Control of the Cardiac Muscarinic K+ Channel by
-Arrestin 2*
Z.
Shui,
I. A.
Khan,
T.
Haga
,
J. L.
Benovic§, and
M. R.
Boyett¶
From the School of Biomedical Sciences, University of Leeds, Leeds
LS2 9JT, United Kingdom, the
Department of
Neurochemistry, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo,
Tokyo 113-0033, Japan, and the § Kimmel Cancer Institute,
Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, December 6, 2000, and in revised form, January 8, 2001
 |
ABSTRACT |
Control of the cardiac muscarinic
K+ current (iK,ACh) by
-arrestin 2 has
been studied. In Chinese hamster ovary cells transfected with m2
muscarinic receptor, muscarinic K+ channel, receptor kinase
(GRK2), and
-arrestin 2, desensitization of iK,ACh
during a 3-min application of 10 µM ACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The
effect of
-arrestin 2 was lost if cells were not co-transfected with
GRK2. Resensitization (recovery from desensitization) of iK,ACh in cells transfected with
-arrestin 2 was
significantly slowed (time constant increased from 34 to 232 s).
Activation and deactivation of iK,ACh on application and
wash-off of ACh in cells transfected with
-arrestin 2 were
significantly slowed from 0.9 to 3.1 s (time to half peak
iK,ACh) and from 6.2 to 13.8 s (time to
half-deactivation), respectively. In cells transfected with a
constitutively active
-arrestin 2 mutant, desensitization occurred
in the absence of agonist (peak current significantly decreased from
0.4 ± 0.05 to 0.1 ± 0.01 nA). We conclude that
-arrestin
2 has the potential to play a major role in desensitization and other
aspects of the functioning of the muscarinic K+ channel.
 |
INTRODUCTION |
The cardiac muscarinic K+ current
(iK,ACh)1 is
responsible, at least in part, for the negative chronotropic,
inotropic, and dromotropic effects of vagal stimulation on the heart
(1-3). In the heart, ACh released from vagal nerves binds to the m2
muscarinic receptor (a G protein-coupled receptor) causing the
dissociation of a trimeric Gi-protein into
and 
subunits and the free 
subunits bind to and activate the
muscarinic K+ channel (4). As in other G protein-coupled
receptor systems, the free 
subunits also bind to and activate
receptor kinase and the activated receptor kinase binds to and
phosphorylates the agonist-bound receptor (on the third intracellular
loop in the case of the m2 muscarinic receptor) (5-7). The
phosphorylation of G protein-coupled receptors, including the m2
muscarinic receptor, leads to receptor desensitization (5-7). The
chronotropic, inotropic, and dromotropic effects of ACh on the heart
fade in the presence of ACh (2, 8-10) and this is likely to be, in
part at least, the result of a fade of iK,ACh as a result
of desensitization (2, 11). In rat atrial cells and in a mammalian cell
line (transfected with m2 muscarinic receptor, muscarinic
K+ channel, and receptor kinase) we have previously
obtained evidence that the phosphorylation of the receptor by receptor
kinase is responsible for short-term desensitization of
iK,ACh (12, 13).
