Stimulation of unitary T-type
Ca2+ channel currents
by calmodulin-dependent protein kinase II
Paula Q.
Barrett1,
Hong-Kai
Lu1,
Roger
Colbran2,
Andrew
Czernik3, and
Joseph J.
Pancrazio4
1 Department of Pharmacology, University of Virginia
School of Medicine, Charlottesville, Virginia 22903;
2 Department of Molecular Physiology and Biophysics, Vanderbilt
University, Nashville, Tennessee 37232; 3 Laboratory of
Molecular and Cellular Neuroscience, Rockefeller University, New York,
New York 10021; and 4 Center for Bio/Molecular Science and
Engineering, Naval Research Laboratory, Washington, District of
Columbia 20375
 |
ABSTRACT |
The effect of
Ca2+/calmodulin-dependent protein kinase II (CaMKII)
stimulation on unitary low voltage-activated (LVA) T-type Ca2+ channel currents in isolated bovine adrenal
glomerulosa (AG) cells was measured using the patch-clamp technique. In
cell-attached and inside-out patches, LVA channel activity was
identified by voltage-dependent inactivation and a single-channel
conductance of ~9 pS in 110 mM BaCl2 or
CaCl2. In the cell-attached patch, elevation of bath
Ca2+ from 150 nM to 1 µM raised intracellular
Ca2+ in K+-depolarized (140 mM) cells and
evoked an increase in the LVA Ca2+ channel probability of
opening (NPo) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank
sweeps, a rise in the frequency of channel opening in nonblank sweeps,
and a 30% reduction in first latency. No apparent changes in the
single-channel open-time distribution, burst lengths, or openings/burst
were apparent. Preincubation of AG cells with lipophilic or peptide
inhibitors of CaMKII in the cell-attached or excised (inside-out)
configurations prevented the rise in NPo elicited by elevated Ca2+ concentration.
Furthermore, administration of a mutant recombinant CaMKII
exhibiting cofactor-independent activity in the absence of elevated
Ca2+ produced a threefold elevation in LVA channel
NPo. These data indicate that CaMKII activity is
both necessary and sufficient for LVA channel activation by
Ca2+.
glomerulosa cells; low voltage-activated channels
 |
INTRODUCTION |
IN ADRENAL
GLOMERULOSA (AG) cells, sustained Ca2+ entry through
low voltage-activated (LVA) T-type Ca2+ channels has been
hypothesized to regulate the synthesis and secretion of the steroid
hormone aldosterone (5). Whole cell LVA currents recorded
in AG cells at 37°C using physiological Ca2+ as the
charge carrier (1.2 mM Ca2+) revealed voltage
dependencies of activation (V1/2 =
50 mV) and inactivation (V1/2 =
68 mV) such that
channel open probability (Po) was
<0.3% at membrane potentials
(Vm) negative to
79 mV (5). Since the Vm of AG cells
established by physiological K+ solutions ranged from
98
to
79 mV (in HEPES-based Ringer solution), these data suggested
little contribution of LVA channels to Ca2+ entry in the
unstimulated steady state (5). Nevertheless, a small shift
in the LVA channel half-activation potential could result in a
substantial increase in Ca2+ entry, even in the absence of
a change in Vm, since the foot of the voltage
dependence of activation curve determines the extent of channel opening
at negative membrane voltages. Therefore, an effective way to regulate
LVA channel activity in AG cells during cell stimulation would be to
effect a hormone-elicited shift in the half-activation potential.
Indeed, in AG cells, activation of protein kinase C (39)
reduces LVA channel activity while activation of Gi
(26) or Ca2+/calmodulin-dependent protein
kinase II (CaMKII) (27) enhances LVA channel activity by
inducing a corresponding shift in the half-activation potential of LVA
Ca2+ channels. Activation of CaMKII evokes a 10-mV
hyperpolarizing shift in the half-activation potential (5,
27), resulting in an augmentation of the channel
Po to 2.2% (5). Thus at a Vm of
74 mV, CaMKII-activated channels are
eight times more likely to be open than under control conditions
(5).
Angiotensin II (ANG II) is the major physiological regulator
of aldosterone secretion. ANG II activates CaMKII (10),
and CaMKII inhibition attenuates ANG II-stimulated aldosterone
secretion (37). Therefore, CaMKII-mediated shifts in LVA
channel gating would appear to mediate the control of Ca2+
entry underlying the physiological control of aldosterone secretion.
In this paper, to begin to delineate the signaling cascade
responsible for the action of CaMKII on whole cell LVA currents, we
evaluated the necessity for additional downstream
Ca2+-dependent effectors in the signaling pathway by
examining the effect of CaMKII on single-channel activity. We
observed that CaMKII activation increased the frequency of channel
opening (NPo) with no apparent change in modal
states or open-state dwell times. Our data suggest that CaMKII
activation is both necessary and sufficient for LVA channel activation
by Ca2+ and that CaMKII activity may be tightly associated
with the plasma membrane close to the LVA Ca2+ channel.
 |
METHODS |
Cell isolation.
Calf AG cells (2-7 days old) were prepared by collagenase
digestion as described previously (10). Thin layers of
zona glomerulosa were prepared from adrenal cortex and collected in a
Ca2+-free Krebs-Ringer bicarbonate (KRB) that contained (in
mM) 120 NaCl, 25 NaHCO3, 3.5 KCl, 1.2 MgSO4,
1.2 NaH2PO4, 1.25 CaCl2, 0.1%
dextrose, and 0.2% bovine serum albumin (BSA), equilibrated with 95%
air-5% CO2 (pH 7.4). The slices were digested for 10 min at 37°C in KRB that contained 0.625 mM CaCl2, 0.1%
BSA, and 0.35 U/ml collagenase (Worthington, Freehold, NJ). The cells
were dispersed mechanically, filtered through a 20-µm nylon mesh
filter (Tetko, Elmsford, NY), collected by centrifugation, and
resuspended in KRB that contained 1.25 mM CaCl2 and 0.2%
BSA. The remaining slices were digested again (10 min at 37°C in KRB
that contained 1.25 mM CaCl2, 0.1%BSA, and 0.35 U/ml
collagenase), and the dispersion and filtration procedures were
repeated. Cells recovered from the second digestion were collected and
further purified on a two-step (30%/56%) discontinuous Percoll
gradient (Pharmacia, Piscataway, NJ) and resuspended in standard KRB
buffer. Cells were either cultured on glass coverslips in serum-free
media (1:1 DMEM/F-12 that contained 200 µg/ml amikacin and 200 µg/ml ticarcillin) for 2 h before experiments or cultured
overnight on glass coverslips in media (1:1 DMEM/F-12 that contained
10% horse serum, 2% fetal calf serum, 200 µg/ml amikacin, and 200 µg/ml ticarcillin) and serum deprived for 6 h before patch
voltage-clamp studies were begun.
