![Simultaneous measurement of receptor-operated TRPC6 currents and PI(4,5)P2 detected by 3-cube FRET. (A) Images of PI(4,5)P2 sensor expressed in HEK293 cells using a 3-cube filter. Phase-contrast image (top left), YFP channel (F542(A); top right), CFP channel (F464(D); bottom left), and FRET channel (F542(D); bottom right) are shown. (B) Diagram of molecules transfected into HEK293 cells. TRPC6, PI(4,5)P2 sensor (CFPmse-PHd [blue box] and YFPmse-PHd [yellow box]), and M1R were expressed. The excitation wavelengths, 427 and 504 nm, were alternately illuminated. (C) Typical example of CCh-induced TRPC6 currents (top) and the corresponding FRET changes (middle). The FR (middle; circles) calculated by 3-cube methods can yield near absolute FRET efficiency (EEFF; right axis). Measured parameters were the duration of Δ10–90% and Δ90–50% of the peak current, the kinetics of FRET decay (τFR), and minimum FR (FRmin). The decline of FRET (FR) was fitted with a single-exponential decay (red solid curve): FR=FRmin+FRΔ×exp(−t/τFR). The bottom panel shows the changes in the fluorescence intensities (a.u.) that passed through the respective filter setting.](https://www.researchgate.net/profile/Ryuji-Inoue/publication/259957296/figure/fig1/AS:11431281177240846@1690416376788/Simultaneous-measurement-of-receptor-operated-TRPC6-currents-and-PI4-5P2-detected-by_Q320.jpg)
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J. Gen. Physiol. Vol. 143 No. 2 183–201
www.jgp.org/cgi/doi/10.1085/jgp.201311033 183
INTRODUCTION
Transient receptor potential classical/canonical (TRPC)
3, C6, and C7 channels are the closest mammalian ho-
mologues of the Drosophila melanogaster TRP channel and
are expressed in various cell types, including smooth
muscle and neurons (Hardie, 2003; Inoue et al., 2006;
Venkatachalam and Montell, 2007). These channels con-
duct cations (Na+, Ca2+) in response to stimulation of
receptors coupled to phospholipase C (PLC), namely
Gq protein–coupled receptors and certain tyrosine ki-
nase receptors (Ramsey et al., 2006). For that reason,
Correspondence to Masayuki X. Mori: m x m o r i @ s b c h e m . k y o t o - u . a c . j p
Abbreviations used in this paper: AVP, arginine8 vasopressin; CCh, car-
bachol; DAG, diacylglycerol; DrVSP, Danio rerio voltage-sensing phospha-
tase; FR, Förster resonance energy transfer ratio; FRET, Förster resonance
energy transfer; HEK, human embryonic kidney; IP3, inositol 1,4,5-tris-
phosphate; M1R, muscarinic type 1 receptor; PA, phosphatidic acid; PHd,
Pleckstrin homology domain; PI(4,5)P2, phosphatidylinositol 4,5-bisphos-
phate; PIP5K, phosphatidylinositol-4-phosphate-5-kinase; PLC, phospholi-
pase C; SPD, self-limiting regulation by PI(4,5)P2–DAG signaling; TRPC,
transient receptor potential classical/canonical.
the currents mediated by these channels are often called
receptor-operated cation currents (Inoue and Kuriyama,
1993; Firth et al., 2007). TRPC3/6/7 channels are also
activated by synthetic membrane-permeable diacylglyc-
erol (DAG) analogues and are thus considered to be
DAG-sensitive or activated channels (Hofmann et al.,
1999; Okada et al., 1999). In a physiological context,
DAG is produced by the hydrolytic activity of PLC,
which is located downstream of the receptors for neu-
rotransmitters and hormones. Therefore, the receptor
stimulation activates TRPC3/6/7 channels through the
production of DAG to generate the receptor-operated
TRPC currents (Beech et al., 2004; Panda et al., 2005;
Hartmann et al., 2008).
PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation
of TRPC6/7 channels
Kyohei Itsuki,1,2 Yuko Imai,1,2 Hideharu Hase,3 Yasushi Okamura,4 Ryuji Inoue,1
and Masayuki X. Mori1,3
1Department of Physiology, School of Medicine, Fukuoka University, Fukuoka 814-0180, Japan
2Faculty of Dental Science, Kyushu University, Fukuoka 812-8581, Japan
3Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University,
Kyoto 615-8501, Japan
4Laboratory of Integrative Physiology, Department of Physiology, Graduate School of Medicine, Osaka University,
Osaka 565-0871, Japan
Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC chan-
nels 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 sug-
gest 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 PLC Pleck-
strin homology domain (PHd), which binds PI(4,5)P2, and used this constant to t 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, conrming 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.
© 2014 Itsuki et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publi-
cation date (see http://www.rupress.org/terms). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
The Journal of General Physiology
184 PIP2 dynamics in receptor-operated TRPC6/7 currents
(Institut für Pharmakologie und Toxikologie, Zürich, Switzer-
land); pCI-neo expression vectors encoding mouse TRPC3 (Gen-
Bank accession no. NM_019510) and TRPC7 (GenBank accession
no. NM_012035) were provided by Y. Mori (Kyoto University,
Kyoto, Japan). Single amino acid mutation in TRPC6 and TRPC7
was generated using the QuikChange Site-Directed Mutagenesis
kit (Agilent Technologies) according to the manufacturer’s in-
structions. To generate bright FRET pairs, super-enhanced YFP or
CFP isolated from a RhoA FRET sensor (provided by M. Matsuda,
Kyoto University, Kyoto, Japan) were modied to give A207K mu-
tants as a monomeric form (CFPmse or YFPmse) (Zacharias et al.,
2002). These modied uorophores were fused to the N-terminal
side of the PLC Pleckstrin homology domain (PHd; provided by
K. Jalink, The Netherlands Cancer Institute, Amsterdam, Nether-
lands) to construct PI(4,5)P2 sensor molecules consisting of CF-
Pmse-PHd or YFPmse-PHd. For DAG detection, CFPmse was fused to
the C-terminal side of PKC (provided by M. Schaefer, Leipzig
University, Leipzig, Germany), yielding PKC-CFPmse. To be an
energy acceptor of membrane-bound PKCE-CFPmse, YFPmse was
attached to the C-terminal side of the GAP-43 myristoyl domain
(Invitrogen) through an octaglycine (G8) linker (Myr-YFPmse).
PI(4,5)P2 and DAG sensor cDNA were each incorporated into an
IRES-reporter region–excluded pIRES2 expression vector (Invit-
rogen). A pEF-BOS expression vector encoding human musca-
rinic type 1 receptor (M1R) was provided by T. Haga (Gakushuin
University, Tokyo, Japan). Human phosphatidylinositol-4-phosphate-
5-kinase (PIP5K; isoform) in pcDNA3.1 vector (Invitrogen) was
provided by S. Kita and T. Iwamoto (Fukuoka University, Fukuoka,
Japan). All PCR products were sequenced entirely.
HEK293 cells (obtained from the ATCC) were maintained in
Dulbecco’s modied Eagle’s medium (Invitrogen) supplemented
with 10% FBS (Gibco) and antibiotics (penicillin and streptomy-
cin; Gibco) at 37°C (5% CO2). For transfection, cells were seeded
on poly--lysine–coated glass coverslips (Matsunami) in 35-mm
culture dishes and transfected with a mixture of plasmid vector–
incorporated DNAs using the SuperFect transfection reagent
(QIAGEN). For FRET-based PI(4,5)P2 detection, HEK293 cells
were cotransfected with 1 µg each of plasmids encoding TRPC3,
6, or 7 together with M1R (or without it, in endogenous musca-
rinic receptor stimulation) and 0.3 µg each of plasmids encoding
CFPmse-PHd and YFPmse-PHd. For DAG detection, 0.3 µg each of
plasmids encoding Myr-YFPmse and PKC-CFPmse were cotrans-
fected instead of the PI(4,5)P2 sensor plasmids. For detection
of local PI(4,5)P2 around the TRPC7 channel, the sequence en-
coding the donor protein (CFPmse) was inserted before the stop
codon of TRPC7. In this case, equal amounts (1 µg) of donor, ac-
ceptor (YFPmse-PHd), and M1R plasmids were used for transfec-
tion. Measurements on transfected cells were made within 24–72 h
after transfection.
A7r5 cells, the cell line derived from rat thoracic aortic smooth
muscle (Brandt et al., 1976), were obtained from the ATCC,
maintained in medium identical to that used for HEK293 cells,
and passaged every 5–7 d. The transfection protocol was essen-
tially the same as the one used with HEK293 cells. A7r5 cells trans-
fected with CFPmse-PHd and YFPmse-PHd were reseeded on poly--
lysine–coated glass coverslips and incubated at 37°C (5% CO2)
for at least 15 min before use. Cells were always used within 2 h
of reseeding.
Solutions and drugs
The standard external solution contained (mM): 140 NaCl, 5 KCl,
1 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4, adjusted
with Tris base; 300 mOsm, adjusted with glucose). The pipette
solution contained (mM): 120 CsOH, 120 aspartate, 20 CsCl,
2 MgCl2, 5 EGTA, 1.5 CaCl2, 10 HEPES, 2 ATP-Na2, 0.1 GTP, and
10 glucose (pH 7.2, adjusted with Tris base; 290–295 mOsm, adjusted
with glucose). CCh (Sigma-Aldrich) was diluted in the standard
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2, or
PIP2) is the major substrate of PLC, and its hydrolysis
produces DAG. PI(4,5)P2 is known to regulate numer-
ous ion channels, modulating electrical signal outputs
from metabotropic receptors in diverse physiological
contexts (Gamper and Shapiro, 2007; Hilgemann, 2007;
Logothetis et al., 2007). However, knowledge concern-
ing the effect of PI(4,5)P2 on TRPC channels is still ac-
cumulating (Jardín et al., 2008; Lemonnier et al., 2008;
Monet et al., 2012). We have recently demonstrated
using Danio rerio voltage-sensing phosphatase (DrVSP)
that reduction or depletion of PI(4,5)P2 inhibits the ac-
tivity of DAG-sensitive TRPC3/6/7 channels, both in an
exogenous expression system and in smooth muscle–
derived cells (A7r5) (Imai et al., 2012; Itsuki et al.,
2012a). The DrVSP-mediated inhibition of TRPC3/6/7
currents was detected even in currents evoked by a
membrane-permeable DAG analogue (OAG), which sug-
gests that reduction in PI(4,5)P2 can inhibit TRPC3/6/7
channel opening regardless of the presence of DAG.