Arrestins act in concert with receptor kinase to bring about
desensitization. Receptor kinase-mediated phosphorylation of the
receptor promotes the binding of an arrestin to the agonist bound
receptor and this causes desensitization by (i) preventing receptor-G
protein interaction and (ii) for the nonvisual arrestins (
-arrestin,
-arrestin 2) only, promoting internalization of the receptor via
clathrin-coated pits (the nonvisual arrestins act as adaptor proteins
and bind both the receptor and clathrin) (5). For the m2 muscarinic
receptor specifically, there is some evidence that arrestins are
involved in desensitization:
-arrestin and
-arrestin 2 bind to
the m2 muscarinic receptor in a phosphorylation-dependent
manner (14). There is evidence that
-arrestin and
-arrestin 2 may
be involved in m2 muscarinic receptor uncoupling: deletion
of a cluster of serine/threonine residues (phosphorylation sites) in
the C-terminal part of the third intracellular loop of the m2
muscarinic receptor greatly reduced the binding of arrestins and in
HEK293 cells abolished desensitization as a result of receptor-G
protein uncoupling (measured as a reduction in the carbachol inhibition
of a isoproterenol-stimulated increase in cAMP as a result of
pretreatment with carbachol) (14, 15). The evidence that
-arrestin
and
-arrestin 2 may also be involved in m2 muscarinic receptor
internalization is less clear: Pals-Rylaarsdam et
al. (14) showed that overexpression of
-arrestin and
-arrestin 2 in HEK-tsA201 cells resulted in an internalization of
the m2 muscarinic receptor via a dynamin-dependent mechanism (arrestin-mediated internalization is known to occur via a
dynamin- and clathrin-dependent mechanism). However, in the
same study Pals-Rylaarsdam et al. (14) showed that
internalization of the m2 muscarinic receptor in HEK-tsA201 cells in
the absence of arrestin overexpression occurred via an unknown pathway
that does not involve arrestins or dynamin. Furthermore, in rat
ventricular cells, Feron et al. (16) reported evidence that
the m2 muscarinic receptor is internalized via caveolae
(clathrin-independent pathway), although we have observed
co-localization of the m2 muscarinic receptor and clathrin after CCh
pretreatment in the same cell type (17).
The aim of the present study was to study the possible role of arrestin
in short-term desensitization of iK,ACh.
-Arrestin 2 was
chosen for study (both
-arrestin and
-arrestin 2 are ubiquitously expressed in tissues (18)).
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
Chinese hamster ovary
(CHO)-K1 cells were cultured and transiently transfected as described
previously (13). Cells were cultured in Ham's F-12 nutrient mixture
supplemented with 10% fetal bovine serum, 100 units/ml penicillin G,
100 µg/ml streptomycin sulfate, and 0.25 µg/ml Fungizone at
37 °C in 95% air and 5% CO2 (all media and chemicals
from Life Technologies Ltd., Paisley, United Kingdom). In all
experiments, cells from one of two cell lines were transiently
transfected with plasmid vectors for Kir3.1 (pEF-GIRK1) and Kir3.4
(pEF-CIR) to form the muscarinic K+ channel heteromultimer
using the calcium phosphate method (13). One cell line was already
stably transfected with plasmid vector for human m2 muscarinic receptor
(pEF-Myc-hm2) and the second cell line was already stably transfected
with plasmid vectors for human m2 muscarinic receptor (pEF-Myc-hm2) and
the G protein-coupled receptor kinase, GRK2, (pEF-GRK2). GRK2 is known
to be present in the heart (19) and it phosphorylates the m2 muscarinic
receptor both in vitro and in vivo (20, 21). The
cell lines will be referred to as clones 1 and 2, respectively. To test
whether the use of a particular clone influenced the results obtained,
experiments were repeated using both clones. The results obtained with
the two clones were indistinguishable and they have been combined, although the clones used are identified in figure legends. Depending on
the experiment, cells of clone 1 were transiently co-transfected with neither, either, or both GRK2 and wild-type
-arrestin
2 (pCMV5-
-arrestin 2). Depending on the experiment, cells of
clone 2 were transiently co-transfected with neither or either
wild-type
-arrestin 2 or constitutively active mutant
-arrestin 2 (pCDNA3-
-arrestin CAM). Finally, all cells were transiently
co-transfected with plasmid vector for the S65T point mutation of green
fluorescent protein (pGFP-S65T; CLONTECH) as a
marker for successfully transfected cells. The final concentrations of
each of the plasmid vectors added during transient transfections were
as follows (in ng/ml): Kir3.1, 400; Kir3.4, 400; GRK2, 400;
-arrestin 2, 400; green fluorescent protein, 200. 10 ml of the
transfecting solution was added to ~1-2 × 106
cells in a 100-mm diameter plastic tissue culture dish. The expression levels of the receptor and GRK2 in the stably transfected cells were
measured and are given in Shui et al. (13). A few hours before electrophysiological experiments, 0.02% EDTA solution was used
to remove the adherent cell layer from the dish. The cells were then
centrifuged for 3 min at 100 × g and resuspended in fresh medium on fragments of glass coverslip.