Recording.
AG cells adherent to noncoated glass coverslips were transferred to a
300-µl volume RC-25 recording chamber (Warner Scientific, New Haven,
CT) that was continuously perfused by gravity at a rate of 0.5 ml/min
(5). Electrical recording was performed at room
temperature. Patch electrodes were pulled from aluminosilicate glass (1724; Garner Glass, Claremont, CA) on a two-stage puller (L/M-3-PA; List-Medical, Darmstadt-Eberstadt, Germany) coated with
Sylgard (Dow Corning, Midland, MI) and fire polished. The electrode
resistances were 5-10 M
after heat polishing. Single-channel currents were recorded with an EPC-7 patch-clamp amplifier
(List-Electronic, Darmstadt-Eberstadt, Germany) filtered with an
eight-pole low-pass Bessel filter (Frequency Devices, Haverhill, MA)
set at a cut-off frequency (
3 dB) of 1.0 kHz and digitized at
intervals of 80-100 µs. Pulse generation and data acquisition
were performed using a ZEOS 486 computer with pCLAMP 6.0 software and a
Digidata interface (Axon Instruments, Foster City, CA). Depolarizing
test pulses (
35 mV or
40 mV) of 150-ms duration were delivered
every 4 s from the holding potential of
90 mV unless otherwise indicated.
Solutions.
A high-K+ external solution was used to lower the
Vm to 0 mV (13, 16). The external
solution contained (in mM) 110 potassium aspartate, 20 potassium
chloride, 10 HEPES, 10 EGTA, 5 dextrose, and 1 magnesium chloride (pH
adjusted to 7.4 with KOH). The Ca2+ concentration in the
external solution was adjusted to 50 nM, 150 nM, 500 nM, and 1 µM
depending on the experiments. Free Ca2+ in the external
solution was calculated using the ligand binding program, EQCAL
(Biosoft, Ferguson, MO). In the excised configuration, 1 mM ATP, 200 µM GTP, and 0.2 µM CaM were added to the external solution unless
otherwise specified. The pipette solution contained (in mM) either 110 BaCl2 and 10 HEPES or 110 CaCl2 and 10 HEPES (pH adjusted to 7.4 with CsOH).
Data analysis.
Idealized current traces were constructed from digitized records using
the Transit 2.0 suite of programs (44). Channel openings or closings were identified using a slope detector to localize transitions between levels and a relative amplitude criterion to remove
spurious transitions (44). Capacitative and leakage currents were adequately eliminated from the records by ensemble averaging records free of channel openings and subtracting this average
from all the records. Idealized records were concatenated into a single
file, using the Convide program provided by Dr. A. M. VanDongen. The concatenated file was used for
NPo and burst analysis by Transit. Open channel
current amplitudes for current-voltage (I-V) relationships
were measured by manually fitting cursors to well-resolved channel
openings. The amplitudes of detected events were binned and fitted to
one or the sum of two Gaussian distributions using SigmaPlot for
Windows version 5 (SPSS, Richmond, CA) {f = A1/sqrt(2
)
1[e
(x
µ1/
1)2/2] + A2 /sqrt(2
)
2[e
(x
µ2/
2)2/2]};
where
1 and
2 = SD of
Gaussian distribution 1 and 2 respectively; µ1 and
µ2 = mean of Gaussian distribution 1 and 2 respectively; A1 and
A2 = amplitude term 1 and 2. For 27 nM
Ca2+ (µ = 0.51 ± 0.06 pA,
=
0.15 ± 0.006, A = 10.51); for 1.2 µM Ca2+ (main conductance: µ = 0.47 ± 0.33 pA,
=
0.173 ± 0.003, A = 38.16 ± 0.66; subconductance: µ = 0.20 ± 0.12 pA,
=
0.021 ± 0.0018, A = 2.83 ± 0.27). Where
appropriate, data are given as means ± SE, and statistical
significance was determined using Student's t-test with
P < 0.05 considered significant. A statistical comparison of the proportion of blank sweeps under control and treatment conditions was accomplished by testing the difference between
proportions (45), with P values <0.05
considered significant.
Measurement of intracellular
Ca2+
concentration.
AG cells were plated on collagen-coated glass coverslips and loaded as
previously reported (10) for 30 min at room temperature with 4 µM fura PE3-AM (Teflabs, Austin, TX), a
Ca2+-sensitive fluorescent dye with limited cell leakage.
Cells were incubated in KRB for 90 min at 37°C to allow for the
intracellular generation of the Ca2+-sensitive form of the
dye. Coverslips were mounted in a 500-µl recording chamber (Medical
Systems, Greenvale, NY), and adherent AG cells were perfused by gravity
with an external patch solution that fixed extracellular
Ca2+ at 50 nM or 1 µM. Fluorescence emission at
400 nm
was measured in response to alternate excitation at 340 or 380 nm using
a Deltascan 4000 spectrofluorometer (Photon Technology International,
South Brunswick, NJ) and digitized at a rate of 5 Hz. Single cell
intracellular Ca2+ concentration
([Ca2+]i) 340/380 fluorescence ratios were
converted to nanomolar [Ca2+]i values
according to standard calibration protocols (43). All
recordings were performed at room temperature.
Preparation of T286D-CaMKII
.
T286D-CaMKII
, which introduces an acidic aspartate residue in place
of Thr-286, was expressed in Sf9 insect cells using baculovirus and
then purified as described for the wild-type kinase (2). The purified baculovirus-expressed enzyme expressed 8-25%
Ca2+-independent activity toward exogenous substrates,
thereby partially mimicking the effect of Thr-286
autophosphorylation. The purified kinase (specific
activity of 3-6
µmol · min
1 · mg
1 in the
presence of Ca2+/CaM) was frozen in small aliquots in
liquid N2 and then stored at
80°C. Thawed aliquots were
diluted 100-fold in external patch solution such that the final
concentration of the T286D-CaMKII
subunit was ~60 nM.
 |
RESULTS |
Properties of the LVA Ca2+ channel in
AG cells.
LVA Ca2+ channels displayed diverse patterns of small
openings in response to weak test depolarizations. Figure
1 shows current records selected to
illustrate the different types of gating behavior observed in a
cell-attached patch of a neonatal calf AG cell where only a single
channel appears to be active. Figure 1A illustrates the
effect of holding potential and 1C the effect of driving
force on the elicited currents. With 110 mM BaCl2 in the
pipette, 150-ms depolarizations to
40 mV from a holding potential of
90 mV evoked currents of small amplitude. Consistent with whole cell
currents in AG cells that show slow activation and inactivation at
potentials negative to
30 mV (26), channel opening
exhibited a long first latency and slow voltage-dependent inactivation.
