?1-Adrenoceptor stimulation potentiates L-type Ca2?
current through Ca2??calmodulin-dependent PK II
(CaMKII) activation in rat ventricular myocytes
Jin O-Uchi*†, Kimiaki Komukai‡, Yoichiro Kusakari*, Toru Obata§, Kenichi Hongo‡, Hiroyuki Sasaki§,
and Satoshi Kurihara*
*Department of Physiology (II),‡Division of Cardiology, and§Division of Molecular Cell Biology, The Jikei University School of Medicine, 3-25-8
Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan
Communicated by Clara Franzini-Armstrong, University of Pennsylvania School of Medicine, Philadelphia, PA, April 29, 2005 (received for review
November 22, 2004)
?1-Adrenoceptor stimulation (?1ARS) modulates cardiac muscle
contraction under physiological conditions by means of changes in
Ca2?current through L-type channels (ICa,L) and Ca2?sensitivity of
the myofilaments. However, the cellular mechanisms of ?1ARS are
not fully clarified. In this study, we investigated the role of
Ca2??calmodulin-dependent PK II (CaMKII) in the regulation of ICa,L
during ?1ARS in isolated adult rat ventricular myocytes by using
the perforated patch–clamp technique. CaMKII inhibition with 0.5
?M KN-93 abolished the potentiation in ICa,L observed during
?1ARS by 10 ?M phenylephrine. In the presence of PKC inhibitor
(10 ?M chelerythrine), the potentiation of ICa,Lby phenylephrine
rine (>1 ?M) increased the amount of autophosphorylated CaMKII
(active CaMKII) significantly, and this increase was abolished by
CaMKII inhibition or PKC inhibition. Also, we investigated changes
in the subcellular localization of active CaMKII by using immuno-
fluorescence microscopy and immunoelectron microscopy. Before
?1ARS, active CaMKII was exclusively located just beneath the
plasmalemma. However, after ?1ARS, active CaMKII was localized
located. From these results, we propose that CaMKII, which exists
near transverse tubules, is activated and phosphorylated by ?1ARS
and that CaMKII activation directly potentiates ICa,Lin rat ventric-
cardiac muscle ? perforated patch–clamp ? PKC ? phenylephrine
studies indicate that ?1-adrenoceptor stimulation (?1ARS)
causes a positive inotropic effect in most mammalian ventricular
myocytes (1–3). The proposed mechanisms underlying the pos-
itive inotropic effect are as follows: prolongation of action
potential duration by inhibition of K?currents (4) and an
increase in myofibrillar responsiveness to Ca2?(1) by intracel-
lular alkalinization (5) and?or phosphorylation of contractile
Although Ca2?current through L-type channel (ICa,L) is an
important determinant of the Ca2?transients that trigger con-
traction, the contribution of ICa,L to the positive inotropic
response to ?1ARS is not fully clarified. In studies (4, 7) using
the whole-cell patch–clamp technique with Ca2?buffer present
in the pipette solution, phenylephrine did not affect ICa,L.
However, in recent studies using the perforated patch–clamp in
the absence of Ca2?buffer, ICa,L, as well as contractile force and
the Ca2?transient, were potentiated in rat ventricular myocytes
(8, 9). Therefore, we postulate that either intracellular Ca2?or
Ca2?-dependent intracellular regulatory mechanisms might be
involved in the potentiation of ICa,L during ?1ARS in rat
Ca2??calmodulin-dependent PK II (CaMKII) is involved in
various kinds of Ca2?-dependent actions both under physiolog-
he ?1-adrenoceptor has an important role in the regulation
of mammalian cardiac muscle contraction (1, 2). Recent
ical and pathophysiological conditions in mammalian ventricular
myocytes (10). The purpose of this study is to investigate the
possible involvement of CaMKII in ?1-adrenoceptor mediated
modulation of ICa,Lin ventricular myocytes. Also, we focused on
the role of PKC as one of the possible kinases in ?1ARS (2).
Here, we provide evidence that CaMKII activation is essential
for the potentiation of ICa,Lduring ?1ARS and that activated
CaMKII is localized in proximity of transverse tubules (T-
tubules), where L-type Ca2?channels are located (11).