Under Gq protein–coupled receptor stimulation, PI(4,5)P2
hydrolysis (breakdown) by the receptor-activated PLC
largely contributes to the production of DAG. There-
fore, such a complex relationship of PI(4,5)P2 and DAG
suggests that TRPC3/6/7 channel activity may be regu-
lated in a self-limiting manner.
The effects of PLC-coupled receptor-driven channel
regulation via enzymatic hydrolysis of PI(4,5)P2 are not
known. To improve our understanding, we simultane-
ously measured PI(4,5)P2 or DAG dynamics and recep-
tor-operated TRPC currents evoked by carbachol (CCh;
a muscarinic receptor agonist) or vasopressin (a vasocon-
strictor). To this end, we measured the levels of PI(4,5)P2
and DAG using a quantitative Förster resonance energy
transfer (FRET)-based sensor alongside detection of
TRPC6 and TRPC7 currents, which are more sensi-
tive than TRPC3 currents to reduction in PI(4,5)P2 in
human embryonic kidney (HEK)293 cells and smooth
muscle–derived cells. We found that the temporal FRET
dynamics of PI(4,5)P2 reduction closely correlate with
the time course of activation and inactivation of receptor-
operated TRPC6/7 channel currents. We also constructed
a kinetic model of receptor-driven PI(4,5)P2–DAG signal-
ing. This model, calibrated with DrVSP-derived func-
tional dissociation constants for PI(4,5)P2 binding to
TRPC3/6/7 channels, closely resembled our experi-
mental results. Our study, combining experimental and
computer simulation data, revealed the crucial role of
receptor-stimulated PI(4,5)P2 hydrolysis in TRPC6/7 cur-
rents. Part of the work presented here has appeared in
abstract form (Itsuki et al., 2012b).
MATERIALS AND METHODS
Plasmids and cells
The pcDNA3 expression vector encoding human TRPC6 (Gen-
Bank accession no. NM_004621) was provided by T. Hofmann
Itsuki et al. 185
is denoted as “464,” and that for acceptor uorescence (542 nm)
as “542.” The excitation lter for donor excitation (427 nm) is
denoted as “D,” and that for acceptor excitation (504 nm) as “A.”
Background intensity, captured using the corresponding lter
setting with nontransfected cells, was subtracted from specimen
uorescence signals. Finally, the FRET ratio (FR) was calculated
according to the “3-cube” method (Erickson et al., 2001):
FR F R F R F R F= − ⋅
( )
⋅ − ⋅
( )
D1 A D2
542 D 464 D 542 A 542 D( ) ( ) ( ) ( )
/ , (1)
where RD1 = F542(D)/F464(D), RD2 = F542(A)/F464(D), and RA =
F542(D)/F542(A). Constants of RD1, RD2, and RA were predeter-
mined using measurements from single cells expressing only
donor- (CFPmse) and acceptor- (YFPmse) tagged molecules, respec-
tively. From the FR values, we can compute the effective FRET ef-
ciency (EEFF):
E FR
EFF
= −
( )
⋅
( ) ( )
1 YFP CFP
mse mse
ε ε
427 427/ , (2)
where YFPmse and CFPmse are the molar extinction coefcients
for the FRET cube excitation lters obtained using the 427-nm
excitation band-pass lter. We determined the ratio in brackets to
be 0.11, based on maximal extinction coefcients for YFPmse and
CFPmse (Mori et al., 2011).
Establishment of a relationship between PI(4,5)P2
concentration and FRET
Here, we calculated the relationship between FRET and PI(4,5)P2.
To this end, we considered two different cases: (1) FRET between
TRPC7 channel-anchored CFPmse and YFPmse-PHd, where YFPmse-
PHd can transit between a soluble cytoplasmic state and a PI(4,5)P2-
bound membrane state; and (2) FRET between CFPmse-PHd and
YFPmse-PHd, where both probes transition between a soluble and
membrane-bound state.
Case 1 is described by:
FR
FR K
PI P
max d PHd
≈ =
+
( )
( )
F
1
14 5 2
,
,
(3)
where F is the fraction of PHd bound to PI(4,5)P2, Kd(PHd) is the
dissociation constant of PHd bound to PI(4,5)P2 (reported as
2.0 µM; Lemmon et al., 1996; Hirose et al., 1999), and FRmax is the
maximum FR at an innitely high concentration of PI(4,5)P2 that
induces all the uorophore-fused PHd probes to bind to the
plasma membrane. FRmax is a purely theoretical value, because
physiological PI(4,5)P2 levels (5–40 µM) are insufcient to local-
ize all PHd proteins to the plasma membrane (Bunce et al., 1993;
McLaughlin and Murray, 2005). Nevertheless, although FRmax is
impossible to demonstrate, overexpression of PIP5K can greatly
increase the cellular levels of PI(4,5)P2 by approximately two- to
threefold (Winks et al., 2005). We observed that cells overexpress-
ing PIP5K demonstrated 1.2 times higher FR than control cells
with resting PI(4,5)P2 levels (Fig. 5 A). We thus calculated the
FRmax value by multiplying the resting FR by this correction factor
of 1.2. Eq. 3 was used to simulate FR dynamics in cells express-
ing TRPC7-CFPmse and YFPmse-PHd, as shown in Eq. 25 (Table 3)
and Fig. 7 D. Conversion of the FRET between CFPmse-PHd and
YFPmse-PHd to PI(4,5)P2 concentration (i.e., case 2) is described
in the Results.
Estimation of expressed fluorophore-tagged PHd proteins
We determined that the average concentration of uorophore-
tagged PHd in single cells was 1.6 µM. This value was calculated
based on the intensity from uorescein (Sigma-Aldrich) as follows.
external solution from its stock concentration (100 mM in H2O).
To conrm monovalent cationic currents, NMDG solution (150 mM
N-methyl--glucamine chloride, 10 mM HEPES, and 1 mM CaCl2,
with pH 7.4 adjusted with HCl) was applied at the end of each
stimulus. RHC80267 (EMD Millipore) was dissolved in DMSO
(Wako Chemicals USA). Stock solutions of Arginine8 vasopressin
(AVP; 100 µM; MP Biomedicals) and nifedipine (10 mM; EMD
Millipore) were dissolved in H2O and DMSO, respectively. AVP
and nifedipine were freshly prepared in the standard external so-
lution to nal concentrations of 1 and 5 µM, respectively, before
applying to A7r5 cells. During experiments, HEK293 and A7r5
cells were continuously perfused with external solution and gravity-
fed at a ow rate of 0.25 ml/min. The perfusion was turned on and
off using electromagnetic solenoid microvalves (The Lee Co.).
Simultaneous measurements of TRPC currents and FRET
Electrophysiology. The whole-cell patch-clamp technique was used
for current detection. Patch electrodes with a resistance of 4–6 M
(when lled with internal solution) were made from 1.5-mm boro-
silicate glass capillaries (Sutter Instrument). Series resistance
errors were compensated >60%. Voltage generation and current
signal acquisition were accomplished using a patch-clamp amplier
(AxoPatch 200B; Axon Instruments) with an A/D D/A converter
(Digidata 1200; Axon Instruments). Sampled data were low-pass
ltered and digitized at 1 kHz using pClamp 9.0 (Axon Instru-
ments) and analyzed using custom-written software (MATLAB;
MathWorks). The currents were recorded at a holding potential
of 50 mV. For activation of DrVSP, depolarizing step pulses
(from 20 to 180 mV, 500-ms duration) were delivered every 20 s.
The ratio of the currents before and after DrVSP activation was
used to quantitate DrVSP-mediated inhibition, “r (I).” Before its
calculation, the leak that was dened by the current in NMDG-
containing solution was subtracted. All experiments were performed
at room temperature (22–25°C).
FRET detection. Fluorescence from voltage-clamped cells was
detected using a microscope (60 × 0.9 N.A. objective; TE300 Eclipse;
Nikon) equipped with a two-channel simultaneous beam-splitter
(Dual-View2; Photometrics) and a high sensitivity EMCCD cam-
era (Evolve512; Photometrics). Excitation light ltered at 427/10
and 504/12 nm was alternately introduced via an optical ber
from a lamp house equipped with a high speed excitation wave-
length selector (75 W xenon lamp; OSP-EXA; Olympus). Epiuo-
rescence from the cells was preltered using a multiband dichroic
mirror (449–483 and 530–569 nm) contained in the microscope,
and then further separated in the beam-splitter (at 505 nm) and
ltered at 464/23 nm (detection of the donor uorescence) or
542/27 nm (detection of the acceptor uorescence). Optical l-
ters were obtained from Semrock, except the splitter (Chroma
Technology Corp.). The duration of camera exposure was 100 ms
and occurred within 150-ms periods of illumination at each exci-
tation wavelength. Images were captured with an EM gain of 300
and then digitized as 512 × 512 pixels by 16-bit arrays in the micro-
scope software (Micro-manager v.1.4). The image pixel resolution
was 0.26 µm. Averaged intensities from the whole-cell region
(typically 20 × 20 to 40 × 40 square pixels) were analyzed to calcu-
late FRET using a custom-written MATLAB program. The electro-
physiology and FRET measurements were synchronized using
brief triggers from the A/D D/A converter linked to the excita-
tion light shutter. All of the data in this paper were recorded from
the rst application of any of the agonists.
Calculation of FRET. Fluorescence signal output obtained from a
given sample is denoted by the descriptor FX(Y), where X and Y are
the uorescence lter settings for the emission and excitation light,
respectively. The emission lter for donor uorescence (464 nm)
186 PIP2 dynamics in receptor-operated TRPC6/7 currents
(Fig. S3). Incomplete matching of the DG model to the experi-
mental FR (Fig. S4). The quick recovery of FR under the AVP
stimulation (Fig. S5). Fitting the SPD model to TRPC7 current
demonstrated less matching to FR dynamics and vice versa
(Fig. S6). Effect of an inactive ATP analogue in the patch pipette
on the receptor-operated TRPC7 currents (Fig. S7). The online
supplemental material is available at http://www.jgp.org/cgi/
content/full/jgp.201311033/DC1.