Atrial Cell Isolation--
Adult rats were killed by stunning
and cervical dislocation. Atrial cells were prepared as described
previously (22).
Electrophysiology--
CHO cells were placed in a recording
chamber mounted on a Nikon Diaphot microscope. 470-490-nm light was
used to excite the green fluorescent protein in successfully
transfected cells. The green fluorescent light was passed through a
515-nm filter for observation. Cells with a middle level of green
fluorescence were chosen for study. Experiments were carried out in the
whole cell configuration of the patch clamp technique at room
temperature (22-25 °C). Extracellular solution contained (in
mM): KCl, 140; MgCl2, 1.8; EGTA, 5; HEPES, 5;
pH 7.4. 10 µM ACh was added to the extracellular solution
when required. Pipette solution contained (in mM):
potassium aspartate, 120; KCl, 20; KH2PO4, 1;
MgCl2, 2.8 (free Mg2+, 1.8); EGTA, 5; HEPES, 5;
Na3GTP, 0.1; Na2ATP, 3; pH 7.4. Whole cell
currents were recorded with an Axopatch-1D amplifier and acquired with
pClamp software (Axon Instruments Inc., Foster City, CA).
Currents were filtered at 2 kHz with an 8-pole Bessel filter and
sampled every 1 ms. Decline in currents was fitted with a single
exponential function with a least squares method using SigmaPlot
(Jandel Corp., San Rafael, CA). Statistical tests (one way
analysis of variance) were carried out using SigmaStat (Jandel Corp.).
 |
RESULTS |
Effect of Expression of
-Arrestin 2 on Desensitization of
IK,ACh--
In the present study, as in our previous study
(13), we have recorded iK,ACh in CHO cells transfected with
the m2 muscarinic receptor and the muscarinic K+ channel
(as well as other proteins). In the previous study, the cell-attached
and inside-out configurations of the patch clamp technique were used
and the basic properties of the channel (single channel conductance,
current-voltage relationship, single channel open time, dependence on
receptor and agonist) were the same as those of the muscarinic
K+ channel in heart cells (13). In the present study, the
whole cell configuration of the patch clamp technique was used; 10 µM ACh was applied using a rapid solution changer for 3 min to activate iK,ACh maximally and current was recorded
at a holding potential of
60 mV in a bathing solution containing 140 mM K+. Fig. 1,
A and C, shows examples of iK,ACh
recorded from CHO cells during an application of ACh. Mean traces from
8 cells are shown. In both cases, iK,ACh was activated
and deactivated on application and wash-off of ACh, respectively.
During the 3-min application of ACh, there was a fade of
iK,ACh as a result of short-term desensitization. In the
present study, iK,ACh was recorded from rat atrial cells
under conditions identical to those used for the recording of
iK,ACh from CHO cells in order that the muscarinic K+ channel system reconstructed in CHO cells can be
compared with the native system in heart. Fig. 6A shows a
typical recording of iK,ACh in a rat atrial cell.
iK,ACh in CHO cells was qualitatively similar to that in
rat atrial cells. However, there were differences. The semi-logarithmic
plots in Fig. 1, B and D, show that the fade of
iK,ACh in CHO cells as a result of desensitization was
monoexponential, whereas it can be seen from Fig. 6A that in
rat atrial cells it is biexponential. It is known that in heart cells,
short-term desensitization is comprised of two independent phases: a
fast phase that develops over ~20 s and a slower phase that develops over several minutes (23). Both of these phases are evident in Fig.