Materials and Methods
Cells, Solutions, Chemicals, and Antibodies. Single ventricular myo-
cytes were enzymatically isolated from adult male Wistar rats
(300–400 g) and suspended in Tyrode’s solution (136.9 mM
NaCl?5.4 mM KCl?1 mM CaCl2?0.5 mM MgCl2?0.33 mM
NaH2PO4?5 mM Hepes?5 mM glucose, pH 7.40), adjusted
with NaOH (5).
One ?M bupranolol (Kaken Pharmaceutical, Tokyo) was
present in the perfusion solution throughout the experiments to
mM CsCl, 10 mM NaCl, 0.5 mM MgCl2, 5 mM Hepes, and 1 mM
CaCl2, and pH was adjusted to 7.20 with CsOH. All reagents
were purchased from Sigma, unless otherwise indicated. KN-93
and KN-92 were obtained from Calbiochem, and 1,2-bis(2-
aminophenoxy)ethane-N,N,N?,N?-tetraacetic acid (BAPTA)–
acetoxymethyl ester was obtained from Molecular Probes. Anti-
phospho-CaMKII (anti-active CaMKII, rabbit polyclonal IgG
raised against threonine phosphorylated peptide corresponding
to the phosphorylated Thr-256 region of the mammalian
CaMKII) was obtained from Promega (12, 13). Anti-total
CaMKII (mouse monoclonal IgG raised against amino acids
303–478 of CaMKII of mouse origin) was obtained from Santa
Cruz Biotechnology (12). Alexa-546-conjugated anti-rabbit sec-
ondary antibody from Molecular Probes. Wheat germ agglutinin
(WGA)–FITC from Biomeda (Foster City, CA) and 15 nm of
gold-conjugated goat anti-rabbit IgG from Amersham
Measurement of ICa,L. Perforated patch–clamp was used to mea-
sure ICa,L by using an EPC-8 amplifier (HEKA Electronik,
Lambrecht?Pfalz, Germany) (14–16). For measuring ICa,L, hold-
ing potential was set at ?40 mV, and a 200-msec depolarizing
pulse to 0 mV was applied every 10 sec. The current–voltage
relationship was obtained by using a series of test pulses between
?30 and ?60 mV in 10-mV increments. Current amplitude was
Abbreviations: ?1ARS, ?1-adrenoceptor stimulation; BAPTA, 1,2-bis(2-aminophe-
noxy)ethane-N,N,N?,N?-tetraacetic acid; CaMKII, Ca2??calmodulin-dependent PK II; ICa,L,
Ca2?current through L-type channel; SA, standard area; T-tubules, transverse tubules;
WGA, wheat germ agglutinin.
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
June 28, 2005 ?
vol. 102 ?
defined as the difference between the peak current and the
nifedipine in the perfusate almost completely blocked this
current (data not shown), indicating that the measured current
was ICa,L. All experiments were performed at room temperature
Western Immunoblotting. Contents of total and phosphorylated
CaMKII (active CaMKII) were determined by Western immu-
noblotting using whole-cell protein extracts (12). Cells were
treated with various concentrations of phenylephrine (0–100
?M) for 15 min and then protein extracts were prepared. For
testing the effect of chemicals (prazosin, KN-93, and cheleryth-
rine) used in electrophysiological experiments, cells were ex-
posed to Tyrode’s solution containing these chemicals for 10 min
before application of phenylephrine and then to the same
solutions containing 10 ?M phenylephrine for 15 min. Samples
(50 ?g per well) were electrophoresed in 12% SDS?PAGE gel,
and exposed to primary antibodies against active CaMKII and
total CaMKII. Immunoreactive bands were visualized by en-
hanced chemiluminescence using the ECL-plus detection kit
(Amersham Biosciences) and quantified by using densitometry
(ATTO, Tokyo). Analysis of the change in total or active
CaMKII due to phenylephrine exposure was carried out by
comparing the band intensity with that of the control (nonex-
Immunofluorescence Microscopy. After treatment with 100 ?M
phenylephrine for 15 min, myocytes were fixed in 100% acetone
active CaMKII and WGA-FITC (overnight), followed by Alexa-
546-conjugated anti-rabbit secondary antibody for 1 h (17).