RESULTS
Simultaneous measurement of PI(4,5)P2 and TRPC currents
The PHd of PLC binds both PI(4,5)P2 and inositol
1,4,5-trisphosphate (IP3) (Hirose et al., 1999). In the rest-
ing state, however, most of the uorophore-tagged PHds
are located at the plasma membrane, making it possible
to measure PI(4,5)P2 at the plasma membrane (van der
Wal et al., 2001; Jensen et al., 2009; Yudin et al., 2011)
(Fig. 1 A). Using this approach, we rst demonstrated the
simultaneous measurements of PI(4,5)P2 and receptor-
operated TRPC6 current. The FRET pairs, which con-
sisted of donor (CFPmse) or acceptor (YFPmse) fused to the
PHd, were coexpressed with TRPC6 channel and M1R
in mammalian HEK293 cells (Fig. 1 B). Fig. 1 C shows a
typical example of the simultaneous measurement of a
TRPC6 current and PI(4,5)P2 levels in a HEK293 cell
after stimulation with 10 µM CCh. Soon after CCh ap-
plication, an inward TRPC6 current (Fig. 1 C, top) and
FRET reduction (middle) were observed concurrently.
To evaluate the kinetics of the current activation and
inactivation, we measured the time required for recep-
tor-operated TRPC6 current to increase from 10 to 90%
of its peak amplitude and then to decay from 90 to 50%
(Fig. 1 C, top). We quantitatively examined the real-time
Fluorescein was dissolved in 50 mM borate buffer (pH 9.1). The
uorescence intensities in small droplets of this uorescein solution
were measured, under the same conditions as the cells overex-
pressing YFPmse-PHd. The respective intensities were standardized
by the quantum yields for uorescein (0.92) and YFPmse (0.57),
and then the average [YFPmse-PHd] in living cell was determined
to be 0.8 µM. Equal amounts of CFPmse-PHd and YFPmse-PHd plas-
mids were transfected, so total amounts of CFPmse-PHd and
YFPmse-PHd proteins could be extrapolated from this value.
Modeling and fitting statistics
Kinetic models of PI(4,5)P2 hydrolysis pathway by PLC were for-
mulated as ordinary differential equations, and the concentra-
tions of PI(4,5)P2 and DAG derived from these were incorporated
into the channel-operation models. Simulations were performed
in Excel (Microsoft) using the forward Euler method with a time
step of 0.05 s. Individual steps were translated into differential
equations based on the proposed kinetic scheme. Fitting the mod-
els to the experimental data of TRPC6/7 currents was performed
by a Generalized Reduced Gradient algorithm of the Solver func-
tion in Excel. Details of model formulations and model tting to
the experimental data are described in the Results.
The errors between the experimental and the back-calculated
FR by model tting to the currents (Figs. 6 and 7) were evaluated
by the standard deviations of residual as follows:
SD Norm. Norm.
σ
( )
= −
( )
−
( )
=
∑FR FR n
bk i
i
n2
01/ ,
where Norm.FR and Norm.FRbk denote the normalized experi-
mental and back-calculated FR at the i points. n indicates the total
number of time points (20/s).
Online supplemental material
Plotting the currents versus FRmin/FRresting relationship (Fig. S1).
DrVSP-mediated inhibition at the single-channel level (Fig. S2).
Diffused PI(4,5)P2 was incorporated in the self-limiting regula-
tion by PI(4,5)P2–DAG signaling (SPD) model-based simulation
Figure 1. Simultaneous measurement of receptor-operated
TRPC6 currents and PI(4,5)P2 detected by 3-cube FRET.
(A) Images of PI(4,5)P2 sensor expressed in HEK293 cells
using a 3-cube lter. Phase-contrast image (top left), YFP
channel (F542(A); top right), CFP channel (F464(D); bottom
left), and FRET channel (F542(D); bottom right) are shown.
(B) Diagram of molecules transfected into HEK293 cells.
TRPC6, PI(4,5)P2 sensor (CFPmse-PHd [blue box] and
YFPmse-PHd [yellow box]), and M1R were expressed. The
excitation wavelengths, 427 and 504 nm, were alternately
illuminated. (C) Typical example of CCh-induced TRPC6
currents (top) and the corresponding FRET changes (mid-
dle). The FR (middle; circles) calculated by 3-cube meth-
ods can yield near absolute FRET efciency (EEFF; right
axis). Measured parameters were the duration of 10–90%
and 90–50% of the peak current, the kinetics of FRET
decay (FR), and minimum FR (FRmin). The decline of
FRET (FR) was tted with a single-exponential decay (red
solid curve):
FR FR FR FR= + × −
min exp t
∆( / ).
τ
The bot-
tom panel shows the changes in the uorescence intensi-
ties (a.u.) that passed through the respective lter setting.
Itsuki et al. 187
uorescence (lled triangles; F542(A)) stayed largely con-
stant (Fig. 1 C, bottom). These congurations ensured
the FRET reduction and low level of quenching of sen-
sor proteins during the recordings. Kinetics of FR re-
duction (FR) and the minimum amount of FR (FRmin)
under the receptor stimulation were obtained by tting
to the FRET data with a single-exponential decay func-
tion (Fig. 1 C, middle).
alteration in PI(4,5)P2 level using the 3-cube FRET mea-
surement (described in Materials and methods). FRET
changes over time between CFPmse-PHd and YFPmse-PHd
(Fig. 1 C, middle) were calculated from the respective
uorescence intensities (Fig. 1 C, bottom). The donor
uorescence (Fig. 1 C, open circles; F464(D)) and reso-
nance uorescence upon donor excitation (black circles;
F542(D)) displayed inverted changes, whereas acceptor
Figure 2. Correlation between the TRPC6/7 currents and the decay of PI(4,5)P2. (A and B) Example traces of TRPC6 currents (top)
and FRET of PI(4,5)P2 sensor (CFPmse-PHd and YFPmse-PHd) (bottom) upon stimulation with 100 µM CCh of either endogenous
(A) or overexpressed M1R (B). (C and D) Same as in A and B, but in cells expressing TRPC7. (E) Summary of currents and FRET
changes. TRPC6/7 current increase (10–90%) and decay (90–50%), kinetics of FRET reduction (FR), and degree of FRET reduc-
tion (FRmin) were accelerated in a CCh concentration and M1R expression-dependent manner. The data depicted by the stripe and
the white bars show without channel expression (transfected only PI(4,5)P2 sensor) and endogenous receptor simulation, respectively.
Numbers in parentheses indicate the number of cells measured, here and throughout. (F and G) Time courses of the 10–90% and
90–50% of receptor-operated currents were plotted against the simultaneously measured FR (F, TRPC6; G, TRPC7). These data were
obtained from various concentrations of CCh or level of M1R expression. The slope with a linear t highlights a relationship between
the time courses and FR.
188 PIP2 dynamics in receptor-operated TRPC6/7 currents
the FR reduction with a near zero FRET efciency (FRmin =
1.15 ± 0.1 and EEFF = 0.016 ± 0.01; n = 11). Intriguingly,
the time to reach the peak current was remarkably
shortened compared with endogenous muscarinic re-
ceptor stimulation at the same concentration of CCh
(100 µM; +M1R = 2.8 ± 0.7 s; endo = 63 ± 8 s). Similar
tendencies in the FR reduction and the peak time were
observed when TRPC7 was expressed instead of TRPC6,
except for the shorter activation and inactivation time
for TRPC7 current (Fig. 2, C and D). In addition, TRPC7’s
FR was slightly delayed and FRmin was also slightly at-
tenuated compared with that of TRPC6. (Data from the
different strength of receptor stimulation was summa-
rized in Fig. 2 E.)
To elucidate the functionality of PI(4,5)P2 hydrolysis,
we focused on the kinetic relationships between TRPC6/7
currents and FRET reduction. For that purpose, the log–
log plots for the activation or the inactivation of TRPC6/7
currents and the values of FR were made using data ob-
tained at varying levels of agonist stimulation, with and
PI(4,5)P2 dynamics at different levels of receptor
stimulation and TRPC6/7 currents
We then explored the effect of PI(4,5)P2 reduction on
TRPC6 or TRPC7 channel currents at varying levels of
receptor stimulation. Stimulation of endogenous mus-
carinic receptors in HEK293 cells with 100 µM CCh
evoked prolonged TRPC6 current and a small amount
of reduction in FR (Fig. 2 A). The time for TRPC6 cur-
rent activation (10–90%) and inactivation (90–50%)
were 32 ± 7 s and 39.2 ± 6.7 s (n = 6), respectively. In ac-
cordance with the current time course, FR was length-
ened to 55.3 ± 4.5 s, and FRmin stayed near the resting
level. Reduction in FR was only 18 ± 6%. Previous stud-
ies have shown that depletion of PI(4,5)P2 can be in-
duced by overexpression of M1R (Xie et al., 2011; Dickson
et al., 2013). Consistent with these studies, overexpress-
ing M1R and treating the cells with a high concentration
of CCh (100 µM) greatly accelerated the TRPC6 current
and FRET reduction (Fig. 2 B) (10–90% = 1.6 ± 0.3 s;
90–50% = 6.2 ± 0.9 s; FR = 4.5 ± 0.9 s), and enhanced
Figure 3. Incompatible correlation between receptor-operated TRPC6/7 currents and DAG production. (A) Principle of DAG detec-
tion by PKC probe FRET. Increments in FRET caused by the translocation of PKC-CFPmse to the plasma membrane were detected by
a coexpressed membrane-resident acceptor protein (Mry-YFPmse) in HEK293 cells. (B) Whole-cell TRPC6 currents (top) and FRET
changes caused by DAG increments, “FRdag” (bottom, green circles), recorded from endogenous muscarinic receptor stimulation with
100 µM CCh. The rise of FRET was tted to the exponential equation: FR FR FR FR
dag dag
max
dag dag
t= − × −
( )
∆exp /
τ
(green solid curve).
(C) Traces of currents and FRdag in M1R-overexpressing cells with 100 µM CCh. (Left) TRPC6 currents. (Right) TRPC7 currents. Pro-
longed DAG production was observed. Purple zones indicate inconsistencies between current inactivation and DAG production. The
inset in the TRPC7 panel shows FRdag changes over a longer time scale (300 s). (D) Summary of FRdag levels at the respective current
points observed in the robust receptor stimulation (+M1R and 100 µM). The black and gray bars denote expression of TRPC6 and
TRPC7 channels, respectively. (E) Time courses of initial phase of current increase (10–30%) were plotted against kinetics of DAG
production (FRdag).