In a few records, repeated events were observed. Many records had no
channel activity, indicating a low probability of opening at
40 mV.
In some cell-attached patches (15%), larger conductance openings
(presumably L-type) were observed, but in no patches were these
openings frequent or observed alone. The complete disappearance of
small conductance openings at a holding potential of
40 mV during an
interpulse interval of 6 s (Fig. 1A, right)
and their full recovery upon repolarization to
90 mV (data not shown)
indicated the presence of potential-dependent inactivation. As shown in
Fig. 1B, the amplitude histogram of the single-channel
events at
35 mV was well fitted to one Gaussian distribution with a
mean value of
0.51 ± 0.061 pA. Replacing BaCl2
(closed circles) with CaCl2 (open circles) in the patch
pipette solution yielded equivalent values for the amplitude of
single-channel openings (Fig. 1C). Construction of a
single-channel I-V curve from current amplitudes measured at
various test potentials defined a slope conductance of 8.7 pS in 110 BaCl2 (Fig. 1C). This value compared favorably with conductance values reported previously for native LVA
Ca2+ channels recorded in glomerulosa cells (8 pS, 8.3 pS)
(29, 30), chick sensory neurons (8.5 pS)
(13), ventricular myocytes (8 pS) (32), adult
hippocampal neurons (8.7 pS) (11), and for the recently
cloned
1H T-type Ca2+ channel (9 pS) (46).
Overall, 1) the long first latency at
35 mV
(30); 2) the small and equivalent conductance
in Ba2+ and Ca2+ (32, 41); and
3) complete voltage-dependent inactivation at
40 mV
(13, 29) is consistent with the documented behavior of
T-type Ca2+ channels.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Characteristics of unitary T-type Ca2+
channel currents. Consecutive cell-attached recordings from T-type
Ca2+ channels. A: effect of holding potential on
single-channel openings. Voltage protocol is shown above current
records. Currents were sampled at 12.5 kHz and filtered at 1 kHz. Data
show potential-dependent inactivation. B: amplitude
histogram of single-channel openings. Detected events fitted to 1 Gaussian distribution (see METHODS) defining a conductance
level of 0.51 ± 0.061 pA. C: unitary current-voltage
relationship. Data obtained from at least 4 patches at each test
potential (5 measurements each) using 110 BaCl2
( ) or 110 CaCl2 ( ) as the
charge carrier. Straight line is linear regression fit of
BaCl2 data yielding slope conductance of 8.7 pS
(r = 0.99).
|
|
Effect of extracellular
Ca2+ elevation on
LVA Ca2+
channels.
Exposure to 140 mM extracellular K+ reduces membrane
potential to zero (16) and allows intracellular
Ca2+ levels to be altered by changes in extracellular
Ca2+ (18). In AG cells, half-maximal
activation of CaMKII is attained by 1.5 µM Ca2+ at a
saturating concentration of CaM (700 nM) (10). LVA channel activity was examined with extracellular Ca2+ set to 150 nM
and 1.0 µM using Ca2+-EGTA buffers. With the use of these
buffers, [Ca2+]i in the bulk cytosol rose
from 62 ± 3 nM, a value similar to that observed in unstimulated
AG cells incubated in physiological KRB (3.5 mM K+), to
473 ± 41 nM (mean ± SE, n = 10), a value
similar to that attained on 5 mM K+ depolarization
(10).
The recordings illustrated in Fig. 2 were
typical of most cell-attached patches, and visual inspection shows that
elevation of extracellular Ca2+ induced an increase in
T-type Ca2+ channel activity. As illustrated in Fig.
2A, at low external Ca2+, small conductance
openings evoked by depolarizing pulses to
35 mV were generally brief
and infrequent. Because of this low frequency of channel opening,
channel kinetics were analyzed from records compiled from 10 cells that
had only a single channel appearing to be active in 100 measured
consecutive sweeps. At
35 mV with 110 mM Ba2+ as the
charge carrier, channels opened with a first latency of 68 ± 2 ms
(mean ± SE), a frequency of 8.8 ± 1.7 openings/s and an
amplitude of 0.51 ± 0.06 pA. Shown at the bottom of the current records, an ensemble average of 1,000 sweeps displayed a peak current
amplitude of 0.023 pA. By contrast, elevation of extracellular Ca2+ to 1.0 µM evoked a greater than threefold increase
in the mean frequency of opening to 30.8 ± 4.7 openings/s
(P
0.05) and an ~30% reduction in first latency to 48 ±1 ms (P
0.05). Whereas the main single-channel
amplitude observed in the presence of elevated Ca2+ was
similar to that observed in low Ca2+, a subconductance
state was also identified in the records (see Fig. 2A). The
resulting amplitude histogram was well fitted with the sum of two
Gaussian distributions with mean amplitudes of the main and
subconductance states equal to 0.47 pA and 0.20 pA, respectively. While
subconductance states have been previously reported for LVA channels
(8, 36), we focused subsequent analysis on the properties
of the main conductance state that constituted >93% of the detected
events. The ensemble averaged current after extracellular
Ca2+ elevation attained a peak current of 0.048 pA, a value
that was approximately twofold greater than that observed in low
extracellular Ca2+. Although this averaged current
displayed little channel inactivation during the 150-ms test pulse to
35 mV, channel inactivation was complete within the 6 s of
interpulse holding at
40 mV (see Fig. 1A). Very slow
inactivation of LVA channels during weak test depolarizations has been
previously observed (11, 17, 30).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of extracellular Ca2+ on unitary
Ca2+ channel currents. A: cell-attached
recordings selected to show diverse patterns of openings elicited upon
raising extracellular Ca2+ from 150 nM to 1.0 µM in 140 mM K+-depolarized cells. Traces below the single-channel
recordings are the average current obtained from 100 consecutive sweeps
from each of 10 cells. B: channel probability of opening
(NPo) analysis from same patches recorded in
A. Data show consistent increase in
NPo evoked by 1.0 µM Ca2+
(Ca2+-dependent potentiation).