Immunostaining was visualized with an LSM-510 laser scanning
confocal microscope (Zeiss). Control experiments performed by
using secondary antibody without primary antibody showed no
Immunoelectron Microscopy. For cryoimmunoelectron micros-
copy, isolated myocytes were fixed in 2% paraformaldehyde in
0.1 M phosphate buffer (pH 7.4) infused with 2.3 M sucrose
containing 20% polyvinylpyrrolidone at 4°C and frozen in liquid
nitrogen. Ultrathin cryosections were cut and processed for
immunolabeling (18, 19). Rabbit anti-active CaMKII IgG as a
primary antibody and 15 nm of gold-conjugated goat anti-rabbit
IgG as a secondary antibody were used. Samples were examined
with an H-7500 transmission electron microscope (Hitachi,
Tokyo) at an accelerating voltage of 100 KV. Gold-particle
density was determined at the level of the Z lines (representing
a T-tubule location) and at the plasmalemma, as described in ref.
20, with some modifications. Briefly, the density of gold particles
within 500 ? 500-nm2areas of the section was determined for
areas located at the level of the Z lines between the myofibrils
(233 areas, presumably indicating T-tubule location) and in
proximity of the plasmalemma (83 areas) by using sections from
four representative single cells before or after ?1ARS, respec-
tively. The counts were normalized by the particles density on
areas of the section within the myofibrils (293 areas, taken as
Statistical comparisons were carried out by using one-way or
one-way repeated measured ANOVA followed by Bonferroni
post hoc test with the significance level set at P ? 0.05.
Effect of 10 ?M Phenylephrine on ICa,L Measured by Using the
Perforated Patch–Clamp Technique. Fig. 1 shows a representative
result of the effect of 10 ?M phenylephrine on ICa,L. The effect
of phenylephrine on the Ca2?responsiveness of the contractile
element in rat ventricular myocytes is almost saturated at this
concentration (5). As reported (8, 9), we observed a transient
decrease followed by a sustained increase of ICa,Lamplitude. As
shown in Fig. 1A, ICa,Ltransiently decreased for up to 2 min after
the application of phenylephrine, and then it gradually increased
and reached another steady-state level at ?15 min after this
application. The amplitudes of ICa,Lat 15 and 20 min were not
statistically different (114.8 ? 14.5% of control and 117.9 ?
13.5% of control, n ? 12, P ? 1.00), and thus, the effect of 10
?M phenylephrine on ICa,Lpotentiation reached a steady state
at 15 min. The amplitude of ICa,Lreturned to the control level at
10 min after removal of phenylephrine (101.4 ? 6.2% of control,
n ? 5, P ? 0.678). The initial transient decrease in ICa,Lat 2 min
(91.4 ? 6.0% of control, n ? 12, P ? 0.05) and the later increase
at 15 min (114.8 ? 14.5% of control, n ? 12, P ? 0.001) (Fig. 1C)
were small but significant. The increase in ICa,Loccurred without
changes in the shape of the current-voltage relationship (Fig.
1B). These negative and positive effects of phenylephrine on
ICa,L were both completely blocked by the ?1-adrenoceptor
antagonist prazosin (1 ?M) (n ? 12, data not shown). In the
phenylephrine. ICa,Lwas elicited by a 200-msec depolarizing pulse from a holding potential of ?40 to 0 mV (cell capacitance, 76.1 pF) every 10 sec. (Bar indicates
(B) Mean current–voltage relationships (n ? 10) of ICa,Lbefore (E) and 15 min after application of 10 ?M phenylephrine (?). (Bars indicate SD.)*, P ? 0.05;**,
P ? 0.01, compared with the current before phenylephrine at each voltage. (C) Time-dependent changes of ICa,Lafter the application of 10 ?M phenylephrine
(F, n ? 12) and in the absence of phenylephrine (‚, n ? 10). The amplitude of current at each time was normalized to the current before the application of
phenylephrine. (Bars indicate SD.)*, P ? 0.05;**, P ? 0.01;***, P ? 0.001, compared with the current before phenylephrine. (D) Time-dependent changes in
ICa,Lafter the application of 10 ?M phenylephrine in cells preincubated with 20 ?M BAPTA–acetoxymethyl ester (}, n ? 10). The amplitude of current at each
time was normalized by the current before the application of phenylephrine. (Bars indicate SD.)
Effect of 10 ?M phenylephrine on ICa,Lusing perforated patch. (A) Representative time course of ICa,Lamplitude changes during application of 10 ?M
O-Uchi et al.PNAS ?