Itsuki et al. 189
inactivation while DAG levels were still increasing (Fig. 3 C,
right, and summarized in D). This inconsistency may be
explained by the idea that PKC-mediated phosphoryla-
tion inhibits channel opening, which has been pro-
posed for TRPC3 (Soboloff et al., 2007).
However, for TRPC6 channel, there was no signicant
difference in the current inactivation (90–50%) be-
tween cells overexpressing PKC (TRPC6, M1R, and DAG
sensor–expressing, 7.5 ± 2.5 s; n = 5) and control (TRPC6
and M1R, 6.2 ± 0.9 s; n = 11). In addition, a previous
report has shown that PKC exerts a negative feedback
effect via phosphorylation of Ser448 of TRPC6 (Bousquet
et al., 2010). Using the phosphorylation-insensitive mu-
tant TRPC6S448A and its corresponding mutant TRPC7S394A,
we tested whether the PKC-mediated phosphorylation
of these residues is involved in the current decay. Under
the robust receptor stimulation, unexpectedly, we did
not detect any clear difference in the 90–50% time
(100 µM CCh: TRPC6wt, 11.4 ± 2.4 s; TRPC6S448A, 12.7 ±
2.7 s; TRPC7wt, 1.8 ± 1.1 s; TRPC7S394A, 1.9 ± 0.9 s; n = 8).
Furthermore, the basal phosphorylation site of TRPC6
has been identied at Ser814 (Bousquet et al., 2011),
which may play a role in the channel function. We also
tested the mutant, TRPC6S814A, but it failed to show any
signicant differences, including 90–50% (10.1 ± 3.5 s;
n = 7), which is consistent with the previous report
(Bousquet et al., 2011). Our data do not exclude the
possibility that PKC-mediated phosphorylation inhibits
channel opening, because of the variety of cell and
measurement conditions. Nevertheless, these results
suggest that the DAG production level and PKC phos-
phorylation–mediated channel modulation may contrib-
ute less to the inactivation of TRPC6/7 channels at the
robust stimulation.
A simulation model was built for further understand-
ing of dual regulation by PI(4,5)P2 and DAG, as described
in the latter section. For that purpose, we presented the
relation between the current increase and the produc-
tion of DAG. Plotting the early phase of the current
increase (10–30%) versus DAG production kinetics
(FRdag) exhibited a clear correlation, with a smaller slope
for the TRPC7 compared with the TRPC6-expressing cells
(Fig. 3 E). This result suggests that the TRPC7 channel
is highly sensitive to the increment of DAG. We thus
set the DAG sensitiveness in the initial parameter
as TRPC7 > TRPC6 in the model simulation (Table 4,
rows 21 and 22).
Functional dissociation constants of PI(4,5)P2 binding
to TRPC3/6/7 channels
These results have shown the substantial importance
of the dissociation of PI(4,5)P2 to the inactivation of
TRPC6/7 channel currents, but the afnity of PI(4,5)P2
to these channels is not yet known. To obtain this pa-
rameter, DrVSP, which functions as a membrane-resident
voltage-controllable phosphoinositides phosphatase, was
without M1R overexpression (Fig. 2, F and G). These
log–log plots showed a clear correlation between FR
and the current time courses (10–90% [left panels] and
90–50% [right panels]) for both TRPC6 and TRPC7
currents. The linear relationship that appeared in
a plot of the relation of 90–50% to FR in TRPC7-
expressing cells showed signicantly steeper slopes
(slope = 1.33) than that in TRPC6-expressing cells (slope =
0.76) (Fig. 2, F and G). This steepness may reect
higher TRPC7 sensitivity to reduction in PI(4,5)P2 than
TRPC3 or TRPC6 (Imai et al., 2012). In addition to the
FRET decay, the extent of the reduction or depletion
of PI(4,5)P2 levels (FRmin) also showed a similar ten-
dency to the time course of current activation or inacti-
vation of each channel (Fig. S1). These plots, however,
were slightly more scattered than those of FR. Such a
scattering was probably caused by cell-to-cell variability in
the released IP3 in response to the hydrolysis of PI(4,5)P2
(Irvine and Schell, 2001). The simultaneous detection of
PI(4,5)P2 and TRPC currents demonstrated that the ac-
tivation and inactivation time courses related to both
the kinetics and the extent of PI(4,5)P2 reduction.
Simultaneous detection of DAG and receptor-operated
TRPC channel current
TRPC3/6/7 channels are DAG-sensitive ion channels,
but the manner in which DAG dynamics correlate with
these TRP channels’ activity remains largely unknown.
To address this question, DAG production was con-
currently monitored with receptor-operated TRPC6/7
channel currents. The detection of DAG dynamics re-
lies on membrane translocation of DAG-activated PKC
in response to increasing DAG levels at the plasma
membrane (Violin et al., 2003) (Fig. 3 A). We used Ca2+-
insensitive PKC as a uorescence donor molecule to
exclude Ca2+-dependent translocation of PKC (Sinnecker
and Schaefer, 2004). Upon stimulation with CCh through
the endogenous muscarinic receptors, TRPC6 current
and DAG production were initiated almost simultane-
ously (Fig. 3 B). This parallel response is consistent with
TRPC6 being a DAG-sensitive channel. Furthermore,
during the inactivation of the TRPC6 channel current,
DAG level also declined. This synchronicity indicated
that when the strength of receptor stimulation was weak,
the production of DAG levels seemed to be a critical fac-
tor to the current appearances.
Contrary to the synchronicity of the TRPC6 current
and DAG dynamics in the weak receptor stimulation,
when the cells overexpressed M1R, the simultaneous
measurement was revealed to be inconsistent. The acti-
vation of TRPC6 channel current paralleled DAG pro-
duction, whereas the inactivation of the channel did
not; there was no decline in DAG production (Fig. 3 C,
left, purple zone). This lack of temporal consistency
between the current decay and DAG levels was even
more prominent in the TRPC7 channel, which exhibited
190 PIP2 dynamics in receptor-operated TRPC6/7 currents
activation was also insufcient to reach a zero FRET
level (FR = 1.46 ± 0.07; n = 14 at 120 mV). We thus spec-
ulated that this partial current inhibition may simply be
because of the incomplete depletion of PI(4,5)P2. This
idea raises a challenging question: how is TRPC3/6/7
inhibition by DrVSP activation observed at the single-
channel level? To answer this question, DrVSP was acti-
vated during receptor stimulation in the cell-attached
patch mode. Robust depolarization led to a brief, but
almost complete, inhibition of the TRPC6 channel dur-
ing the bursting activity (Fig. S2). This observation en-
abled us to use a standard ligand-binding isotherm for
PI(4,5)P2–TRPC3/6/7 channel binding. The calcula-
tion of PI(4,5)P2 concentration from the reduction in
FR was done by a boundary function (described in
Materials and methods). Because the FRET between
CFPmse-PHd and YFPmse-PHd proteins is cancelled by
detachment of either the donor or acceptor uoro-
phore-fused PHd proteins from the membrane, FR
reduction could be approximated as a cooperative
square law of the membrane-bound fraction of PHd
(Eq. 3) as follows:
FR
FR
K
PI P
max
dPHd
≈ = +
( )
( )
F2
2
2
1 1 4 5
/[ , ] , (4)
where Kd(PHd) is the dissociation constant of PI(4,5)P2
binding to the PHd. The left side of Eq. 4 is always posi-
tive, and solving PI(4,5)P2 yields:
used to reduce intrinsic PI(4,5)P2 (Okamura et al.,
2009). Our previous report demonstrated that reduc-
tion of PI(4,5)P2 by activation of DrVSP led to concomi-
tant inhibition of TRPC3/6/7 currents (Imai et al., 2012).
We simultaneously measured the voltage-dependent
stepwise controls of FRET between CFPmse-PHd and
YFPmse-PHd, and the DrVSP-mediated inhibition. To evoke
the currents, the DAG lipase inhibitor, RHC80267, was
used. This compound is suitable to produce stabilized
inward currents, mainly caused by elevating the resting
level of DAG (Albert et al., 2005). The step-pulse proto-
col, from 20 to 180 mV with a duration of 500 ms, en-
ables the current inhibition and FRET reduction to be
observed simultaneously (Fig. 4 A).
By plotting the current inhibition “r(I)” and FRET re-
duction “r(FR)” against the depolarizing pulses that ac-
tivate DrVSP, the various sensitivities of the TRPC3,
TRPC6, and TRPC7 channels were quantied (Fig. 4 B).
The inhibition of the TRPC3 current (Fig. 4 B, left, circles)
was relatively insensitive, compared with the reduction in
FRET (triangles). In contrast, the inhibition of TRPC7
was highly sensitive to the reduction in FRET (Fig. 4 B,
right). TRPC6 exhibited a similar level of gradual changes
in r(I) and r(FR) (Fig. 4 B, middle). This result conrms
our previous observations showing differential sensitivi-
ties of TRPC3/6/7 channels to PI(4,5)P2 reduction,
with an order of TRPC7 > C6 > C3 (Imai et al., 2012).
However, the inhibition of TRPC3 and TRPC6 currents
was not complete, and the FRET reduction by DrVSP
Figure 4. TRPC current inhibition and PI(4,5)P2
reduction in response to the protocol for measur-
ing the voltage dependence of DrVSP activation.
(A) TRPC6, CFPmse-PHd, YFPmse-PHd (PI(4,5)P2
sensor), and voltage-sensing phosphatase (DrVSP)
were coexpressed in HEK293 cells. Gradual current
inhibition and reduction in PI(4,5)P2 caused by the
step-pulse protocol (left; from 20 to 180 mV; dura-
tion of 500 ms; repeated every 25 s). A DAG lipase
inhibitor (RHC80267; 100 µM) was applied to in-
duce the currents (gray horizontal bar). The ratio
of current inhibition, r(I), and FRET reduction,
r(FR), upon the depolarization pulses was used to
quantify the channel activity and PI(4,5)P2 changes
after DrVSP activation (right). (B) The voltage de-
pendence of current inhibition (left axis; circles)
and FR reduction (right axis; triangles) after DrVSP
activation in cells expressing TRPC3 (left), TRPC6
(middle), and TRPC7 (right) channels.
