe; Ryuji Inoue; Masayuki X Mori
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-05-01
Journal Detail:
Title:  Channels (Austin, Tex.)     Volume:  6     ISSN:  1933-6969     ISO Abbreviation:  Channels (Austin)     Publication Date:    2012 May-Jun
Date Detail:
Created Date:  2012-08-22     Completed Date:  2012-12-07     Revised Date:  2013-07-12    
Medline Journal Info:
Nlm Unique ID:  101321614     Medline TA:  Channels (Austin)     Country:  United States    
Other Details:
Languages:  eng     Pagination:  206-9     Citation Subset:  IM    
Department of Physiology, School of Medicine, Fukuoka University, Japan.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Cell Line, Transformed
Ciona intestinalis
Diglycerides / metabolism
Ion Channel Gating
Phosphatidylinositol 4,5-Diphosphate / metabolism
Phosphoinositide Phospholipase C / metabolism
Phosphoric Monoester Hydrolases / metabolism*
Species Specificity
TRPC Cation Channels / metabolism*
Zebrafish Proteins / metabolism*
Reg. No./Substance:
0/Diglycerides; 0/Phosphatidylinositol 4,5-Diphosphate; 0/TRPC Cation Channels; 0/TRPC3 cation channel; 0/TRPC6 protein, human; 0/Trpc7 protein, mouse; 0/Zebrafish Proteins; EC 3.1.3.-/Phosphoric Monoester Hydrolases; EC 3.1.3.-/VSP protein, zebrafish; EC 3.1.3.-/voltage-sensor-containing phosphatase, Ciona intestinalis; EC Phospholipase C

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): Channels (Austin)
Journal ID (iso-abbrev): Channels (Austin)
Journal ID (publisher-id): CHAN
ISSN: 1933-6950
ISSN: 1933-6969
Publisher: Landes Bioscience
Article Information
Download PDF
Copyright © 2012 Landes Bioscience
Print publication date: Day: 01 Month: 5 Year: 2012
pmc-release publication date: Day: 01 Month: 5 Year: 2012
Volume: 6 Issue: 3
First Page: 206 Last Page: 209
ID: 3431592
PubMed Id: 22760061
Publisher Id: 2012CHANNELS0025R
Publisher Item Identifier: 20883
DOI: 10.4161/chan.20883

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides
Kyohei Itsuki12
Yuko Imai12
Yasushi Okamura3
Kihachiro Abe2
Ryuji Inoue1
Masayuki X. Mori1*
1Department of Physiology; School of Medicine; Fukuoka University; Fukuoka, Japan
2Faculty of Dental Science; Kyushu University; Fukuoka, Japan
3Laboratory of Integrative Physiology; Department of Physiology; Graduate School of Medicine; Osaka University; Suita, Osaka, Japan
*Correspondence to: Masayuki X. Mori, Email:

DrVSP and CiVSP: Time-solved Tool for Channel Regulation

Recognition of the importance of ion channel regulation by phosphoinositides (PIPs), especially phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), continues to grow.1 In earlier studies, the effects of PtdIns(4,5)P2 application were examined directly, or a PtdIns(4,5)P2 antibody, PtdIns(4,5)P2 scavenger (poly-L-lysine) or a pharmacological inhibitor (wortmannin or quercetin) was used to reduce the membrane PtdIns(4,5)P2 content. These methods remain solid and are well established, but their utility is limited to assessing steady-state PtdIns(4,5)P2 levels. By contrast, methods for using a chemically inducible PIP control system and voltage-sensing phosphatase (VSP) are becoming a standardized means of surveying dynamic PIP regulation. Chemical induction of PIPs is well described in a recent review article.2 In this addendum, therefore, we have focused on the advantages of using VSP to study channel regulation.