|
|
Figure 2B shows that elevation of extracellular
Ca2+ consistently increased channel activity. Plotted is
the percent open time calculated from 100 consecutive sweeps for each
of 10 cells before and after Ca2+ was elevated. Although
there is considerable variation in the NPo among
control cells, Ca2+ increased NPo in
all cell-attached patches from 200 to 600%. This increase in
NPo was primarily the consequence of a reduction in the number of blank sweeps (557-245 blanks/1,000 sweeps,
P < 0.001) and an increase in the number of openings
in nonblank sweeps (3.4 ± 0.6 to 6.5 ± 0.8 openings/sweep;
P
0.05). An analysis of NPo by
sweep indicated a continuous range of NPo values
in low and high extracellular Ca2+ rather than values that
grouped near a few distinct values. Thus unlike N-type
Ca2+ channels that are induced by epinephrine to switch
gating modes, Ca2+ elevation does not appear to increase
channel activity by altering the modal behavior of the T-type
Ca2+ channel (7). Figure
3 shows a log-log plot of an open-state dwell-time distribution that was well fit to the sum of two
exponentials (r > 0.98), suggesting the existence of
two open states with open-time constants that differed from each other
by nearly an order of magnitude (
1 = 0.31 ± 0.04 ms;
2 = 2.29 ± 0.64 ms). In low
extracellular Ca2+, ~62% of the detected events
(1,354/1,000 sweeps) were identified by the fast open-time constant
with the remainder characterized by the slower time constant. Raising
the bath Ca2+ concentration increased the number of
detected openings to 4,734 (per 1,000 sweeps) but neither prolonged the
fast or slow open-time constants (
1 = 0.26 ± 0.02 ms;
2 = 2.21 ± 0.35 ms) nor increased the relative proportion of openings identified by the slow open-time constant (70%:
1; 30%:
2). Thus the
Ca2+-induced increase in NPo could
not be explained by a prolongation of the duration of open events.
Rather, the present data suggested that the Ca2+-induced
increase in NPo was associated with a greater
than threefold increase in the number of openings assigned to each
open-state distribution.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of extracellular Ca2+ on dwell-time
distributions. Log-log histograms of linearly binned open times
obtained with a digital sampling interval of 0.08 ms. The best-fit 2 exponential is shown superimposed with components. A: at 150 nM, extracellular Ca2+ 01 (fast) = 0.3 ± 0.01 ms, events/bin = 360 ± 45;
02 (slow)= 2.3 ± 0.6 ms, events/bin = 30 ± 10; r = 0.95. B: at 1.0 µM,
extracellular Ca2+ 01 (fast) = 0.3 ± 0.01 ms, events/bin = 1,855 ± 129, r = 0.97; 02 (slow) = 2.2 ± 0.6 ms,
events/bin = 95 ± 18, r = 0.98. Data show
Ca2+ elevation increases frequency of fast and slow
events.
|
|
Visual inspection of the single-channel records also showed that
many of the events did not occur in isolation but were separated by
short- and long-lived closures, a characteristic of bursting activity.
As expected, the distribution of dwell times in the closed state was
multiexponential. However, because the Po of the
channel was low in both low and high Ca2+ and the last
closing could not be distinguished from inactivation, many
long-duration channel closures were missed and could not be adequately
quantified. Nevertheless, a Ca2+-induced reduction in a
slow closed/inactivated state time constant or proportion of
closed/inactivated state events would be expected to underlie the
observed Ca2+-induced increase in the number of openings in
nonblank sweeps. We examined records for burst activity with the use of
critical closed-time estimates based on criteria by Colquhoun and
Sigworth (5a) (2.4 and 2.0 ms: low and high Ca2+). With the
use of these estimates, only 6.5% of the events would be expected to
be misclassified. As anticipated from the previous kinetic analysis of
unitary events, high Ca2+ exposure increased the observed
number of bursts from 534 to 1,741. This increase in burst number was
not accompanied by an increase in the mean number of openings/burst
(1.8 ± 1.7: low Ca2+; 2.2 ± 2.2: high
Ca2+) or a change in the distribution of burst lengths.
Thus whether analyzed as unitary or bursting events, Ca2+
increased T-type Ca2+ channel activity by effecting an
increase in event frequency rather than by prolonging the length of events.
Mediation by
Ca2+/CaM-dependent protein
kinase II.
In the next series of experiments, we determined whether CaMKII
activity underlies the potentiating effect of extracellular Ca2+ elevation. Previous studies from our
laboratory have shown that in AG cells, activation of CaMKII induces a
hyperpolarizing shift in the voltage dependence of activation of the
T-type Ca2+ channel (27). An increase in event
frequency is consistent with the CaMKII-evoked shift in the voltage
dependence of activation of whole cell currents that occurs without a
change in the
of deactivation of whole cell T-type tail currents.
Figure 4 shows that inhibition of
CaMKII activity prevents the potentiating effect of extracellular
Ca2+ on single-channel activity in cell-attached patches.
CaMKII activation was blocked by preincubation of AG cells with KN-62
(3 µM, 1 h), a lipophilic inhibitor that competes with CaM for
its binding site on the kinase. Representative single-channel
recordings from AG cells treated with KN-62 (Fig. 4A) showed
that KN-62 did not prevent Ca2+ channel openings elicited
on depolarization to
35 mV in low Ca2+ but blocked the
elevation in channel activity by high extracellular Ca2+.
In untreated cells, an analysis of the Po
indicated that 1.0 µM Ca2+ increased
NPo 4.2-fold from 1.4 ± 0.3 to 5.9 ± 0.9% (P
0.05) in 12 cell-attached patches. This increase
in NPo compared with an insignificant effect in
cells treated with KN-62 [2.07 ± 0.3 vs. 2.6 ± 0.5%;
n = 8; not significant (n.s.)]. By contrast,
pretreatment with the inactive analog of KN-62, KN-04, which differs
from KN-62 by only a single carbonyl residue, did not prevent the
increase in channel activity (Fig. 4A) evoked by
Ca2+ (2.0 ± 0.8 vs. 6.2 ± 1.4%;
n = 7; P < 0.05). These data suggested that the potentiating effect of Ca2+ on LVA
Ca2+ channel opening depended on CaMKII activation.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of Ca2+/calmodulin-dependent
protein kinase (CaMKII) inhibition on Ca2+-dependent
potentiation. A: cell-attached recordings selected to show
the effect of Ca2+ elevation when CaMKII activation is
inhibited (KN-62) or preserved (KN-04). Voltage protocol is shown above
current records. B: NPo analysis of
cells bathed in extracellular Ca2+ of 150 nM or 1.0 µM
Ca2+ that were untreated or pretreated with KN-62 (active
analog) or KN-04 (inactive analog). Data show active analog selectively
inhibits potentiated Ca2+ channel activity.
|
|
The role of CaMKII in the regulation of LVA Ca2+ channels
was further investigated using excised inside-out patches. Unlike high
voltage-activated (HVA) channels where activity ceases within minutes of patch excision, T-type Ca2+ channels can remain
active in this configuration (3, 22). Because excision
into low Ca2+ (150-nM free) increased baseline channel
activity to submaximal stimulatory levels, bath Ca2+ was
reduced to 50 nM, close to the measured intracellular Ca2+
level. This observed "run up" of single-channel activity on patch excision into 150-nM free Ca2+ confirms the disequilibrium
that exists between extracellular Ca2+ and intracellular
Ca2+ in the cell-attached configuration. In the presence of
1 mM ATP, 200 µM GTP, and 0.2 µM CaM, LVA channel activity
persisted during the entire 20-min recording period. Depolarization to
35 mV evoked unitary currents of small amplitude that were not
different from those recorded in the cell-attached configuration (Fig.