Itsuki et al. 191
where FRmax is the maximum FR value at an innite con-
centration of PI(4,5)P2. According to the resting FR of
PIP5K-overexpressing cells, that value was estimated as
1.2-fold higher than the resting FR of the control cells
(Fig. 5 A). After solving Eq. 5, r(I) versus the estimated
PI(4,5)P2 concentration (Est, PI(4,5)P2) plots were tted
using the Hill equation (Hill, 1910) (Fig. 5 B). Assum-
ing a dissociation constant of PI(4,5)P2 PHd = 2 µM
(Hirose et al., 1999), the functional dissociation con-
stants of PI(4,5)P2 binding to TRPC3, TRPC6, and TRPC7
channels were estimated at 1, 2, and 5 µM, respectively.
These factors were incorporated into K3 as the initial
parameter for simulation in channel regulation by
PI(4,5)P2 (see below).
Modeling TRPC channel activity coupled to
PI(4,5)P2–DAG signaling
Having determined the functional constants of PI(4,5)P2
binding to TRPC channels, we attempted to simulate
channel activity and compare the results with the experi-
mental data. Our model consists of three components.
Part 1 covers the minimal PI(4,5)P2-to-DAG reaction,
including the process of PI(4,5)P2 recovery (Table 1,
Eqs. 6–17). Part 2 is the calculation of the open proba-
bility (Po) and the resultant current based on the dy-
namics of DAG and PI(4,5)P2 concentrations (Table 2,
Eqs. 18–20). Part 3 is a back-calculation of normalized
FR of PI(4,5)P2 sensor according to the concentrations
of PI(4,5)P2 and IP3 (Table 3; Eqs. 21–26). Details of the
respective components are described in full below.
(Part 1) Scheme of the minimum essential PI(4,5)P2–DAG
reaction. The goals of this simulation were to reproduce
Est K FR
FR
FR
FR
dPHd
max max
, , ,PI P4 5 1
2
( )
= − −
⋅
( )
(5)
Figure 5. Functional dissociation constants of PI(4,5)P2 bind-
ing to TRPC3/6/7 channels. (A) Comparison of FR or EEFF in the
resting condition between cells expressing TRPC6 channel and
CFPmse-PHd and YFPmse-PHd (control), and those overexpressing
PIP5K (+PIP5K). The FR of cells overexpressing PIP5K increased
on average by 1.2-fold compared with control cells. *, P < 0.05;
unpaired t test. (B) Steady-state plots for estimating the functional
Kd of PI(4,5)P2 binding to TRPC3/6/7 channels. Horizontal axis
indicates the estimated PI(4,5)P2 concentration based on the con-
version from FR to PI(4,5)P2, according to Eq. 5.
Figure 6. PI(4,5)P2 reaction model and
channel gating models (DG and SPD) used
for simulations. (A) Minimal PI(4,5)P2–DAG
reaction scheme (top). Hydrolysis of PI(4,5)P2
(local) is the rst step in this model (ki). The
produced DAG can serve as a substrate for
DAG kinase, DAG lipase, and DAG acety l-
transferase. For simplicity, we refer only to
DAG kinase (kii). PA, phosphatidic acid. Fur-
ther catalytic steps for the producing of CDP-
DAG and PI were bound to directly generate
PI(4)P (kiii). The PI(4,5)P2 recovery from
PI(4)P by PIP5K is referred to as “kiv”. PI(4,5)P2
and DAG concentrations are linked to the
three-state DG (bottom left) and the four-
state SPD (bottom right) models. (B) Com-
parison of TRPC6 current data processed
through the DG and SPD simulation models
with parameters of the accelerated PLC kinet-
ics. The top panel shows that a rapid decay of
TRPC6 current was seen with the SPD model
(red trace), but not with the DG model (or-
ange trace). The current amplitudes were
normalized to their peak currents (Norm.cur-
rent). The bottom panel shows the simulated
dynamics of PI(4,5)P2 (solid line) and DAG
(dashed line) concentrations.
192 PIP2 dynamics in receptor-operated TRPC6/7 currents
products of CDP-DAG and phosphatidylinositol (Fig. 6 A,
kiii, and Table 1, Eq. 12). For recovery of PI(4,5)P2, we
considered two pathways: (1) resynthesis of PI(4,5)P2
from PI(4)P by PIP5K (Fig. 6 A, kiv, and Table 1, Eq. 13)
and (2) diffusion of PI(4,5)P2 from global to local chan-
nel area (Eqs. 14 and 15). The idea and necessity of the
diffusion pathway are described in the Discussion and
the legend of Fig. S4. The amount of scavenged PI(4,5)P2
arising from the binding of PHd proteins was solved by
Eq. 16 (Table 1). The total available amount of PI(4,5)P2
to transfer to Part 2 (channel gating) was calculated by
Eq. 17 (Table 1).
(Part 2) Channel gating. Two channel gating models were
constructed. The rst, a simple channel gating model,
only consists of DAG binding and unbinding for channel
opening and closing, and it lacks the inhibitory regulation
the experimental data and elucidate the functional role
of the reduction in PI(4,5)P2 caused by agonist-induced
receptor-operated currents. Thus, we focused on the ac-
tivation of PLC by Gq protein–coupled receptors as fol-
lows. The initial step is hydrolysis of PI(4,5)P2 by PLC,
which generates DAG and IP3 (Fig. 6 A, ki, and Table 1,
Eqs. 6–8). Activation of PLC starts at the rst time step
(0.05 s) after time zero. In the factor of PLC activity, we
also included the adjusted factors of receptor desensiti-
zation (Table 1, Eq. 9) and the time-dependent accelera-
tion of PLC activity by receptor stimulation (Eq. 10). The
latter factor has been demonstrated as Ca2+-dependent
positive feedback in PLC activity (Horowitz et al., 2005).
The next step is DAG phosphorylation by DAG kinase to
generate phosphatidic acid (PA) (Fig. 6 A, kii, and Table 1,
Eq. 11). The third step is production of PI(4)P, a pre-
cursor of PI(4,5)P2, from PA, skipping the intermediate
TABLE 1
Equations for minimal PI(4,5)P2–DAG reaction scheme
Equation No.
d PI 4 5 P d PI 4 5 P d PI 4 5 P
2
local
2
local
2
loca
, , ,
( )
=
( )
−
( )
ll
i t f t
De dt· · ·
( ) ( )
k6
d DAG d DAG d PI 4 5 P De dt d DAG
2
tot
i t f(t)
[ ]
=
[ ]
+
( )
⋅ ⋅ ⋅ −
[ ]
⋅,( )
k kkii dt⋅7
d IP d IP d PI 4 5 P De dt d IP
3 3 2
tot
i t f(t) 3
[ ]
=
[ ]
+
( )
⋅ ⋅ ⋅ −
[ ]
⋅,( )
k kkv dt⋅8
De 1 Rd f exp t Sd f exp t
f t rd sd( ) _ / _ /= − ⋅ −
( )
( )
+ ⋅ −
( )
τ τ
Rd f fraction of rapid desensitization
_ :
Sd f fraction of slow desensitization_ :
time constant for the rapid
rd
τ: ddesensitizatio
n
time constant
sd
τ: for the slow desensitization
9
k k
i t i
pg t
B PLC
B PLC basal PLC activi
( )
•
_
_ :
= ⋅
tty
pg power parameter of G PCR rec
q
: eeptor stimulatio
n
10
d PA d PA d DAG dt d PA dt
ii iii
[ ]
=
[ ]
+
[ ]
−
[ ]
· · · ·k k
11
d PI 4 P d PI 4 P d PA dt d PI 4 P
iii iv
( )
=
( )
+
[ ]
−
( )
· · ·k k ·· dt 12
d PI 4 5 P d PI 4 P dt d PI 4 5 P
2
recovered
iv 2
, ,
( )
=
( )
⋅ ⋅ −
( )
k
recovered
i t f t
De dt· · ·
( ) ( )
k13
d PI 4 5 P d PI 4 5 P d PI 4 5 P
2global 2global 2gl
, , ,
( )
=
( )
−
( )
oobal i g t f t
i g
De dt
ra
· · ·
:
_ ( ) ( )
_
k
ktte constant for global PLC activity
14
d PI 4 5 P d PI 4 5 P d PI 4 5 P
2diffused in 2global 2
, , ,
_
( )
=
( )
−
( )
()
−
local
1 errf· sspot sprt 4 dcoef dt d PI 4 5 P2diffused in
/ · · , ·
_
( )
( )
( )
−
( )
De dt
errf erro
i t f t
k( ) ( )
· ·
: rr function
dcoef diffusion c: ooefficient of PI 4 5 P
spot
2
,
( )
:: distance between local and global domain
15
d PI 4 5 P d PHd d PHd 1 1
2scavenged PHd r el IP3
, ( /(
_
( )
=
[ ]
−
[ ]
( )
⋅ + KKd PHd 2 local
d PI 4 5 P
,/( ,
( )
d PI 4 5 P d PI 4 5 P
2recovered 2dif
+
( )
+
( )
, , ffused in_ ))) 16
d PI 4 5 P d PI 4 5 P d PI 4 5 P
2tot 2local 2recove
, , ,
( )
=
( )
+
( )
rred 2diffused in 2scavenged PHd
d PI 4 5 P d PI 4 5 P+
( )
−
( )
, ,
_ _
17
Itsuki et al. 193
diffusible in the cytoplasm and binds to PHd proteins
with higher afnity (0.1 µM) than PI(4,5)P2 (Hirose et al.,
1999). The factor of FRET reduction caused by the bind-
ing of IP3 to PI(4,5)P2 sensor was incorporated into the
back-calculation of FR dynamics (Table 3, Eqs. 21–24).
Although it was difcult to calculate the absolute ef-
ciency of FRET, because of various expression levels of
uorophore-fused PHd, we could estimate the normal-
ized FR (Norm.FR) changes by the resting level of FR. The
alternations in Norm.FR, caused by CFPmse-fused TRPC7
channel versus YFPmse-PHd and CFPmse-PHd versus YFPmse-
PHd, were solved by Eqs. 25 and 26 (Table 3), respectively.
Insufficient matching of the DG model
First, we examined whether a model without PI(4,5)P2
regulation (DG model) was able to reproduce the exper-
imental data of the simultaneous measurements. Fitting
to the TRPC6 currents was accomplished by minimizing
the sum of the squared errors with 19 free parameters,
by PI(4,5)P2 reduction (termed the “DG model” in Fig. 6 A,
bottom left). The second is a realistic model that takes
into account the inhibitory effect caused by a reduction
of PI(4,5)P2 that directly induces transition of the chan-
nel to an inactive state from any closed or open state.