So far VSPs from two aquatic species, Ciona intestinalis (Ci) and Danio rerio (Dr), have been identified. Ci- and DrVSP exhibit only small differences, mainly in their voltage-sensitivities and expression levels, and their catalytic activities (PI5-phosphatase) and substrate phosphoinositide (PI) specificities are nearly identical.3 Nonetheless, we think that DrVSP may be somewhat better suited for studying PI-mediated regulation of ion channels. This is because gating currents indicate the level of DrVSP expression in HEK cells to be two to three times higher than that of CiVSP, and because the voltage-sensitivity of DrVSP is shifted rightward by about 50 mV (V1⁄2 values from the Q-V curves are 94 and 44 mV for DrVSP and CiVSP, respectively).4 Considering that the resting membrane potential is around −40 to −10 mV in HEK cells,5-7 the enzymatic phosphatase activity driven by the resting potential is more negligible with DrVSP. In addition, the rather steep Q-V curve for DrVSP would be expected to yield more dramatic effects. When we combined DrVSP with TRPC channels in HEK cells, a depolarizing pulse to +100 mV for 500 ms was sufficient to produce maximum channel inhibition through depletion of PtdIns(4,5)P2 (referred to as VMI, VSP-mediated inhibition was evaluated based on the residual current (r) before and after the depolarization). However, such robust depolarizations can have unfavorable effects on channel regulation, e.g., the voltage-dependent inhibition relief observed with high voltage-gated Ca channels.8 To exclude this possibility, we confirmed our results using various voltage-sensing DrVSP derivatives [VMI (r)-V curve], as shown in Figure 1A. Note that the V-shifted DrVSPs all inhibited TRPC currents to the same degree as wild-type DrVSP. These data support our conclusion and further emphasize the advantages of using DrVSP to regulate the activities of a variety of ion channels, including voltage-gated channels.9,10

Dynamics of Inhibition and Recovery from the Inhibition by VSP Activation

VSP has enormous potential for use as a molecular tool with which to clarify the PtdIns(4,5)P2 sensitivity and binding kinetics of ion channels, even during periods when ionic currents are flowing. In Figure 1B, we present an atypical example of the inhibition of carbachol (CCh)-induced TRPC6 currents (upper) via VSP activation and subsequent current recovery. In contrast to the typical and averaged data presented in our recent study,11 in 2 of 11 cells tested, we observed responses like those depicted in the figure [i.e., there was a clear bell- or U-shaped relationship between stimulus number and VMI (r) (middle) and τ-recovery from the inhibition (bottom)]. The bell-shaped r curve was closely related to the peak CCh-induced current. Moreover, τ-recovery accelerated as the CCh-induced current and the value of r became larger. This observation raises the possibility that even during agonist-stimulated macroscopic activity in living cells, VMI magnitude may provide a clue to the level of PtdIns(4,5)P2 binding to the channels, as well as to the kinetics of evoked changes in PtdIns(4,5)P2 binding. The latter would also reflect to some degree PtdIns(4,5)P2 re-synthesis, which would be expected to influence the observed response. In an earlier study, Hardie et al., showed dynamics of living PtdIns(4,5)P2 which was accompanied with the light response in Drosophila photoreceptors by measuring currents through the PtdIns(4,5)P2-sensitive Kir2.1 channel.12 However, we suggest the use of ectopic VSP is an alternative or a more convenient approach because VSP does not itself produce ionic flow other than gating currents.

Self-Limited Ion Channels

Because bioelectric signals are largely attributable to the flow of ions, it is critically important to maintain ion channel activities for appropriate durations. To shorten the duration of ion flow, channels, particularly those contributing to excitation, often possess self-limiting regulatory systems. For instance, Figure 2A to C illustrate the mechanisms of self-limiting regulation found in voltage-gated sodium channels, high voltage-gated calcium channels and inotropic ATP receptors. The mechanism underlying the self-limiting regulation of TRPC3/C6/C7 channels (Fig. 2D) clearly differs from the other examples shown. TRPC3/C6/C7 channels are intracellular ligand-gated channels assembled as homo- or heterotetramers, and are activated by DAG produced through a reaction catalyzed by G protein- or receptor tyrosine kinase-coupled PLC. But when DAG is produced from its substrate PtdIns(4,5)P2, the resultant reduction in membrane PtdIns(4,5)P2 content independently inhibits channel activation. Thus both activation and inhibition are simultaneously induced by PLC-catalyzed degradation of PtdIns(4,5)P2. As a result, the time required for the response to reach the first current peak is more susceptible to modulation than the other modes of self-limiting regulation.