5). Elevation of Ca2+ to 500 nM greatly increased Ca2+ channel activity.
NPo rose 3.8-fold from 0.8 ± 0.2 to
3.0 ± 0.8% (n = 10, P
0.05) upon
Ca2+ stimulation, demonstrating that
Ca2+-induced potentiation can be reconstituted in the
membrane patch. In the excised configuration, the highly specific,
nonpermeant CaMKII inhibitor, autocamtide 2-related inhibitory peptide
(AIP) (20), could be used to reduce kinase activity. This
synthetic peptide (KKALRRQEAVDAL) is modeled on the autonomy site
(Thr-286) in the autoregulatory domain (RQETV) of CaMKII
but has an
Ala substitution for Thr (15). Inclusion of this peptide
in the external solution at 1 µM [a concentration ~25 times the
IC50 for CaMKII inhibition (20)] prevented
the increase in Ca2+ channel activity elicited by
Ca2+ elevation (0.8 ± 0.2 vs. 0.9 ± 0.2;
n = 11; n.s.). The observed inhibition of LVA channel
activity induced by AIP was specific to stimulated activity since this
inhibitor of the kinase did not reduce basal Ca2+ channel
activity recorded in 50 nM Ca2+. Together, these data
suggest that CaMKII is contained within the domain of the excised
patch, that regulation occurred locally, and that the participation of
a distant kinase cascade is not obligatory.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of CaMKII activation or inhibition on
Ca2+ channel activity in the excised patch. A:
excised-patch recordings selected to show effect of Ca2+
elevation (50-500 nM) and CaMKII inhibition effected by
autocamtide 2-related inhibitory peptide (AIP). Voltage protocol is
shown above records. B: NPo analysis
of recordings from inside-out patches bathed in 50 nM or 500 nM with or
without 1 µM AIP. Data show selective inhibition of CaMKII blocks
Ca2+ from potentiating channel activity.
|
|
Although our studies to this point indicated the importance of CaMKII
activation in the regulation of T-type Ca2+channel
activity, they did not rule out the participation of other Ca2+-dependent effectors. To address this concern, the
efficacy of preactivated CaMKII on single-channel activity was assessed
in the excised patch in the absence of Ca2+ elevation. We
used mutant recombinant CaMKII
, in which residue 286, the threonine
phosphorylation site, was replaced by an aspartate (T286D-CaMKII
).
This substitution produces the same functional effect on the kinase as
the autophosphorylation of Thr-286 in the autoinhibitory domain and
generates partial activity in the absence of activation by CaM and
Ca2+ (see METHODS) by increasing the affinity
of CaMKII for CaM by ~100-fold (31) and permitting
Ca2+-independent (autonomous) phosphorylation of substrates
following dissociation of Ca2+/CaM (reviewed in Ref.
1). While keeping bath Ca2+ fixed at 50 nM,
Ca2+ channel activity was recorded before and after the
addition of T286D-CaMKII (~60 nM final). As illustrated in the
representative recordings, single-channel activity was greatly
increased by the addition of T286D-CaMKII (Fig.
6A).
Within 5 min of kinase exposure, NPo rose
2.8-fold from a mean value of 0.5 ± 0.1 to 1.4 ± 0.2% (n = 9, P
0.05). This effect
was dependent on ATP (ATP free: 0.38 ±0.2; n = 6;
n.s.) and specific to the addition of active kinase because neither the
addition of vehicle (0.4 ± 0.1; n = 5; n.s.) nor
heat-inactivated kinase (0.6 ± 0.2; n = 5; n.s.) were effective in increasing NPo. As expected,
subsequent addition of noninactivated kinase and ATP resulted in
enhanced channel activity. Thus CaMKII activity was necessary and
sufficient to increase NPo in the absence of
activation of other Ca2+-dependent effectors.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of constitutively active CaMKII on
Ca2+ channel activity. Asp replaced Thr-286 in recombinant
CaMKII , rendering the kinase active in the absence of
Ca2+ elevation. A: excised-patch recordings
selected to show effect of active or heat-inactivated T286D-CaMKII
(~60 nM) in 50 nM Ca2+. Voltage protocol is shown above
records. B: NPo analysis of
recordings taken before and 1-5 min after the addition of
T286D-CaMKII, vehicle, or heat-inactivated T286D-CaMKII.
* P < 0.5 vs. T1 activity. Data show
T286D-CaMKII is sufficient to increase channel activity.
|
|
 |
DISCUSSION |
Ca2+-dependent
potentiation.
The modulation of Ca2+ channel currents by Ca2+
has been documented for at least two major types of HVA
Ca2+ channels (L-type, P/Q-type) (9, 12, 14, 18, 25,
28, 48, 50). Here, we describe at the single-channel level
potentiation of LVA Ca2+ channel currents by
Ca2+. With Ca2+ or Ba2+ as the
permeant ion, elevation of bath Ca2+ increased LVA
Ca2+ channel Po in
K+-depolarized cell-attached patches and excised patches in
the inside-out configuration. Potentiation of LVA channel activity by
Ca2+ contrasts with the well-known effect of
Ca2+ to inactivate L-type and P/Q-type Ca2+
channels, but it mimics the less familiar effect of Ca2+ to
potentiate L-type Ca2+ channel activity (18, 28,
48). In the present study, a biphasic effect of Ca2+
on the activity of LVA channels was not observed. We report that Ca2+ increased Ca2+ channel
Po by 200-600% primarily as a result of
1) an evoked increase in channel availability, as indicated
by the reduction in null sweeps, and 2) by an elicited
increase in the conditional probability of opening, as judged by an
increase in the Po of nonblank sweeps with only
a small change in first latency. In contrast, the characteristics of
feed-forward regulation of L-type Ca2+ channels by
Ca2+ include both an increase in the frequency of channel
opening and an additional, but modest, increase in the duration of the open state (18). In the present study, the
Ca2+-dependent enhancement of T-type channels was not
accompanied by a change in the conductance of the channel or by a
prolongation of either open state but, rather, by an increase in the
frequency of channel opening. The mechanism by which the frequency of
channel opening is achieved may involve the modulation of one or more closed or inactivated states that can precede channel opening, as
recently described by Serrano et al. (40). Thus a detailed kinetic description underlying this effect must await further studies.