6A. It has been shown that the fast phase is the result of a
change in the channel, whereas the slower phase is the result of a
change in the receptor (23, 24). The fast phase of desensitization may
involve a dephosphorylation of the muscarinic K+ channel
(23-26), and a cytosolic protein and a G protein-independent pathway
(27). Alternatively, it may be caused by the nucleotide exchange and
hydrolysis cycle of the G protein (28). Based on the time course of
desensitization (Fig. 1), the fast phase of desensitization observed in
heart cells is absent in CHO cells (perhaps the underlying cellular
machinery is absent) and the desensitization of iK, ACh in
CHO cells is equivalent to the slower phase in heart cells.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of expression of
-arrestin 2 on desensitization of
iK,ACh. A, iK,ACh during 3 min
application of 10 µM ACh (shown by the bar) in
CHO cells transfected with GRK2 only (clone 2). Mean current from 15 cells shown. B, semi-logarithmic plot of the fade of
iK,ACh in A. The difference between the current
during the application of ACh and the calculated asymptote is plotted
on a logarithmic scale against time. The data are fitted with an
exponential function with the time constant shown. C,
iK,ACh in CHO cells transfected with GRK2 and -arrestin
2 (clone 2). Mean current from eight cells shown. D,
semi-logarithmic plot of the fade of iK,ACh in
C. E, mean + S.E. amplitude of desensitization of
iK,ACh in rat atrial cells. The amplitude of
desensitization in rat atrial cells was measured as 100 × [(iK,ACh(20 s) iK,ACh(end))/iK,ACh(20
s)] and the amplitude of desensitization in CHO cells was
measured as 100 × [(iK,ACh(peak) iK,ACh(end))/iK,ACh(peak)], where
iK,ACh(peak), iK,ACh(20 s), and
iK,ACh(end) are the peak current amplitude at the start of
the ACh application and the currents amplitudes 20 s after the
start and at the end of the ACh application, respectively. n
numbers are shown in parentheses. All cells were transfected with the
m2 muscarinic receptor and muscarinic K+ channel.
|
|
Fig. 1, A and C, shows iK,ACh in CHO
cells transfected with (in addition to the receptor and channel) GRK2
alone (Fig. 1A) or GRK2 and
-arrestin 2 (Fig.
1C). As compared with cells transfected with GRK2 alone
(Fig. 1A), in cells transfected with GRK2 and
-arrestin 2 (Fig. 1C), activation and deactivation of iK,ACh was slowed (see below), the peak amplitude of iK,ACh was
not significantly different (p = 0.8) and the fade of
iK,ACh as a result of desensitization was greater. The
amplitude of desensitization (see Fig. 1 legend for measurement) in
four groups of CHO cells is shown in Fig. 1E: (i) CHO cells
not transfected with either GRK2 or
-arrestin 2, (ii) CHO cells
transfected with
-arrestin 2, (iii) CHO cells transfected with GRK2,
and (iv) CHO cells transfected with GRK2 and
-arrestin 2. Desensitization was least in cells not transfected with either GRK2 or
-arrestin 2. Desensitization was significantly greater in cells
transfected with GRK2 alone (p < 0.05), but not with
-arrestin 2 alone (p = 0.3). Desensitization was
greatest in CHO cells transfected with both GRK2 and
-arrestin 2, in
this cell group desensitization was significantly greater than that in
other cell groups (p < 0.05 in each case). In summary,
the results show that desensitization was increased by transfection with GRK2 and
-arrestin 2, but not with
-arrestin 2 alone. For comparison, Fig.
1E also shows the amplitude of the equivalent phase of
desensitization in rat atrial cells (see Fig. 1 legend for measurement).