This model is referred to as the “SPD model” (Fig. 6 A,
bottom right). Open probabilities (Po) expressed as a
function of agonist concentration, calculated according
to the DG and SPD models, are described in Eqs. 18 and
19, respectively (Table 2). Finally, whole-cell currents
were calculated using Eq. 20.
(Part 3) Back-calculation of FR from the simulated PI(4,5)P2
concentration. As described in the previous section, the
observed FR changes were converted to PI(4,5)P2 con-
centrations, according to Eq. 5. Here, we attempted to
do the opposite, using these equations to explore FRET
dynamics. Receptor stimulation produces DAG and IP3
after PLC-mediated hydrolysis of PI(4,5)P2. IP3 is highly
TABLE 2
Equations for channel gating
Comment to the equation Equation No.
Open probability in DG model: P K K K
o 1 2
2
2
1
d DAG d DAG 1=
[ ]
+
[ ]
+
( )
−
· / / 18
Open probability in SPD model:
P
K K K K K K
o
1 2
2
2 1 2
2
3 2
d DAG d DAG d DAG d[PI 4 5 P
=
[ ]
+
[ ]
+
[ ]
( )
· / / ( · / )·( / , ]] )
· / , / / ,
tot
2 3 2 tot 3 2
d PI 4 5 P d DAG d PI 4 5 P+
( )
( )
[ ]
+
( )
K K K
ttot
1
1 2
1
and are dissociation constants for DA
( )
+
−
K K GG to TRPC channel
is dissociation constant for PI 4 5 P
3
.
,K
( )
22to TRPC channel.
19
TRPC6 / C7 currents: I N g P V V
N
g
h rev
= −
( )
· · ·
:
:
o
number of channels
single cchannel conductance
holding potential 5 mV
V
V
h:−
( )
0
rrev : reversal potential mV0
( )
20
TABLE 3
Equations for the back-calculated FR
Comment to the equation Equation No.
Fraction of IP3 bound PHd (PHd): d PHd 1 d PHd PHd
IP3b IP3 tot
( )
= −
[ ] [ ]
/21
Concentration of
membrane bound PHd:
d PHd PHd d PHd
rem tot rel
[ ]
=
[ ]
−
[ ]
22
Concentration of cytosolic PHd
upon PI(4,5)P2 reduction: d PHd 1 1 d PI 4 5 P 1 PHd
rel d PHd 2 tot tot
[ ]
= −
( )
+
( )
( )
⋅
[ ]
/ / ,
,
K23
Concentration of IP3 bound PHd: d PHd 1 d IP 1 d PHd
IP3 d IP3 3 rel
[ ]
=
[ ]
+
( )
( )
⋅
[ ]
/ /
,
K24
Norm.FR between TRPC7-CFPmse and
YFPmse-PHd: Norm d PHd 1 1 d PI 4 5 P 1 1
IP3b d PHd 2 tot d
. / / , /
,
FR K K=
( )
⋅ +
( )
( )
( )
⋅ + ,, ( )
/ ,
PHd 2 tot
1
PI 4 5 P
( )
( )
−
0
PI 4 5 P PI 4 5 P concen
2
tot
2
, : ,
( )
( )
( )
0
ttration at time zero
25
Norm.FR between CFPmse-PHd
and YFPmse-PHd: Norm d PHd 1 1 d PI 4 5 P 1 1
IP3b d PHd 2 tot
2
. / / , /
,
FR K K=
( )
⋅ +
( )
( )
( )
⋅ + dd PHd 2 tot
21
PI 4 5 P
,( )
/ ,
( )
( )
()
−
0
26
194 PIP2 dynamics in receptor-operated TRPC6/7 currents
the delayed receptor-operated currents, but it is not to-
tally suitable for the rapid case. As we demonstrated,
when the parameter for PLC activity (ki) was set to an
accelerated kinetics (ki = 0.7), a marked TRPC6 current
inactivation emerged only in the SPD model but not in
the DG model (Fig. 6 B, top).
Fitting the SPD model to the experimental data
The SPD model was then tested. The same basic tting
strategy as in the DG model was used. After tting the
SPD model to the experimental receptor-operated cur-
rent data, the computed PI(4,5)P2 data were compared
with the experimental FR dynamics. In this case, in ad-
dition to the 19 parameters, the dissociation constant of
PI(4,5)P2 binding was incorporated at K3 (Table 4, row
23). The tting of the SPD model to the whole-cell
TRPC6 currents did indeed show overlapping of the
dynamics of the experimental Norm.FR and the back-
calculated Norm.FR, with a smaller SD value than in the
the initial values of which are listed in Table 4. The -
delity of the model was assessed by a similarity of FR
between the experimentally measured FR and simu-
lated FR, which was obtained from the back-calculation
of the resultant PI(4,5)P2 concentrations by tting to
the currents, according to the equations described in
the section on model Part 3 and Table 3.
When the currents were induced by the weak recep-
tor stimulation through endogenous muscarinic recep-
tors, the experimental FR was substantially compatible
with the calculated FR from the simulated PI(4,5)P2
changes (Fig. S4 A; SD = 0.12). In contrast, tting the
currents in M1R-overexpressing cells to the DG model
highly deviated from the experimental FR changes
(Fig. S4 B; SD = 0.63). Furthermore, the DAG produc-
tion was quite more transient than that observed in the
PKC-based FRET dynamics (Fig. S4 B, bottom panel,
dashed line). Therefore our tting examination indi-
cates that the DG model may be useful for mimicking
TABLE 4
Parameters and initial conditions for the tting
Row Parameters (unit) Setting Initial value of TRPC6 endo/C6 + M1R/
A7r5/C7 + M1R
Source or comments
1 PI(4,5)P2 (µM) at resting Free 20/20/20/20 Bunce et al., 1993; McLaughlin and Murray,
2005
2ki_PLC (s1) Free 0.04/1/1/1 Rational to PI(4,5)P2 reduction with our data
3kii_DAG kinase (s1) Free 0.03/0.03/0.08/0.03 Rational to DAG changes with our data
4kiii_PA to PIP reactions (s1) Free 0.01/0.01/0.01/0.01 Appropriate for PI(4,5)P2 synthesis
5kiv_PIP5K (s1) Free 0.1/0.1/0.25/0.1 Appropriate for PI(4,5)P2 resynthesis
6kv_IP3 phosphatase (s1) Free 0.5/0.5/0.5/0.5 Appropriate for IP3 hydrolysis
7rd (s) Free 5/2/1/4 Appropriate for Receptor desensitization
8sd (s) Free 50/10/5/10 Same as above
9 Rd_f (no unit) Free 0.5/0.5/0.5/0.5 Same as above
10 Sd_f (no unit) Free 0.5/0.5/0.5/0.5 Same as above
11 spot (distance global to local; µm) Free 4/4/3/4 5 times more than the diffusion coefcient of
PI(4,5)P2
12 dcoef of PI(4,5)P2 (µm2/s) Free 0.8/0.8/0.8/0.8 Golebiewska et al., 2008
13 Ratio of local ki / global kiFree 1/4/2/5 Approximate from the uneven FRET reduction
(Fig. S3)
14 PI4P (µM) Fixed 10/10/10/10 Brown et al., 2008
15 Activation delay (no unit) Free 0.01/0.001/0.001/0.001 Appropriate for receptor activation
16 Activation power (no unit) Free 0.5/0.3/0.7/0.3 Same as above
17 Vrev (mV) Fixed 0/0/0/0
18 Vhold (mV) Fixed 50/50/50/50
19 No. of channels Free 100–7,000 Appropriate for the current density
20 Channel conductance (pS) Fixed 35/35/35/70 Hofmann et al., 1999; Lemonnier et al., 2008
21 K1 (Kd for DAG1; µM) Free 60/60/35/10 Effective OAG concentrations are 10 to 100 µM
in Hofmann et al., 1999; Okada et al., 1999; Imai
et al., 2012
22 K2 (Kd for DAG2; µM) Free 30/30/10/10 Same as above
23 K3 (Kd for PI(4,5)P2; µM) Free 2/2/5/5 This paper, Fig. 4 D
24 Expressed PHd (µM) Fixed 1.6/1.6/1.6/1.6 This paper, Materials and methods
25 Kd PI(4,5)P2 of PHd (µM) Fixed 2.0/2.0/2.0/2.0 Hirose et al., 1999
26 Kd IP3 of PHd (µM) Fixed 0.1/0.1/0.1/0.1 Hirose et al., 1999
Itsuki et al. 195
Such rapid FRET recovery is expected with the rapid
desensitization of vasopressin receptors, and was repro-
duced by coexpressing vasopressin type 1A receptors
with TRPC6 or TRPC7 channel in HEK293 cells (Fig. S5).
When tting these AVP-evoked TRPC6/7-like currents,
the simulation parameters for rapid desensitization
(Table 4, rows 7 and 8) also overlapped the Norm.FR
dynamics (Fig. 7 C, middle, blue line; SD = 0.12). These
results indicate that the SPD model is useful even in the
context of physiological cells.
Contrary to these positive results, the back-calculated
FRET demonstrated less similarity to the experimental
FRET achieved by tting to TRPC7 currents observed in
HEK293 cells and vice versa (Fig. S6; SD = 0.18; n = 4).
In addition, TRPC7 currents often demonstrated a pla-
teau or biphasic response when M1R was overexpressed,
but the FRET did not clearly show such irregular dynam-
ics. We reasoned that the FRET detection by uorophore-
fused PHd was too slow to respond to the PI(4,5)P2
dynamics compared with the time course of TRPC7 cur-
rent, recorded by the electrophysiological method. To
improve this issue, we redesigned the FRET pairs to de-
tect PI(4,5)P2 changes in the local vicinity of the TRPC7
DG model (SD = 0.04; n = 4). Furthermore, similar over-
lapping was clearly evident in TRPC6 currents in M1R-
overexpressing cells (SD = 0.05; n = 4; Fig. 7 B, middle).
The tting expressed a prolonged existence of DAG,
persisting well beyond the current decay (Fig. 7 B, bot-
tom, solid dashed line). Such prolonged existence of
DAG was seen experimentally after robust receptor
stimulation (Fig. 3 B) and in recent work by Falken-
burger et al. (2013). The correlation between the ex-
perimental and modeled data supports the description
of TRPC6 currents and PI(4,5)P2 dynamics in the SPD
model (tted parameters are summarized in Table 5).