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absence of phenylephrine, the amplitude of ICa,Lwas stable for
up to 15 min (Fig. 1C).
We explored the effect of intracellular Ca2?buffering on ICa,L
in response to 10 ?M phenylephrine (Fig. 1D). After establishing
perforated patch, a cell was incubated with 20 ?M BAPTA–
acetoxymethyl ester, the cell permeable form of BAPTA, for 10
min and we confirmed that the cell did not contract during the
pulse to evoke ICa,L(16). In this condition, phenylephrine did not
show either a significant transient decrease in ICa,L at 2 min
(97.2 ? 4.4% of control, n ? 10, P ? 1.00) or a sustained increase
at 15 min (101.3 ? 17.1% of control, n ? 10, P ? 1.00). Thus,
intracellular Ca2?is a key factor for the potentiation in ICa,L
induced by phenylephrine. The different responses of ICa,Lto
phenylephrine recorded by conventional patch and perforated
patch can be explained by the intracellular Ca2?concentration.
The Role of CaMKII in the Regulation of ICa,Lby ?1ARS.Recentstudies
have reported that CaMKII is involved in various Ca2?-
dependent effects both under physiological and pathophysiolog-
ical conditions in mammalian ventricular myocytes (10). Thus,
we investigated the role of CaMKII in the regulation of ICa,Lby
?1ARS by using KN-93, a synthetic CaMKII inhibitor. At a
concentration of 0.5 ?M, KN-93 selectively inhibits CaMKII
without affecting other PKs (21). A 10-min exposure to 0.5 ?M
KN-93 significantly decreased ICa,Lfrom 9.12 ? 3.27 to 5.50 ?
3.39 pA?pF, corresponding to 39.1 ? 25.6% decrease (n ? 12,
P ? 0.001) without changing the shape of the current–voltage
relationship (n ? 10) (see also ref. 22). In the presence of KN-93,
10 ?M phenylephrine produced only a sustained decrease of
ICa,L(Fig. 2A). At 15 min after application of 10 ?M phenyl-
ephrine, the amplitude of ICa,Lsignificantly decreased to 66.7 ?
22.0% of the value before application of phenylephrine in the
presence of KN-93 (n ? 12, P ? 0.001) without changing the
shape of the current-voltage relationship (n ? 10). In contrast,
0.5 ?M KN-92, an inactive KN-93 analogue, did not show
significant effects on the biphasic change of ICa,L caused by
?1ARS (Fig. 2A). We also investigated the effect of 10 ?M
phenylephrine in the presence of another CaMKII inhibitor,
autocamtide-2 inhibitory peptide, a membrane-permeable and a
highly specific peptide type inhibitor of CaMKII (23). When we
used 10 ?M autocamtide-2 inhibitory peptide, 10 ?M phenyl-
ephrine produced only a sustained decrease of ICa,L without
Thus, CaMKII inhibition abolished the potentiation of ICa,L
The Role of PKC in the Regulation of ICa,LDuring ?1ARS. PKC may be
involved in the ?1-adrenoceptor-mediated modulation of cardiac
K?channels, intracellular alkalinization and myofibrillar re-
sponsiveness to Ca2?(2). Therefore, we investigated the role of
PKC in the regulation of ICa,Lby ?1ARS by using chelerythrine
as a PKC inhibitor. We found that 10 ?M chelerythrine selec-
tively inhibits PKC without affecting other PKs (24) and effec-
tively blocks the effect of phenylephrine on the Ca2?respon-
siveness of the contractile element (5). Exposure to 10 ?M
chelerythrine for 10 min significantly decreased ICa,Lto a new
steady state from 7.42 ? 1.61 to 3.78 ? 1.23 pA?pF, correspond-
ing to a 49.7 ? 13.2% decrease (n ? 9, P ? 0.001) without
changing the shape of the current–voltage relationship. In the
presence of chelerythrine, 10 ?M phenylephrine produced only
a sustained decrease of ICa,L as observed in the presence of
CaMKII inhibitors (Fig. 2B). At 15 min after application of 10
?M phenylephrine, the amplitude of ICa,Ldecreased to 42.3 ?
10.9% of the value before application of phenylephrine in the
presence of chelerythrine (n ? 9, P ? 0.001) (Fig. 2B) without
changing the shape of the current–voltage relationship (n ? 9).