TRPC channels are often activated by neurohormones released from autonomic nerves and can be thought of as being downstream of the autonomic nervous system (ANS). The ANS is important for maintenance of the stable internal physiological conditions often referred to as “homeostasis.” This makes the self-limiting regulation of TRPC channels interesting in part because the resultant regulation of the global effects of the ANS appears to arise from the molecular level.

Channel Regulation Linked to Enzymatic Reaction

Enzymatic reactions are often involved in cell signaling through, for example, an increase in the concentration of a product. On the other hand, the functionality of substrate reduction or depletion is easily masked by the reduction in the productivity of the catalytic reaction. Consequently, signaling systems mediated by enzymatic reactions might be expected to exhibit physiologically bimodal biochemical responses. The self-limiting regulation linked to PLC activity could thus provide profound mechanistic insight of channel modulation as well as rediscovering subtle feature of enzymatic functionality. Furthermore, when this regulation is disrupted (which can be seen experimentally in the case of CCh-induced currents in the presence of an excess of the substrate analog dic8-PtdIns[(4,5)P2], the decay phase of the currents is prolonged (Fig. 2B), and the ensuing buildup of cytosolic Na+ and/or Ca2+ can have significant pathophysiological effects that could underlie the development of such ailments as vascular hypertension, cardiac hypertrophy and renal failure.

Overall, our results emphasize that TRPC3/C6/C7 channels are subject to self-limiting regulation that is related to their close association with the PtdIns(4,5)P2-PLC-DAG cascade and is distinct from the self-limiting mechanisms observed in voltage-gated channels. The specific physiological importance of this type of regulation, as compared with other modes of channel regulation, will be an important area of investigation in the future.


Previously published online:


This work was supported by Grants-in-aid for Young Scientist from the Japan Society for the Promotion of Sciences, The Naito Foundation and Central Research Institute of Fukuoka University (to M.X.M.).


1. Hilgemann DW. Local PIP(2) signals: when, where, and how?Pflugers ArchYear: 2007455556710.1007/s00424-007-0280-917534652
2. Ueno T,Falkenburger BH,Pohlmeyer C,Inoue T. Triggering actin comets versus membrane ruffles: distinctive effects of phosphoinositides on actin reorganizationSci SignalYear: 20114ra8710.1126/scisignal.200203322169478
3. Okamura Y,Murata Y,Iwasaki H. Voltage-sensing phosphatase: actions and potentialsJ PhysiolYear: 20095875132010.1113/jphysiol.2008.16309719074969
4. Hossain MI,Iwasaki H,Okochi Y,Chahine M,Higashijima S,Nagayama K,et al. Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatasesJ Biol ChemYear: 2008283182485910.1074/jbc.M70618420018375390
5. Beyder A,Rae JL,Bernard C,Strege PR,Sachs F,Farrugia G. Mechanosensitivity of Nav1.5, a voltage-sensitive sodium channelJ PhysiolYear: 201058849698510.1113/jphysiol.2010.19903421041530
6. Søgaard R,Ljungstrøm T,Pedersen KA,Olesen SP,Jensen BS. KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacologyAm J Physiol Cell PhysiolYear: 2001280C8596611245603
7. Saleem F,Rowe IC,Shipston MJ. Characterization of BK channel splice variants using membrane potential dyesBr J PharmacolYear: 20091561435210.1111/j.1476-5381.2008.00011.x19068078
8. Currie KP. G protein modulation of CaV2 voltage-gated calcium channelsChannelsYear: 2010449750910.4161/chan.4.6.1287121150298
9. Falkenburger BH,Jensen JB,Hille B. Kinetics of PIP2 metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cellsJ Gen PhysiolYear: 20101359911410.1085/jgp.20091034520100891
10. Suh BC,Leal K,Hille B. Modulation of high-voltage activated Ca(2+) channels by membrane phosphatidylinositol 4,5-bisphosphateNeuronYear: 2010672243810.1016/j.neuron.2010.07.00120670831
11. Imai Y,Itsuki K,Okamura Y,Inoue R,Mori MX. A self-limiting regulation of vasoconstrictor-activated TRPC3/C6/C7 channels coupled to PI(4,5)P₂-diacylglycerol signallingJ PhysiolYear: 201259011011922183723
12. Hardie RC,Gu Y,Martin F,Sweeney ST,Raghu P. In vivo light-induced and basal phospholipase C activity in Drosophila photoreceptors measured with genetically targeted phosphatidylinositol 4,5-bisphosphate-sensitive ion channels (Kir2.1)J Biol ChemYear: 2004279477738210.1074/jbc.M40752520015355960
13. Mori MX. Interaction between the Voltage-Dependent Sodium Channel and Calmodulin. Thesis, (PhD) 2000; The Graduate University for Advanced Studies.
14. Mori MX,Erickson MG,Yue DT. Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channelsScienceYear: 2004304432510.1126/science.109349015087548
15. Werner P,Seward EP,Buell GN,North RA. Domains of P2X receptors involved in desensitizationProc Natl Acad Sci U S AYear: 199693154859010.1073/pnas.93.26.154858986838