The semi-logarithmic plots in Fig. 1, B and D,
show that in CHO cells transfected with GRK2 alone (Fig. 1B)
and GRK2 and
-arrestin 2 (Fig. 1D) the fade of
iK,ACh as a result of desensitization occurred with similar
time constants of 69.5 and 53.7 s, respectively (based on
recordings from 15 and eight cells, respectively). The time constants
of desensitization were also similar for CHO cells not transfected with
either GRK2 or
-arrestin 2 (54.5 s; based on recordings from nine
cells) or CHO cells transfected with
-arrestin 2 alone (40.9 s;
based on recordings from eight cells). The time constant of the
equivalent phase of desensitization in rat atrial cells was 144.0 s
(based on recordings from 10 cells).
Effect of Expression of
-Arrestin 2 on Activation and
Deactivation of iK,ACh--
Close inspection of Fig. 1
shows that activation and deactivation of iK,ACh on
application and wash-off of ACh was slowed in CHO cells transfected
with GRK2 and
-arrestin 2 as compared with activation and
deactivation in CHO cells transfected
with GRK2 alone. This is clearly shown by Figs.
2 and
3.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of expression of
-arrestin 2 on activation of
iK,ACh. A, activation of iK,ACh
on application of 10 µM ACh in rat atrial cells
(n = 5) and in CHO cells transfected with GRK2 only
(clone 2; n = 15) or GRK2 and -arrestin 2 (clone 2;
n = 8). The traces shown are mean currents
(from number of cells above) and have been normalized to the peak
current on application of ACh. The application of ACh is shown by the
bar, and the start of application is indicated by the
dashed line. B, mean + S.E. value of the time to
half-peak iK,ACh on application of ACh in rat atrial cells
and in CHO cells transfected without either GRK2 or -arrestin 2 (clone 1), with GRK2 only (clones 1 and 2), with -arrestin 2 only
(clone 1) and with GRK2 and -arrestin 2 (clones 1 and 2).
n numbers are shown in parentheses. All CHO cells were
transfected with the m2 muscarinic receptor and muscarinic
K+ channel.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of expression of
-arrestin 2 on deactivation of
iK,ACh. A, deactivation of iK,
ACh on wash-off of 10 µM ACh in rat atrial cells
(n = 5) and in CHO cells transfected with GRK2 only
(clone 2; n = 15) or GRK2 and -arrestin 2 (clone 2;
n = 8). The traces shown are mean currents
(from number of cells above) and have been normalized to the current at
the end of the application of ACh. B, mean + S.E. value of
the time to half-deactivation of iK,ACh on wash-off of ACh
in rat atrial cells and in CHO cells transfected without either GRK2 or
-arrestin 2 (clone 1), with GRK2 only (clones 1 and 2), with
-arrestin 2 only (clone 1) and with GRK2 and -arrestin 2 (clones
1 and 2). n numbers are shown in parentheses. All
CHO cells were transfected with the m2 muscarinic receptor and
muscarinic K+ channel.
|
|
Fig. 2A compares the activation of iK,ACh on
application of ACh in CHO cells, transfected with GRK2 alone or GRK2
and
-arrestin 2, and rat atrial cells. Mean traces from 5 to 15 cells are shown. Fig. 2B shows the mean time to half-peak
iK,ACh on application of ACh. The time to half-peak
iK,ACh in CHO cells transfected with GRK2 and
-arrestin
2 was significantly longer than in CHO cells transfected with GRK2
alone (p < 0.001), whereas the time to half-peak
iK,ACh in atrial cells was not significantly different from
that in CHO cells transfected with GRK2 alone (p = 0.08). Fig. 2B also shows the mean time to half-peak
iK,ACh in CHO cells not transfected with either GRK2 or
-arrestin 2 and in CHO cells transfected with
-arrestin 2 only,
these data show that the effect of
-arrestin 2 on activation was
independent of the absence or presence of overexpressed GRK2 (they also
show that overexpressed GRK2 had no effect on activation).
The effect of transfection of GRK2 and
-arrestin 2 on deactivation
of iK,ACh in CHO cells is shown in Fig. 3. Fig.