Activation of CaMKII During ?1ARS. Our electrophysiological ex-
is mediated by both CaMKII and PKC. Although PKC has been
suggested to be involved in the ?1-adrenoceptor signal-
transduction pathway (2), there are no reports that CaMKII is
activated by ?1ARS in rat ventricular myocytes. The amount of
CaMKII is low compared with other PKs; consequently, it is
difficult to determine CaMKII activity directly in cardiac muscle
autophosphorylation site of CaMKII, Thr-286, and an enhanced
chemiluminescence system to measure CaMKII activity in
whole-cell lysates in response to ?1ARS (12, 13). Active CaMKII
increased significantly at concentrations of ?1 ?M phenyleph-
rine (Fig. 3A Bottom), although total CaMKII was not altered
(Fig. 3A Top; data not shown). The increase of active CaMKII
level induced by 10 ?M phenylephrine was completely blocked
by the ?1-adrenoceptor antagonist prazosin (1 ?M), showing
that this effect was mediated by ?1-adrenoceptor (Fig. 3B). The
selective CaMKII inhibitor 0.5 ?M KN-93 that we used in the
perforated patch experiments partially blocked the basal
CaMKII activity in the absence of phenylephrine and also
completely blocked the increase in the active CaMKII level by 10
?M phenylephrine (Fig. 3C).
To determine whether PKC could be a regulator of CaMKII
activation during ?1ARS, the effect of phenylephrine in the
presence of chelerythrine on CaMKII activity was determined.
Chelerythrine (10 ?M) did not produce significant changes in
the CaMKII activation before phenylephrine (86.6 ? 25.1% of
control, n ? 7, P ? 0.39). However, the increase in active
(102.2 ? 24.3% of control, n ? 7, P ? 1.00), as in the case of
KN-93, indicating that PKC is involved in the activation of
CaMKII during ?1ARS (Fig. 3D).
Immunocytochemical Localization of Active CaMKII by ?1ARS. It has
been reported that CaMKII may directly phosphorylate the
L-type Ca2?channel and regulate the positive feedback system
that facilitates ICa,Lunder physiological conditions (17, 26). Our
biochemical experiments showed that CaMKII is significantly
activated by ?1ARS in adult rat isolated ventricular myocytes. To
10 ?M phenylephrine in the presence of 0.5 ?M KN-93 (‚, n ? 12) or 0.5 ?M
KN-92 (Œ, n ? 7). After establishing a new steady state for ICa,Lby 10-min
application of KN-93 or KN-92, the effect of 10 ?M phenylephrine on ICa,Lwas
observed in the continuous presence of KN-93 or KN-92. The amplitude of the
current at each period was normalized to the current before the application
of phenylephrine. (Bars indicate SD.)*, P ? 0.05;**, P ? 0.01;***, P ? 0.001,
compared with the normalized current in the presence of KN-92 at each time.
Phe, phenylephrine. (B) Time-dependent changes of ICa,Lafter the application
in the absence of chelerythrine (F, n ? 12). The amplitude of current at each
period was normalized to the current before the application of phenyleph-
the current after the application of 10 ?M phenylephrine in the absence of
chelerythrine at each time.
Effect of CaMKII inhibition and PKC inhibition on ICa,Lin the presence
www.pnas.org?cgi?doi?10.1073?pnas.0503569102O-Uchi et al.
investigate whether CaMKII could directly regulate the L-type
Ca2?channel during ?1ARS, we determined the localization of
active CaMKII in isolated ventricular myocytes by using immu-
nofluorescence microscopy, as shown in Fig. 4. We used WGA-
FITC, a marker of sarcolemma including T-tubules (27), and
anti-active CaMKII antibody to establish the intracellular local-
ization of active CaMKII (Fig. 4 C and D). Before ?1ARS, active
CaMKII was detectable at the plasmalemma (Fig. 4 A and E) as
reported (17). After ?1ARS (100 ?M phenylephrine) active
CaMKII was still present at the plasmalemma (Fig. 4B), but it
was also clearly visible along transverse bands that coincide with
the location of WGA-FITC. This result suggests that a higher
level of active CaMKII was localized at or near the T-tubules
after ?1ARS than in the resting state (Fig. 4F).