[Figure ID: F1]

Figure 1. DrVSPs on TRPC currents. (A) top: Exemplar of the voltage-dependence of VMI (r) of TRPC3/C6/C7 currents observed in HEK cells. TRPC6 currents were evoked by external application of the DAG lipase inhibitor RHC80267 (100 μM). r indicates the residual current after depolarization. The red arrow shows the transient inhibition elicited by the depolarization. Bottom: VMI of TRPC6 currents plotted against depolarization pulse amplitude applied in the presence of the indicated DrVSP mutants (n = more than 4). Note that the Q-V curves of the mutants are also shifted leftward [V1/2(OFF] values for R153Q, T156R and I165R are 16, 73 and 60 mV, respectively).4 (B) top: Atypical inhibition trace obtained from HEK cells co-transfected with TRPC6 and wild-type DrVSP. Brief depolarizations (+100 mV, 500 ms) were applied every 10 sec (protocol displayed in top), and currents were evoked by CCh (100 μM). Middle and bottom: r and τ-recovery are plotted against stimulus number from the upper trace. The blue dashed line in the middle panel suggests DrVSP-available PtdIns(4,5)P2 (speculative). The typical trace, averaged r, and averaged τ-recovery data were shown in ref. 11.

[Figure ID: F2]

Figure 2. Self-limiting regulatory systems in ion channels. Schemes for channel opening (Cho). (A): Voltage-gated sodium channels (rNaV1.2) in vertebrates open (+) and quickly inactivate (–) in response to depolarization (ψ1 and ψ2: difference in the potentials coordinate the V-shaped current trace). (B): High voltage-gated Ca channels (CaV1.2) open upon membrane depolarization (ψ1) and inactivate due to negative feedback regulation wherein Ca2+ permeating through the channels inactivates the channels (calcium-dependent inactivation). (C): Ligand-gated cys-loop receptors (P2X1) are activated and then desensitized by the concentration-dependent binding of an agonist (a). Copyright (1996) National Academy of Sciences, USA (D): TRPC3/C6/C7 channels are activated by DAG, a product of PtdIns(4,5)P2, and the resultant reduction in PtdIns(4,5)P2 independently inhibits channel opening. Strength linkages with G protein-coupled receptors alter the kinetics in TRPC6 currents (tight and loose linkages are represented by the red and black traces, respectively). The respective current traces were obtained from refs. 13, 14, 15 and 11. (E): Currents obtained under self-limiting conditions exhibit faster inactivation than those obtained under unlocked conditions induced by application of excessive PtdIns(4,5)P2 through the patch-pipette (TRPC7 currents obtained from ref. 11).

Article Categories:
  • Article Addendum

Keywords: Keywords: Ca2+ signal, PIP2, TRP channel, TRPC3, TRPC6, TRPC7, VSP, ion channel regulation, phosphoinositides, receptor-operated ion channels, smooth muscle physiology.

Previous Document:  Mutations in the basic loop of the Zn binuclear cluster of the UaY transcriptional activator suppres...
Next Document:  A comparative study of the central effects of melanocortin peptides on food intake in broiler and la...