LVA channels in AG cells exhibited a conductance of 8.7 pS, a unitary
current amplitude of
0.51 pA at
35 mV, and slow but full
steady-state potential-dependent inactivation negative to
35 mV. LVA
Ca2+ channels characterized in glomerulosa cells, cardiac
myocytes, and neurons have similar properties measured with comparable
solutions (3, 11, 13, 29, 30, 32, 33). At least two groups of open times could be distinguished: one characterized by a fast (0.3 ms) and the other a slower (2.2, 2.3) open-state time constant. Our
finding of a bimodal distribution of open times, which is suggestive of
two open states, has not been uniformly observed for the T-type
Ca2+ channel (3, 4, 8, 13). While it is
conceivable that the algorithm used to detect channel openings
permitted greater sensitivity, it is also possible that the variable
observation of two open states may relate to heterogeneity within
the T-type Ca2+ channel gene family and/or
posttranslational modifications. Since the cloning of the
1G (36), two new genes encoding T-type
Ca2+ channels have been identified (
1H and
1I) that share 90-80% sequence identity across
putative transmembrane segments (6). Although at the whole
cell level these channel paralogs have been shown to have very similar
voltage-dependent properties (24), to date
characterization at the single-channel level has been minimal (6,
24, 35, 36, 46). Therefore, it remains possible that the
single-channel behavior reported here may be unique to a subtype within
the T-type Ca2+ channel family.
Modulation by CaMKII.
In the cell-attached patch, we observed that Ca2+-dependent
potentiation was prevented by pretreatment with KN-62. This inhibitor of the CaM-kinase family competes with Ca2+/CaM for binding
and thus displays a more selective inhibitory activity than drugs like
the trifluoperazines that bind directly to CaM and thereby
inhibit all CaM-dependent enzymes (21). Nonetheless, similarities in the CaM binding site among CaM-dependent kinases explains the reported inhibition of both CaMKII and CaMKIV by KN-62
(20). Thus, despite the fact that the inactive analog of
KN-62, KN-04, was without effect, the inhibition of
Ca2+-dependent potentiation by KN-62 cannot be interpreted
as strong evidence for the unique participation of CaMKII.
In the excised configuration, Ca2+-dependent potentiation
was inhibited by AIP, a peptide that interacts at two distinct
substrate binding sites on CaMKII. AIP competes noncompetitively with
exogenous substrates for phosphorylation and competitively with the
endogenous substrate, the autophosphorylation domain of the kinase
(19). As such, AIP inhibited CaMKII selectively in the
absence and presence of CaM. At 1 µM, the concentration of inhibitor
used in this study, AIP completely inhibited CaMKII activity without
effecting other multifunctional protein kinases such as CaMKIV,
cAMP-dependent protein kinase, and protein kinase C
(20). Inhibition of Ca2+-dependent
potentiation by AIP in the excised patch and the duplication of
Ca2+ channel activation evoked by the addition of
constitutively active recombinant CaMKII (T286D-CaMKII) provide strong
evidence for the regulation of T-type Ca2+ channel
activity by CaMKII in the AG cell. Moreover, because Ca2+-induced potentiation of T-type Ca2+
channel activity required ATP and active kinase, this regulation differed mechanistically from the regulation of L- and P/Q-type Ca2+ channels by CaM recently reported (23,
49). In these studies, the direct binding of CaM (to motifs on
the COOH-terminal tail of the Ca2+ channel subunits
1C or
1A) was observed to promote
inactivation and facilitation of these high voltage-activated
Ca2+ channels independently of kinase activation. Because
in our study Ca2+ channel activation also could be evoked
by T286D-CaMKII in the absence of elevated Ca2+, the
participation of an additional Ca2+-dependent effector
downstream of CaMKII in the regulatory cascade was ruled out.
Therefore, in the AG cell, activation of CaMKII is both necessary and
sufficient to initiate a feed-forward regulation of T-type
Ca2+ channels by Ca2+.
The fact that modulation of LVA Ca2+ channels by
Ca2+ elevation was recapitulated in inside-out excised
patches implies that at least some of the AG cell CaMKII remained
associated with the excised patch. This could in principal occur via
the association of CaMKII with plasma membrane proteins or with lipids.
Several recent studies have provided evidence for similar localization of CaMKII close to sites of Ca2+ entry. For example,
neuronal CaMKII binds to the NR-2B subunit of
N-methyl-D-aspartic acid (NMDA)-type glutamate
receptors, a ligand and voltage-gated Ca2+ channel
(42). Thus CaMKII is appropriately located to be very rapidly activated by Ca2+ entering through this receptor
and, by phosphorylating NMDA receptor subunits, may regulate their
function (34). Similarly, in cardiomyocytes, a portion of
the CaMKII appears to colocalize with subunits of the L-type
Ca2+ channel (47), where it presumably plays a
role in the feedback regulation of Ca2+ entry. Thus the
retention of CaMKII on excised AG cell membrane patches strongly
suggests that this kinase is appropriately localized to be uniquely
sensitive to Ca2+ entering through LVA Ca2+
channels and to provide rapid feedback (forward) regulation.
In the AG cell, the selective enhancement of LVA channel activity at
negative potentials by CaMKII activation plays a critical role in
setting the level of steady-state Ca2+ entry and the level
of aldosterone secretion elicited by ANG II stimulation. An increase in
single-channel activity reported here offers insight into the
obligatory signaling events underlying the CaMKII-induced change in
whole cell channel behavior. In chronic heart failure, sustained
activation of the renin-angiotensin system contributes pathologically
to Na+ retention, volume expansion, and the harmful
remodeling of the heart and vasculature. In light of the very recent
findings indicating that blockade of aldosterone receptors in
conjunction with conventional therapies further reduces the incidence
of death among patients with severe heart failure (38),
targeted disruption of this regulatory mechanism may be of significant
clinical utility.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-36977 (to P. Q. Barrett).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: P. Q. Barrett, Dept. of Pharmacology, Univ. of Virginia School of
Medicine, 1300 Jefferson Park Ave., Charlottesville, VA 22903 (E-mail:
pqb4b{at}virginia.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 January 2000; accepted in final form 17 July 2000.
 |
REFERENCES |
1.