3A compares the deactivation of iK,ACh on
wash-off of ACh in CHO cells, transfected with GRK2 alone or GRK2 and
-arrestin 2, and rat atrial cells. Mean traces from 5 to 15 cells
are shown. Fig. 3B shows the mean time to half-deactivation
of iK,ACh on wash-off of ACh. The time to half-deactivation
of iK,ACh in CHO cells transfected with GRK2 and
-arrestin 2 was significantly longer than in CHO cells transfected with GRK2 alone (p < 0.001), whereas the time to
half-deactivation of iK,ACh in atrial cells was
significantly shorter than in CHO cells transfected with GRK2 alone
(p < 0.001). Fig. 3B also shows the mean
time to half-deactivation of iK,ACh in CHO cells not transfected with either GRK2 or
-arrestin 2 and in CHO cells transfected with
-arrestin 2 only, these data show that the effect of
-arrestin 2 on deactivation was independent of the absence or
presence of overexpressed GRK2 (they also show that overexpressed GRK2
had no effect on deactivation).
Effect of Expression of
-Arrestin 2 on Resensitization of
iK,ACh--
Resensitization of iK,ACh
(i.e. the recovery of iK,ACh from
desensitization) was studied by applying test applications of ACh at
various test intervals after control applications of ACh. Fig.
4 shows typical traces of
iK,ACh in response to control (Fig. 4, A and
C) and test (Fig. 4, B and D)
applications of ACh in CHO cells transfected with GRK2 alone (Fig. 4,
A and B) or GRK2 and
-arrestin 2 (Fig. 4,
C and D). When the test application of ACh was
applied soon after the control application, iK,ACh during
the test application of ACh was smaller than the control as a result of
insufficient time for recovery from the desensitization that developed
during the control application of ACh. As the recovery interval between
the two applications was increased, iK,ACh during the test
application of ACh increased toward the control as a result of
resensitization. The test intervals for full recovery of
iK,ACh in CHO cells transfected with GRK2 alone or GRK2 and
-arrestin 2 were 3 and 10 min, respectively (Fig. 4, B
and D). The time courses of iK,ACh
resensitization in CHO cells transfected with GRK2 alone
(squares) or GRK2 and
-arrestin 2 (circles)
are shown in Fig. 5 from three or four
cells, the peak amplitude of iK,ACh in response to the test
application of ACh has been normalized to the peak amplitude of
iK,ACh in response to the previous control application of
ACh and plotted against the recovery interval between the two
applications. Fig. 5 confirms that resensitization of iK,ACh was slower in CHO cells transfected with both GRK2
and
-arrestin 2 than in CHO cells transfected with GRK2 alone. The time constant of resensitization in the CHO cells transfected with GRK2
alone or GRK2 and
-arrestin 2 was 34 and 232 s,
respectively.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of expression of
-arrestin 2 on resensitization of
iK,ACh. A and B,
iK,ACh during 3 min control (A) and test
(B) applications of 10 µM ACh (shown by
bars) recorded from a CHO cell transfected with GRK2 only
(clone 2). Four test responses are shown superimposed. The peak
currents are indicated by the arrows with the intervals
between control and test applications of ACh. C and
D, iK,ACh recorded from a CHO cell transfected
with GRK2 and -arrestin 2 shown in the same format as A
and B (clone 2). The CHO cells were also transfected with
the m2 muscarinic receptor and muscarinic K+ channel.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 5.