To confirm the subcellular localization of active CaMKII
before and after ?1ARS, immunoelectron microscopy was used.
The number of gold particles over a standard area (SA) at the
plasmalemma and at the level of the Z lines where T-tubules are
located was counted in sections from four representative cells
before and after ?1ARS. Before ?1ARS, the frequency of gold
particles was very low both beneath the plasmalemma (0.10 ?
0.31 particles per SA, 0.39 ? 1.20 in normalized density, n ? 30
areas) and at T-tubules (0.09 ? 0.29 particles per SA, 0.36 ? 1.15
in normalized density, n ? 108 areas) (Fig. 5D). After applica-
various concentration of phenylephrine (n ? 8). Although the level of total CaMKII protein was not changed, the level of active CaMKII significantly increased
at concentrations of phenylephrine ?1 ?M. (Bottom) Bar graphs show the intensity of the active CaMKII band, normalized to the control, indicating the change
KN-93, and chelerythrine, respectively. (Bottom) Increase of the level of active CaMKII during ?1ARS was completely blocked by prazosin (n ? 6), KN-93 (n ? 9),
difference between the two experimental results; CTL, control; Phe, phenylephrine; Pra, prazosin; Che, chelerythrine.
Activation of CaMKII in response to ?1ARS. (A) (Top and Middle) Western immunoblot analyses showing the activation levels of CaMKII in response to
bars in F, 10 ?m.)
Localization of activated CaMKII in response to ?1ARS. Immunofluorescence images of ventricular myocytes labeled with active CaMKII antibody (red;
O-Uchi et al. PNAS ?
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tion of phenylephrine (100 ?M), as shown in Fig. 5 A and B, the
frequency of gold particles (indicating active CaMKII) near
T-tubules was increased (1.21 ? 0.92 particles per SA, 3.95 ?
3.00 in normalized density, n ? 125 areas), showing ?10-fold
enrichment, consistent with the result of immunofluorescence
image analysis (compare Figs. 4F and 5E). In contrast, the
particle density beneath the plasmalemma remained low (0.49 ?
0.80 particles per SA, 1.60 ? 2.61 in normalized density, n ? 53,
P ? 0.27, compared with that before ?1ARS) (Fig. 5C). Thus,
immunogold labeling showed ?2.5-fold enrichment of active
CaMKII in T-tubules compared with the plasmalemma after
?1ARS (Fig. 5E). The particle density in the myofibrils areas was
not significantly different before (0.25 ? 0.48 particles per SA,
n ? 110) and after ?1ARS (0.30 ? 0.52 particles per SA, n ?
There seems to be a discrepancy between the results obtained
by using light and electron microscopy regarding the location of
active CaMKII in the resting cells. The light-microscope images
did not show active CaMKII at T-tubules, whereas electron-
microscopic images detected a very low level at those sites. The
confocal sections are relatively thick compared with the thick-
ness of the T-tubules so that a small portion of each section is
occupied by T-tubule membrane and a weak signal in this
membrane is not detected. In contrast, the plasmalemma runs
from one end of the section to the other, so that the signal from
this membrane is detectable even if weak. In the thin sections for
electron microscopy, plasmalemma and T-tubule membrane are
more equally represented.
In this study, we explored the involvement of CaMKII in the
signal transduction pathway between ?1ARS and the potentia-
tion of ICa,L in rat ventricular myocytes, and we show direct
evidence indicating that CaMKII is activated by ?1ARS and has
an important effect on ICa,L.
Our electrophysiological experiments using the perforated
patch technique confirm the reported (8, 9) potentiation of ICa,L
during ?1ARS in rat ventricular myocytes. When ICa,Lis mea-
sured by using the whole-cell patch–clamp technique, phenyl-
ephrine does not affect ICa,Lat any concentrations (4, 7), and we
can mimic this effect by buffering the intracellular Ca2?with
BAPTA–acetoxymethyl ester. Thus, intracellular Ca2?concen-
tration is essential for the potentiation of ICa,Lduring ?1ARS.
This result is consistent with the report that basal intracellular
Ca2?is sufficient for the initial CaMKII activation under
physiological condition (28).