The SPD model was also examined for TRPC6/7-like
currents in aortic smooth muscle–derived A7r5 cells
(Brueggemann et al., 2006; Maruyama et al., 2006). The
PI(4,5)P2 sensor proteins (CFPmse-PHd and YFPmse-PHd)
were coexpressed in A7r5 cells without exogenous ex-
pression of channels or receptors (Fig. 7 C, inset). By
applying AVP, TRPC6/7-like currents and FRET reduc-
tion were observed similarly to the observations after
stimulation of HEK293 cells with CCh (Fig. 7 C, top and
middle). A noticeable difference was that PI(4,5)P2 re-
covered quickly (5–20 s after the application of AVP).
Figure 7. SPD model tting to experimentally observed TRPC6/7 currents and AVP-evoked TRPC6/7-like currents in A7r5 cells.
(A) Fitting of the receptor-operated TRPC6 current simultaneously measured with PI(4,5)P2 by FRET with the SPD model (top; light
blue trace). The experimental data were obtained from the low strength of receptor stimulation carried by CCh application (100 µM)
to endogenous muscarinic receptor in HEK293 cells. Back-calculated FR of PI(4,5)P2 concentrations at the respective points (bottom;
solid) was normalized against the time zero FR (Norm.FR; middle; light blue), and that was overlaid on the experimental Norm.FR
(middle; circles). The bottom panel displays the tting resultant changes in PI(4,5)P2 (solid), DAG (dashed), and PA (thin dashed).
The similarity of Norm.FR was assessed by SD as described in Materials and methods. (B) Fitting of experimental TRPC6 current data
from M1R-overexpressing cells. The initial parameters are identical to those in A. (C) Fitting of experimental TRPC6/7-like currents
recorded from A7r5 aorta–derived smooth muscle cells. The currents were evoked by 1 µM AVP. The inset in the top panel shows A7r5
cells expressing the PI(4,5)P2 sensor. (D) Fitting of TRPC7 currents. Local PI(4,5)P2 dynamics were detected using a FRET donor linked
to the TRPC7 channel at the end of its C-terminal domain (top; inset). Transient increments in FRET were detected during the plateau
or biphasic response (middle; arrow).
196 PIP2 dynamics in receptor-operated TRPC6/7 currents
uorescence emission (3-cube method) and electro-
physiological measurements on patch-clamped cells.
We accomplished this by using CFPmse- or YFPmse-fused
PHd proteins as a FRET sensor of PI(4,5)P2, or CFPmse-
fused PKC and YFPmse-fused myristoyl membrane–
attached peptides to monitor DAG. CCh or AVP was
used to induce TRPC6/7 currents, which were mea-
sured to monitor the synchronicity of receptor-operated
currents and changes in either PI(4,5)P2 or DAG levels.
The experimental results demonstrated a correlation
between the kinetics of PI(4,5)P2 reduction and the acti-
vation or the inactivation of receptor-operated TRPC6/7
currents. Fitting the experimental data to the DG or
SPD model revealed that the idea of self-limiting regula-
tion by PI(4,5)P2 and DAG is critical for reproducing
receptor-operated TRPC6/7 currents.
Reproducing the currents and FRET reduction
in the SPD model
Our results show that reduction in PI(4,5)P2 by PLC-
mediated hydrolysis appears to be fundamental to the
inactivation of TRPC6/7 channels. This contribution is
even more apparent after robust receptor stimulation,
either by high concentrations of receptor agonist or by
channels. The donor uorophore (CFPmse) directly
fused to the channel was coexpressed with YFPmse-PHd
(Fig. 7 D, inset). Intriguingly, this FRET pair showed
a transient increment of FRET during the plateau or
biphasic current (Fig. 7 D, red arrow). By tting the
TRPC7 currents with the corresponding “down-up-
down” PI(4,5)P2 dynamics (Fig. 7 D, bottom), we achieved
an improved matching of Norm.FR (SD = 0.14) as well.
This transient up-regulation of PI(4,5)P2 reects a
quick replenishment by diffusible PI(4,5)P2 in the SPD
model (Fig. S3). These results show that corresponding
Norm.FR responses in various cell and channel settings
strongly support the delity of the SPD model for re-
producing simultaneous events of receptor-operated
TRPC currents and FR changes.
DISCUSSION
Because PI(4,5)P2 hydrolysis by PLC is the critical event
in receptor-operated TRPC3/6/7 currents, the parallel
observation of PI(4,5)P2 or DAG with channel activity is
fundamental to advancing our understanding of chan-
nel regulation. To address this point, we performed
concerted quantitative FRET measurements of sensitized
TABLE 5
Resultant parameters by tting to TRPC currents with SPD model
Cell HEK293 HEK293 A7r5 HEK293
Transfected plasmids TRPC6/CFPmse-PHd/
YFPmse-PHd
TRPC6/M1R/CFPmse-
PHd/YFPmse-PHd
CFPmse-PHd/YFPmse-
PHd
TRPC7-CFPmse/M1R/
YFPmse-PHd
Receptor agonist (conc. µM) CCh (100) CCh (100) Arg-Vasopressin (1) CCh (100)
The number of tted cells 4 4 4 4
Row Parameters (unit)
1 PI(4,5)P2 (µM) at resting 22.5 ± 0.4 23.0 ± 0.4 22.2 ± 0. 2 14.6 ± 0.1
2ki_PLC (s1) 0.05 ± 0.02 1.2 ± 0.20 1.3 ± 0.42 0.93 ± 0.09
3kii_DAG kinase (s1) 0.04 ± 0.01 0.03 ± 0.02 0.07 ± 0.03 0.02 ± 0.01
4kiii_PA to PIP reactions, (s1) 0.008 ± 0.002 0.006 ± 0.002 0.004 ± 0.001 0.006 ± 0.0001
5kiv_PIP5K (s1) 0.050 ± 0.004 0.07 ± 0.04 0.17 ± 0.083 0.04 ± 0.03
6kv_IP3 phosphatase (s1) 0.7 ± 0.4 0.2 ± 0.1 0.88 ± 0.093 0.84 ± 0.14
7rd (s) 12.0 ± 3.8 1.4 ± 0.6 1.4 ± 0.2 4.4 ± 0.4
8sd (s) 24 ± 11 17 ± 5.2 6 ± 2.4 38 ± 15
9 Rd_f (no unit) 0.65 ± 0.08 0.55 ± 0.03 0.60 ± 0.12 0.46 ± 0.05
10 Sd_f (no unit) 0.35 ± 0.05 0.44 ± 0.02 0.43 ± 0.124 0.53 ± 0.06
11 spot (distance global to local; µm) 0.64 ± 0.09 2.78 ± 0.79 2.83 ± 0.98 1.90 ± 0.29
12 dcoef of PI(4,5)P2 (µm2/s) 0.36 ± 0.08 0.8 ± 0.47 0.5 ± 0.11 0.29 ± 0.06
13 Ratio of local ki / global ki1.1 ± 0.28 4.0 ± 1.6 2.8 ± 0.98 6.0 ± 1.3
14 Activation delay factor 0.03 ± 0.024 0.01 ± 0.002 0.003 ± 0.002 0.03 ± 0.025
15 Activation power factor 0.6 ± 0.33 0.32 ± 0.07 0.7 ± 0.17 0.3 ± 0.12
16 The number of channels 1,644 ± 277 5,808 ± 959 317 ± 121 2,384 ± 1,896
17 K1 (kd for DAG1; µM) 39 ± 6.3 26 ± 2.4 23 ± 7.6 7.0 ± 0.3
18 K2 (kd for DAG2; µM) 29 ± 5.1 22 ± 2.0 8 ± 3.4 6.3 ± 0.25
19 K3 (kd for PI(4,5)P2; µM) 1.9 ± 0.6 3.0 ± 0.66 9 ± 1.5 9.7 ± 0.14
SD 0.04 0.05 0.12 0.14
Sr0.01a82.7% 80.9% 70.1% 56.2%
Parameters are presented as mean ± SEM. (Typical results are displayed in Fig. 7.)
aPercentage of the squared residuals lower than the value of 0.01.
Itsuki et al. 197
experimental data. Furthermore, the SPD model–based
relationship in 90–50% TRPC7 currents to FR resulted
in a slight increase in the steepness to TRPC6 (Fig. 8 B).
This result is similar to the TRPC7 current to FR rela-
tionship observed experimentally (Fig. 8 B, dotted
line). In contrast, the SPD model–based TRPC6 relation-
ship resulted in an even steeper slope compared with
the experimentally observed relationship (Fig. 8 B, solid
line). The difference implies further unknown charac-
teristics that underlie the regulation by PI(4,5)P2 and
DAG in receptor-operated TRPC6/7 currents. Recently,
several reports have addressed an alternation of PI(4,5)P2
regulation by intracellular Ca2+ in KCNQ2/3 (Kosenko
et al., 2012; Falkenburger et al., 2013) and TRPV6 chan-
nels (Cao et al., 2013). According to Kosenko et al.
(2012), calmodulin binding to KCNQ2 channels stabi-
lized PI(4,5)P2 channel binding. The involvement of
Ca2+ regulation to PI(4,5)P2 or DAG regulations requires
further investigation. Nevertheless, because of the con-
sistency of the SPD model simulation with the experi-
mental results, we concluded that the inhibitory effect
of reductions in PI(4,5)P2 would accelerate the channel
inactivation with the increased receptor stimulation.
Proposed function of the self-limiting regulation
in receptor-operated TRPC6/7 currents
Based on the model simulation, we explored several
PLC-dependent features of the self-limiting regulation
by PI(4,5)P2 and DAG. The rst characteristic is the
maximum Po (Pomax). As shown in the left plot in Fig. 8 C,
in both models, Pomax values gradually increased as PLC
overexpression of functional receptors. Here, we simu-
late in a virtual setting the inhibitory role played by a
reduction in PI(4,5)P2 levels in the SPD model in the
context of different levels of PLC activity. In the aver-
aged tted data for TRPC6 currents, PLC activity (ki)
was gradually decreased or increased, as shown in Fig. 8 A.
The simulated TRPC6 currents and FRET by the PHd
clearly demonstrated PLC-dependent dynamics in re-
gard to the amplitudes or kinetics in the currents (Fig.