Braun, AP,
and
Schulman H.
The multifunctional calcium/calmodulin-dependent protein kinase: from form to function.
Annu Rev Physiol
57:
417-445,
1995[ISI][Medline].
2.
Brickey, DA,
Colbran RJ,
Fong YL,
and
Soderling TR.
Expression and characterization of the
-subunit of Ca2+/calmodulin-dependent protein kinase II using the baculovirus expression system.
Biochem Biophys Res Commun
173:
578-584,
1990[ISI][Medline].
3.
Carbone, E,
and
Lux HD.
Single low-voltage-activated calcium channels in chick and rat sensory neurones.
J Physiol (Lond)
386:
571-601,
1987[Abstract].
4.
Chen, CF,
and
Hess P.
Mechanism of gating of T-type calcium channels.
J Gen Physiol
96:
603-630,
1990[Abstract].
5.
Chen, XL,
Bayliss DA,
Fern RJ,
and
Barrett PQ.
A role for T-type Ca2+ channels in the synergistic control of aldosterone production by angiotensin II and K+.
Am J Physiol Renal Physiol
276:
F674-F683,
1999[Abstract/Free Full Text].
5a.
Colquhoun, D,
and
Sigworth FJ.
Fitting and statistical analysis of single-channel records.
In: Single-Channel Recordings (2nd ed.), edited by Sakmann B,
and Neher E.. New York: Plenum, 1995, p. 483-585.
6.
Cribbs, LL,
Lee JH,
Yang J,
Satin J,
Zhang Y,
Daud A,
Barclay J,
Williamson MP,
Fox M,
Rees M,
and
PerezReyes E.
Cloning and characterization of
1H from human heart, a member of the T-type calcium channel gene family.
Circ Res
83:
103-109,
1998[Abstract/Free Full Text].
7.
Delcour, AH,
and
Tsien RW.
Altered prevalence of gating modes in neurotransmitter inhibition of N-type calcium channels.
Science
259:
980-984,
1993[ISI][Medline].
8.
Droogmans, G,
and
Nilius B.
Kinetic properties of the cardiac T-type calcium channel in the guinea-pig.
J Physiol (Lond)
419:
627-650,
1989[Abstract].
9.
Eckert, R,
Chad JE,
and
Kalman D.
Enzymatic regulation of calcium current in dialyzed and intact molluscan neurons.
J Physiol (Paris)
81:
318-324,
1986[Medline].
10.
Fern, RJ,
Hahm MS,
Lu HK,
Liu LP,
Gorelick FS,
and
Barrett PQ.
Ca2+/calmodulin-dependent protein kinase II activation and regulation of adrenal glomerulosa Ca2+ signaling.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F751-F760,
1995[Abstract/Free Full Text].
11.
Fisher, RE,
Gray R,
and
Johnston D.
Properties and distribution of single voltage-gated calcium channels in adult hippocampal neurons.
J Neurophysiol
64:
91-104,
1990[Abstract/Free Full Text].
12.
Forsythe, ID,
Tsujimoto T,
Barnes-Davies M,
Cuttle MF,
and
Takahashi T.
Inactivation of presynaptic calcium current contributes to synaptic depression at a fast central synapse.
Neuron
20:
797-807,
1998[ISI][Medline].
13.
Fox, AP,
Nowycky MC,
and
Tsien RW.
Single-channel recordings of three types of calcium channels in chick sensory neurones.
J Physiol (Lond)
394:
173-200,
1987[Abstract].
14.
Gurney, AM,
Charnet P,
Pye JM,
and
Nargeot J.
Augmentation of cardiac calcium current by flash photolysis of intracellular caged Ca2+ molecules.
Nature
341:
65-68,
1989[ISI][Medline].
15.
Hanson, PI,
Kapiloff MS,
Lou LL,
Rosenfeld MG,
and
Schulman H.
Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation.
Neuron
3:
59-70,
1989[ISI][Medline].
16.
Hess, P,
Lansman JB,
and
Tsien RW.
Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells.
J Gen Physiol
88:
293-319,
1986[Abstract].
17.
Hess, P,
Lansman JB,
and
Tsien RW.
Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists.
Nature
311:
538-544,
1984[ISI][Medline].
18.
Hirano, Y,
and
Hiraoka M.
Dual modulation of unitary L-type Ca2+ channel currents by [Ca2+]i in fura-2-loaded guinea-pig ventricular myocytes.
J Physiol (Lond)
480:
449-463,
1994[Abstract].
19.
Ishida, A,
and
Fujisawa H.
Stabilization of calmodulin-dependent protein kinase II through the autoinhibitory domain.
J Biol Chem
270:
2163-2170,
1995[Abstract/Free Full Text].
20.
Ishida, A,
Kameshita I,
Okuno S,
Kitani T,
and
Fujisawa H.
A novel highly specific and potent inhibitor of calmodulin-dependent protein kinase II.
Biochem Biophys Res Commun
212:
806-812,
1995[ISI][Medline].
21.
Klee, CB.
Interaction of calmodulin with Ca2+ and target proteins.
In: Calmodulin, edited by Cohen P,
and Klee CB.. New York: Elsevier, 1988, p. 35-56.
22.
Kostyuk, PG,
Shuba Ya M,
and
Savchenko AN.
Three types of calcium channels in the membrane of mouse sensory neurons.
Pflügers Arch
411:
661-669,
1988[ISI][Medline].
23.
Lee, A,
Wong ST,
Gallagher D,
Li B,
Storm DR,
Scheuer T,
and
Catterall WA.
Ca2+/calmodulin binds to and modulates P/Q-type calcium channels.
Nature
399:
155-159,
1999[ISI][Medline].
24.
Lee, JH,
Daud AN,
Cribbs LL,
Lacerda AE,
Pereverzev A,
Klockner U,
Schneider T,
and
Perez-Reyes E.
Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family.
J Neurosci
19:
1912-1921,
1999[Abstract/Free Full Text].
25.
Li, L,
Satoh H,
Ginsburg KS,
and
Bers DM.
The effect of Ca(2+)-calmodulin-dependent protein kinase II on cardiac excitation-contraction coupling in ferret ventricular myocytes.
J Physiol (Lond)
501:
17-31,
1997[Abstract].
26.
Lu, HK,
Fern RJ,
Luthin D,
Linden J,
Liu LP,
Cohen CJ,
and
Barrett PQ.
Angiotensin II stimulates T-type Ca2+ channel currents via activation of a G protein, Gi.
Am J Physiol Cell Physiol
271:
C1340-C1349,
1996[Abstract/Free Full Text].