Summary of the effect of expression of
-arrestin 2 on resensitization of
iK,ACh. Mean ± S.E. peak amplitude of
iK,ACh during a test application of ACh (calculated as a
percentage of the peak amplitude of the current during the previous
control application) plotted against the test interval. Data for CHO
cells transfected with GRK2 only (squares; n = 4 cells; clone 2) or GRK2 and -arrestin 2 (circles;
n = 3 cells; clone 2) are shown. The data are fitted
with single exponential functions with time constants of 34 s
(GRK2) and 232 s (GRK2 and -arrestin 2). The CHO cells were
also transfected with the m2 muscarinic receptor and muscarinic
K+ channel.
|
|
Fig. 6 shows resensitization of
iK,ACh in rat atrial cells. A typical trace during a
control application of ACh is shown in Fig. 6A and
superimposed traces of iK,ACh during test applications of
ACh are shown in Fig. 6B. As discussed above, in rat atrial cells, unlike in CHO cells, there are two phases of desensitization of
iK,ACh during a 3-min application of ACh. The two phases
can be seen in Fig. 6, A and B. Resensitization
of iK,ACh will be influenced by both the fast and slower
phases, whereas in this study we are concerned with the slower phase
only. For this reason, resensitization of iK,ACh was
calculated in two ways: (i) peak iK,ACh during the test
application of ACh was expressed as a percentage of peak
iK,ACh during the previous control application of ACh and
plotted against the recovery interval between the two applications; (ii) the amplitude of iK,ACh after the fast phase of
desensitization (20 s after the start of the ACh application) was
expressed as a percentage of the corresponding measurement from the
previous control application of ACh and once again plotted against the recovery interval between the two applications. The time course of
iK,ACh resensitization in rat atrial cells as determined by the two methods is shown in Fig. 6C and is roughly similar.
The time constant of resensitization of iK,ACh in rat
atrial cells was 64 and 75 s for methods i and ii, respectively.
This is intermediate between the time constants of resensitization of
iK,ACh in CHO cells with and without
-arrestin 2 (Fig.
5).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Resensitization of iK,ACh in rat
atrial cells. A and B, iK,ACh
during 3 min control (A) and test (B)
applications of 10 µM ACh (shown by bar)
recorded from a rat atrial cell. Five test responses are shown
superimposed. The peak currents are indicated by the arrows
with the intervals between control and test applications of ACh.
C, mean ± S.E. (n = 3-5) amplitude of
iK,ACh during a test application of ACh (calculated as a
percentage of the amplitude of iK,ACh during the control
application) plotted against the test interval. Both the peak amplitude
of iK,ACh (squares) and the amplitude of
iK,ACh after the fast phase of desensitization (20 s after
the start of the ACh application) are shown. The data are fitted with
single exponential functions with time constants of 64 s (peak
amplitude) and 75 s (amplitude of current after the fast phase of
desensitization).
|
|
Effect of Expression of a Constitutively Active
-Arrestin 2 Mutant on iK,ACh--
Arrestins preferentially bind to the
receptor once the receptor is agonist-bound and has been phosphorylated
by receptor kinase. However, truncation of the C terminus of arrestins
enhances binding to the dephosphorylated receptor, perhaps because the
C terminus via an intramolecular interaction with the N terminus
maintains arrestins in an inactive conformation (29, 30). With the
phosphorylated but not dephosphorylated receptor, arrestins result in a
high affinity agonist-binding state, which has been postulated to
correlate with the desensitization process (31). The C-terminal
truncation mutant,
-arrestin 2-(1-393), is able to result in
such a state even in the case of the dephosphorylated receptor (31).
This suggests that
-arrestin 2-(1-393) is constitutively active and binds regardless of the phosphorylation state of the receptor. Expression of such a constitutively active
-arrestin 2 mutant might
be expected to result in desensitization in the absence of the agonist,
although this has never been tested. Fig.
7A shows iK,ACh recorded from CHO cells transfected with GRK2 only
or GRK2 and the constitutively active
-arrestin 2 mutant. Mean
traces from
7 cells are shown in Fig. 7A. As predicted,
the peak amplitude of iK,ACh was reduced upon transfection
with the constitutively active
-arrestin 2 mutant. Fig.