We chose 10 ?M phenylephrine in our electrophysiological
experiments because the effect of phenylephrine on ICa,L
potentiation is saturated at this concentration (see Fig. 6,
which is published as supporting information on the PNAS web
site). ?1ARS has two opposite effects on ICa,L. (i) Higher
concentration of phenylephrine (?10 ?M) cause a biphasic
response: an initial brief (?2 min) depression, or negative
phase, followed by a potentiation or positive phase; and (ii)
lower concentration of phenylephrine (?1 ?M) cause a
monophasic positive effect (Fig. 6). The positive effect (po-
tentiation) depends on CaMKII activation and PKC activation
because CaMKII inhibition or PKC inhibition abolished the
potentiation of ICa,L(Fig. 2 and Fig. 7, which is published as
supporting information on the PNAS web site). There is less
information about the negative effect of ?1ARS on ICa,L. In
these experiments, we focused on the potentiation of ICa,L
during ?1ARS (positive effect).
Our electrophysiological experiments using CaMKII inhib-
itors demonstrated the important role of CaMKII in the
potentiation of ICa,L(positive phase) during ?1ARS in cardiac
myocytes. Western immunoblot analysis confirmed an increase
in active CaMKII (see also ref. 13) in parallel with the
potentiation of ICa,L. Also, we showed that PKC, which is
activated by the Gq-phospholipase C-diacylglycerol pathway
(2), is involved in the activation of CaMKII during ?1ARS,
based on the similar effects of CaMKII and PKC inhibition on
ICa,Lin the presence of phenylephrine. It has been reported
that, in resting cardiac myocytes, there is significant activation
of CaMKII but active CaMKII can be lost when the intracel-
lular Ca2?concentration is lowered to very low levels by
removal of extracellular Ca2?(17). Basal activity of CaMKII
is determined mainly by the resting intracellular Ca2?level
(and not by the PKC activity). However, after ?1ARS, PKC
activity has an important role in the additional and sustained
gold-active CaMKII before and after ?1ARS. After ?1ARS, a higher intensity of gold labeling was observed directly under T-tubule membranes (A and B), and a
lower intensity of gold labeling was observed just beneath the non-T-tubular surface sarcolemmal membrane (C). (D) No gold labeling was observed directly
(n ? 108 areas counted before ?1ARS and n ? 125 areas counted after ?1ARS) and sarcolemma (n ? 30 areas counted before ?1ARS and n ? 53 areas counted
after ?1ARS) relative to the surface areas on the myofilaments (n ? 110 areas counted before ?1ARS and n ? 183 areas counted after ?1ARS). The calculation
method for normalized gold particles density is described in Materials and Methods. (Bars in E indicate SD.)***, P ? 0.001.
Activated CaMKII is preferentially localized in T-tubules after ?1ARS. (A–D) Immunoelectron microscopy images of myocytes labeled with 15 nm of
www.pnas.org?cgi?doi?10.1073?pnas.0503569102 O-Uchi et al.
activation of CaMKII. PKC can directly phosphorylate the
autophosphorylation site of CaMKII in vitro, thus directly
increasing CaMKII activity (29). This report strongly supports
our hypothesis that there is a physiological linkage between
PKC and CaMKII during ?1ARS.
Immunolabeling at the ultrastructural level demonstrates that
the level of active CaMKII is very low along the plasmalemma
and T-tubules in the resting cells but increases significantly along
the T-tubules, where L-type Ca2?channels are mostly present
(11), after ?1ARS. The correspondence between the distribu-
tions of active CaMKII and L-type Ca2?channels strongly
supports our view that CaMKII directly phosphorylates the
L-type Ca2?channels and potentiates ICa,L in response to
?1ARS. Immunofluorescence microscopy confirms the increase
in active CaMKII at the T-tubules from an undetectable to a
detectable level with stimulation.
It has been reported (10) that CaMKII modulates ICa,Lunder
physiological conditions. Several groups have demonstrated that
Ca2?-dependent ICa,L facilitation is mediated by CaMKII-
dependent phosphorylation of L-type Ca2?channel, and this
mechanism is considered to be related to the positive staircase
of ICa,Linduced by repeated depolarization from physiological
holding potential (17, 26, 30). However, the molecular mecha-
nisms of how ICa,Lis potentiated by the activation of CaMKII
have not been elucidated.
In summary, these experiments show that (i) ?1ARS poten-
tiates ICa,L by PKC and CaMKII activation and (ii) activated
CaMKII is highly localized close to the T-tubules after ?1ARS.