27.
Lu, HK,
Fern RJ,
Nee JJ,
and
Barrett PQ.
Ca2+-dependent activation of T-type Ca2+ channels by calmodulin-dependent protein kinase II.
Am J Physiol Renal Fluid Electrolyte Physiol
267:
F183-F189,
1994[Abstract/Free Full Text].
28.
McCarron, JG,
McGeown JG,
Reardon S,
Ikebe M,
Fay FS,
and
Walsh JV.
Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II.
Nature
357:
74-77,
1992[ISI][Medline].
29.
McCarthy, RT,
Isales CM,
Bollag WB,
Rasmussen H,
and
Barrett PQ.
Atrial natriuretic peptide differentially modulates T- and L-type calcium channels.
Am J Physiol Renal Fluid Electrolyte Physiol
258:
F473-F478,
1990[Abstract/Free Full Text].
30.
McCarthy, RT,
Isales CM,
and
Rasmussen H.
T-type calcium channels in adrenal glomerulosa cells: GTP-dependent modulation by angiotensin II.
Proc Natl Acad Sci USA
90:
3260-3264,
1993[Abstract].
31.
Meyer, T,
Hanson PI,
Stryer L,
and
Schulman H.
Calmodulin trapping by calcium-calmodulin-dependent protein kinase.
Science
256:
1199-1202,
1992[ISI][Medline].
32.
Nilius, B,
Hess P,
Lansman JB,
and
Tsien RW.
A novel type of cardiac calcium channel in ventricular cells.
Nature
316:
443-446,
1985[ISI][Medline].
33.
Nowycky, MC,
Fox AP,
and
Tsien RW.
Three types of neuronal calcium channels with different calcium agonist sensitivity.
Nature
316:
440-443,
1985[ISI][Medline].
34.
Omkumar, RV,
Kiely MJ,
Rosenstein AJ,
Min KT,
and
Kennedy MB.
Identification of a phosphorylation site for calcium/calmodulin-dependent protein kinase II in the NR2B subunit of the N-methyl-D-aspartate receptor.
J Biol Chem
271:
31670-31678,
1996[Abstract/Free Full Text].
35.
Perez-Reyes, E,
Cribbs LL,
Daud A,
Lacerda AE,
Barclay J,
Williamson MP,
Fox M,
Rees M,
and
Lee JH.
Molecular characterization of a neuronal low-voltage-activated T-type calcium channel.
Nature
391:
896-900,
1998[ISI][Medline].
36.
Perez-Reyes, E,
Cribbs LL,
Daud A,
Yang J,
Lacerda AE,
Barclay J,
Williamson MP,
Fox M,
Rees M,
and
Lee JH.
Molecular characterization of T-type calcium channels.
In: Low-Voltage-Activated T-type Calcium Channels, edited by Tsien RW,
Clozel J-P,
and Nargeot J.. Chester, UK: Adis, 1998, p. 290-306.
37.
Pezzi, V,
Clark BJ,
Ando S,
Stocco DM,
and
Rainey WE.
Role of calmodulin-dependent protein kinase II in the acute stimulation of aldosterone production.
J Steroid Biochem Mol Biol
58:
417-424,
1996[ISI][Medline].
38.
Pitt, B,
Zannad F,
Remme WJ,
Cody R,
Castaigne A,
Perez A,
Palensky J,
and
Wittes J.
The effect of spironolactone on morbidity and mortality in patients with severe heart failure.
N Engl J Med
341:
709-717,
1999[Abstract/Free Full Text].
39.
Rossier, MF,
Aptel HBC,
Python CP,
Burnay MM,
Vallotton MB,
and
Capponi AM.
Inhibition of low threshold calcium channels by angiotensin II in adrenal glomerulosa cells through activation of protein kinase C.
J Biol Chem
270:
15137-15142,
1995[Abstract/Free Full Text].
40.
Serrano, JR,
Perez-Reyes E,
and
Jones SW.
State-dependent inactivation of the
1G T-type calcium channel.
J Gen Physiol
114:
185-201,
1999[Abstract/Free Full Text].
41.
Shuba, YM,
Teslenko VI,
Savchenko AN,
and
Pogorelaya NH.
The effect of permeant ions on single calcium channel activation in mouse neuroblastoma cells: ion-channel interaction.
J Physiol (Lond)
443:
25-44,
1991[Abstract].
42.
Strack, S,
and
Colbran RJ.
Autophosphorylation-dependent targeting of calcium/calmodulin-dependent protein kinase II by the NR2B subunit of the N-methyl-D-aspartate receptor.
J Biol Chem
273:
20689-20692,
1998[Abstract/Free Full Text].
43.
Thomas, AP,
and
Delaville F.
The use of fluorescent indicators for measurements of cytosolic-free calcium concentration in cell populations and single cells.
In: Cellular Calcium: A Practical Approach, edited by McCormack JG,
and Cobbold PH.. New York: Oxford Univ. Press, 1991, p. 1-54.
44.
VanDongen, AM.
A new algorithm for idealizing single ion channel data containing multiple unknown conductance levels.
Biophys J
70:
1303-1315,
1996[Abstract].
45.
Walpole, RE,
and
Meyers RH.
Probability and Statistics for Engineers and Scientists. New York: Macmillan, 1978.
46.
Williams, ME,
Washburn MS,
Hans M,
Urrutia A,
Brust PF,
Prodanovich P,
Harpold MM,
and
Stauderman KA.
Structure and functional characterization of a novel human low-voltage activated calcium channel.
J Neurochem
72:
791-799,
1999[ISI][Medline].
47.
Wu, Y,
MacMillan LB,
McNeill RB,
Colbran RJ,
and
Anderson ME.
CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias.
Am J Physiol Heart Circ Physiol
276:
H2168-H2178,
1999[Abstract/Free Full Text].
48.
Yuan, W,
and
Bers DM.
Ca-dependent facilitation of cardiac Ca current is due to Ca-calmodulin-dependent protein kinase.
Am J Physiol Heart Circ Physiol
267:
H982-H993,
1994[Abstract/Free Full Text].
49.
Zuhlke, RD,
Pitt GS,
Deisseroth K,
Tsien RW,
and
Reuter H.
Calmodulin supports both inactivation and facilitation of L-type calcium channels.
Nature
399:
159-162,
1999[ISI][Medline].
50.
Zygmunt, AC,
and
Maylie J.
Stimulation-dependent facilitation of the high threshold calcium current in guinea-pig ventricular myocytes.
J Physiol (Lond)
428:
653-671,
1990[Abstract].
Am J Physiol Cell Physiol 279(6):C1694-C1703
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society