8 A, top) and the kinetics or the extent of FRET reduc-
tion (bottom). We then depicted log–log plots of the
time course of current growth (10–90%) or decay
(90–50%) versus FR (Fig. 8 B, red circles). For com-
parison, we also simulated this plot with the DG model
(Fig. 8 B, black circles) as well as TRPC7 channels. The
log–log plots of 10–90% to FR showed a linear rela-
tionship in both SPD and DG models of TRPC6/7 cur-
rents. These plots closely resemble the experimental
data, which are shown as gray lines in Fig. 8 B (solid and
dashed line for TRPC6 and TRPC7 channels, respec-
tively). In contrast, the linear relationship between cur-
rent decay (90–50%) and FR was reproduced only
when modeled with the SPD model (Fig. 8 B, right, red
symbols), but not with the DG model.
The simulation result indicates that when the strength
of receptor stimulation PI(4,5)P2 is low or the reduction
of PI(4,5)P2 is delayed (FR, >50 s), the DG model may
be useful to reproduce such delayed receptor-operated
TRPC currents. However, the situation alters as receptor
stimulation is strengthened or the reduction of PI(4,5)P2
is accelerated, and the DG model deviates from the
Figure 8. Proposed contribution of PI(4,5)P2
reduction in receptor-operated TRPC6/7 cur-
rents. (A) Representative SPD model simula-
tions for receptor-operated TRPC6 currents
(top) and FRET by PI(4,5)P2 sensor (bottom)
from various PLC activities (ki). ki varied from
0.03 to 1.0 (s1). The same color curves dis-
played in each panel were calculated from
the same ki value. (B) The various kinetics of
PI(4,5)P2 reduction (FR) according to the
changes in PLC activity (ki) impact activation
(left; 10–90%) and inactivation (right; 90–
50%) of TRPC6/7 channel currents. Shown are
the prediction made by the DG model (black)
and the SPD model (red) for TRPC6 (circles)
and TRPC7 channels (triangles). The star in
the panels indicates the result from the ki set-
ting of 0.7. The solid and dashed lines indicate
the current–FR relationships experimentally
observed in TRPC6 and TRPC7, respectively.
The direction of the arrows and its size indicate
the gap from the DG to SPD model. (C) Simu-
lation results of PLC-dependent characteristics
of TRPC6/7 channels by DG (black) and SPD
(red) models. (Left) The relationship between
maximum open probability (Pomax) and ki.
(Middle) The time required to peak currents
(s) and ki. (Right) The total ionic inux and ki.
t = 30 s.
198 PIP2 dynamics in receptor-operated TRPC6/7 currents
Furthermore, PIP5K substantially contributes to the quick
recovery of PI(4,5)P2 after its depletion by DrVSP activa-
tion (Falkenburger et al., 2010; Imai et al., 2012). How-
ever, in case of receptor-operated TRPC6/7 currents,
we observed that a high concentration of ATP or its in-
active analogue AMP-PNP in the patch-pipette solution
had little effect on TRPC6/7 currents (Fig. S7), even in
the plateau or biphasic phase. We speculate that an ad-
ditional PI(4,5)P2 replenishment pathway, such as the
detachment of PI(4,5)P2 from proteins, dispersion
from its clustered complex (van den Bogaart et al.,
2011), translocation of phosphoinositides from the PIS
organelle (Kim et al., 2011), or an unknown mechanism
(Hammond et al., 2012), may be involved.
However, here we focused on the lateral diffusion
of PI(4,5)P2. When we analyzed FRET in regions of
small compartments, a nonuniform reduction in FR
by PI(4,5) P2 sensor upon receptor stimulation was ob-
served (Fig. S3). This variability of a region-specic
PI(4,5)P2 reduction was consistent with an electron
microscopic study showing that PI(4,5)P2 located in
a membrane microdomain (known as caveolae) de-
creased slower than in an undifferentiated membrane
upon receptor stimulation (Fujita et al., 2009). We
have speculated that such nonuniformity of PI(4,5)P2
reduction creates the opportunity for PI(4,5)P2 diffu-
sion to occur. For that reason, we incorporated the
pathway of PI(4,5)P2 replenishment by lateral diffusion
into our model (Part 1). By incorporation of this path-
way, the plateau or biphasic currents after the peak cur-
rent can be enhanced as the gap of PI(4,5)P2 dynamics
between the local and global domain increases (Fig. S3).
In the case of TRP channels in ies, the plateau phase
of the TRP current in response to light adaptation has
been demonstrated to have a key role in phototransduc-
tion (Lo and Pak, 1981; Minke, 2010). Therefore, incor-
poration of replenishment of PI(4,5)P2 in our model
may provide an important contribution to understand-
ing the physiological consequences.
Molecular insight into PI(4,5)P2 and DAG binding
to TRPC channels
The tting result of the SPD model predicted the dis-
sociation constant of DAG binding to TRPC6 channel
to be 20 to 40 µM and to TRPC7 to be <10 µM (Table 5,
rows 17 and 18). This parameter has not been well char-
acterized, despite being critical for DAG-sensitive TRPC
channel activation. In the experiment, receptor-operated
TRPC7 currents demonstrated a higher sensitivity to DAG
production (Fig. 3 E) than TRPC6 currents, which was
consistent with the result predicted by the SPD model,
thus providing a fundamental parameter into the TRPC6/7
channel activation. However, it is still unclear exactly
where these lipids bind to the TRPC3/6/7 channels.
Among the TRP superfamily, heat- and capsaicin-
activated TRPV channels are relatively well characterized
activity increased in the range where the ki values were
small (ki of <0.1). However, Pomax in the SPD model was
sustained or slightly attenuated when the ki values were
>0.1. Kwon et al. (2007) showed that disruption of the
potential PI(4,5)P2-sensing residue of the TRPC6 chan-
nel nearly doubles the maximum current amplitude.
This is consistent with our simulation, where the Pomax at
ki = 0.7 s1 eventually increased by two- to threefold in
the DG model compared with that in the SPD model.
The consistency of our simulation and the previous re-
port strongly supports the functionality of the self-limiting
regulation in TRPC6/7 channels for protecting the ex-
cess cation inux.
The second characteristic we measured was the shift
in peak appearance. When we applied robust receptor
stimulation, the time to peak currents were drastically
shortened compared with the endogenous receptor
stimulation (see PI(4,5)P2 dynamics at different levels...
in Results). Essentially, the similar tendency was repro-
duced in both models (Fig. 8 C, middle). However, the
time to peak shortening was even more striking in the
SPD model (Fig. 8 C, red symbols) than in the DG
model (black symbols). Petersson et al. (2011) compu-
tationally demonstrated that TRPC channel activity in
the nervous system contributes to the temporal integra-
tion of the generation of a lower rate-ring pattern at
distal dendrites. Their simulation result suggests that
the rate of ring may be controlled by the peak time of
TRPC currents. Therefore, TRPC6/7 channels may play a
critical role in determining the neural ring rate, de-
pending on the strength of receptor stimulation.
The third characteristic is the total ionic inux rela-
tion (Fig. 8 C, right). The total ionic inux, which was
calculated by integration of the ow during the recep-
tor stimulation (t = 30 s), was predicted as the bell-shape
response to the PLC activity in the SPD model. There-
fore, the optimum PLC activity appeared neither weak
nor robust, but was in the middle (0.1 < ki < 0.3; Fig. 8 C,
right, red symbols). TRPC3/6/7 channels contribute to
vascular tone, inammation, cell remodeling, and intes-
tinal contraction through increased Na+ or Ca2+ levels
(Tsvilovskyy et al., 2009; Smedlund et al., 2010; Tauseef
et al., 2012; Weissmann et al., 2012; Koenig et al., 2013).
Hence, this bell-shaped relationship may be linked to
these multiple pathophysiological responses.
Collectively, channel inhibition by PI(4,5)P2 reduc-
tion in DAG-sensitive TRPC3/6/7 channels not only ac-
celerates the rate of current decay but also contributes
to suppression of the current amplitude, shortening of
the peak time, and the bell shape of the ionic inux,
leading to the multiple functionality of these channels.
PI(4,5)P2 replenishment mechanism by diffusion
It has been suggested that phosphatidylinositol 4-phos-
phate is critical for replenishing PI(4,5)P2 via its phos-
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and have at least two PI(4,5)P2-binding sites in the
C-terminal domains, proximal and distal regions to the
S6 domain (Prescott and Julius, 2003; Doerner et al.,
2011; Ufret-Vincenty, et al., 2011). In the case of the
TRPC family, earlier studies have also shown that the
distal region of the TRPC6 C-terminal domain contrib-
utes to the current decay (Kwon et al., 2007), whereas
PI(4,5)P2 binding to the PH-like domain in the N-terminal
region reduces TRPC3 current amplitudes (van Rossum
et al., 2005). These results indicate that PI(4,5)P2 bind-
ing may be supported by multiple channel domains/
regions in a complex manner. Furthermore, another
complex point regarding the PI(4,5)P2 regulation is
that various heteromeric TRPC3/6/7 channels can be
formed, including heteromers of TRPC3/6/7 (Goel
et al., 2002) and other subfamilies (Hofmann et al., 2002;
Takai et al., 2004; Ambudkar et al., 2006). Addressing
these points is essential to further extend our under-
standing on the self-limiting regulation by PI(4,5)P2 and
DAG signals observed in TRPC3/6/7 channels.
We thank Masato Hirata (Kyushu University), Michael Schaefer
(Leipzig University), and Moritoshi Sato (The university of
Tokyo) for their helpful advice on the detection of PI(4,5)P2
and DAG. We thank to Yasuo Mori and Nozomi Ogawa (Kyoto
University) for the critical reading of our manuscript. Miho
Sumiyoshi (Fukuoka University) assisted with the molecular bio-
logical preparations.
This work was supported by Grants-in-Aid for Young Scientists
from the Japan Society for the Promotion of Sciences and the
Naito Foundation (both to M.X. Mori).
The authors declare no competing nancial interests.
Author contributions: K. Itsuki and M.X. Mori conceived the
study. K. Itsuki, H. Hase, and M.X. Mori performed the experi-
ments and analyzed the data. Y. Okamura and R. Inoue provided
guidance and support throughout. K. Itsuki, Y. Imai, Y. Okamura,
R. Inoue, and M.X. Mori wrote the paper.
Sharona E. Gordon served as editor.
Submitted: 17 May 2013
Accepted: 8 January 2014
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