We thank Dr. S. Matsuoka (Kyoto University, Kyoto) for his helpful
comments; Ms. N. Tomizawa, Ms. Y. Natake, Ms. E. Kikuchi,
Ms. M. Murata, Ms. M. Nomura, Mr. H. Saito, and Mr. Y. Kimura for
technical assistance; and the staffs of Department of Physiology, Divi-
sion of Molecular Immunology, and Laboratory of Neurophysiology at
The Jikei University School of Medicine for their continuous support
and encouragement. A part of this study was supported by a Grant-in-
Aid from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan (to K.K., Y.K., K.H., and S.K.) and a grant from
the Uehara Memorial Foundation (to S.K.).
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O-Uchi et al.PNAS ?
June 28, 2005 ?
vol. 102 ?
no. 26 ?
PHYSIOLOGY. For the article ‘‘?1-Adrenoceptor stimulation po-
tentiates L-type Ca2?current through Ca2??calmodulin-
dependent PK II (CaMKII) activation in rat ventricular myo-
cytes,’’ by Jin O-Uchi, Kimiaki Komukai, Yoichiro Kusakari,
Toru Obata, Kenichi Hongo, Hiroyuki Sasaki, and Satoshi
Kurihara, which appeared in issue 26, June 28, 2005, of Proc.
Natl. Acad. Sci. USA (102, 9400–9405; first published June 17,
2005; 10.1073?pnas.0503569102), the authors note that the pan-
els in Fig. 4 were incorrectly labeled, due to a printer’s error. The
corrected figure and its legend appear below.
bars in F, 10 ?m.)
Localization of activated CaMKII in response to ?1ARS. Immunofluorescence images of ventricular myocytes labeled with active CaMKII antibody (red;
Table 1. Potency of chemicals and reversibility of their effects
gravitropism, IC50*Inhibition of growth
Inhibition of hypocotyl
HypocotylRoot Hypocotyl length†‡
NA96.8 (0.02 ?M)
60 (0.3 ?M)
98.8 (0.3 ?M)
21.8 (10 ?M)
90.4 (0.1 ?M)
88.7 (2 ?M)
93.8 (0.3 ?M)
NA, not applicable, because chemical has little or no effect on gravitropism. ND, not determined.
*Estimated concentration resulting in 50% inhibition after 24-h gravistimulation.
†Lengths are reported as a percentage of the untreated controls.
‡Length measurements performed at the concentrations indicated.
PLANT BIOLOGY. For the article ‘‘The power of chemical genomics to
Carter, Glenn R. Hicks, Jacob Vasquez, and Natasha V. Raikhel,
USA (102, 4902–4907; first published March 16, 2005; 10.1073?
pnas.0500222102), the authors note that in Table 1, the concentra-
tions are incorrectly presented as ‘‘M’’ instead of ‘‘?M,’’ due to a
printer’s error. The corrected table appears below. In addition, the
authors note the last sentence of the Fig. 3 legend, ‘‘Chemical
now appear online in Table 2, which is published as supporting
information on the PNAS web site. These errors do not affect the
conclusions of the article.
July 26, 2005 ?
vol. 102 ?
no. 30 www.pnas.org
BIOPHYSICS. For the article ‘‘A quantitative assessment of models
for voltage-dependent gating of ion channels,’’ by Michael
Grabe, Harold Lecar, Yuh Nung Jan, and Lily Yeh Jan, which
USA (101, 17640–17645; first published December 10, 2004;
10.1073?pnas.0408116101), the authors note that Fig. 3a was
computed with a tilt angle different from that detailed in the
Results. The corrected curves, presented below, are ?7% smaller
in magnitude than the curves presented in Fig. 3a Left, but their
shape and spacing are preserved. The correction will not alter
the curve shown in Fig. 3a Right. The corrected activation energy
barrier for the paddle model is ?75 kBT, and the corresponding
activation time is 1028ms or 1017years. The conclusions of the
article remain unchanged. The corrected figure and its legend
charge movement for lipid-exposed (a), translation (b), and rotation (c) mod-
model is rugged because of the complicated interfacial geometry.
Total electrostatic solvation energy of the S4 segment and gating
July 26, 2005 ?
vol. 102 ?
no. 30 ?