7B shows the peak amplitude of iK,ACh in CHO
cells transfected with: (i) GRK2 and wild-type
-arrestin 2, (ii)
GRK2 alone, and (iii) GRK2 and the constitutively active
-arrestin 2 mutant. The peak amplitudes of iK,ACh in CHO cells transfected with GRK2 alone or GRK2 and
-arrestin 2 were similar. However, the peak amplitude of iK,ACh was significantly
reduced in CHO cells transfected with GRK2 and the constitutively
active
-arrestin 2 mutant (versus CHO cells transfected
with GRK2 and
-arrestin 2, p < 0.001;
versus CHO cells transfected with GRK2 alone,
p < 0.001).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of expression of a constitutively
active -arrestin 2 mutant on
iK,ACh. A, iK,ACh during a
3-min application of 10 µM ACh (shown by bar)
in CHO cells transfected with GRK2 alone (clone 2; n = 15) or GRK2 and the constitutively active -arrestin 2 mutant (clone
2; n = 7). Mean traces (from number of cells above) are
shown. B, mean + S.E. value of the peak amplitude of
iK,ACh in CHO cells transfected with GRK2 and -arrestin
2, with GRK2 only or GRK2 and the constitutively active -arrestin 2 mutant. n numbers are shown in parentheses.
|
|
 |
DISCUSSION |
In the present study, evidence has been obtained to show that
-arrestin 2 is a multifunctional protein and it affects activation, desensitization, deactivation, and resensitization of
iK,ACh: when
-arrestin 2 was expressed in CHO cells,
activation of iK,ACh on application of ACh was slowed,
short-term desensitization of iK,ACh during the application
of ACh was enhanced, deactivation of iK,ACh on wash-off of
ACh was slowed, and resensitization of iK,ACh after the
wash-off of ACh was slowed.
Short-term Desensitization of iK,ACh--
In the
present study we have studied desensitization of iK,ACh
during a 3-min application of ACh, in CHO cells there was a fade of
iK,ACh with a time constant of 40.9-69.5 s depending on the components transfected (Fig. 1). This is equivalent to the short-term desensitization that develops over minutes in heart cells,
e.g. rat atrial cells as used in the present study.
Kobrinsky et al. (32) have recently studied desensitization
of iK,ACh in neonatal rat atrial cells during a 2-min
application of 100 µM ACh (same phase of desensitization
investigated as in the present study). They have proposed a radical new
hypothesis to explain the desensitization of iK,ACh in the
heart (32). They have suggested that ACh, as well as activating
iK,ACh, activates phospholipase C and this results in the
hydrolysis of phosphatidylinositol bisphosphate and a consequent
decrease in phosphatidylinositol bisphosphate binding to the
muscarinic K+ channel and, thus, channel activity. In part
this hypothesis was based on the observation that addition of the
aminosteriod, U-73122, to inhibit phosphatidylinositol bisphosphate
hydrolysis, abolished the desensitization of iK,ACh
(32). According to this hypothesis the desensitization of
iK,ACh is the result of a change in the channel rather than
the receptor. However, Zang et al. (23) showed that in
guinea pig atrial cells, if the muscarinic K+ channel was
activated by GTP
S, which is known to bypass the receptor and
activate the G protein and thus the channel directly, the phase of
desensitization of iK,ACh that develops over minutes was
abolished, this suggests that this phase of desensitization is a
receptor, rather than channel, phenomenon. Furthermore, Shui et
al. (12) showed that this phase of desensitization of
iK,ACh was observed in whole cell, cell attached, and
perforated outside-out recordings (in which the cytoplasm is retained)
but was lost in inside-out and conventional outside-out recordings (in
which the cytoplasm is lost). It was conjectured that receptor kinase
was lost on loss of the cytoplasm and, consistent with this
explanation, in inside-out recordings desensitization of
iK,ACh was restored on adding exogenous GRK2 (12). This
work suggested that, in heart, this phase of desensitization of
iK,ACh is the result of phosphorylation of the receptor by
receptor kinase (12). Subsequently Shui et al. (13) showed
that in CHO cells transfected with the m2 muscarinic receptor,
muscarinic K+ channel, and GRK2 short-term desensitization
of iK,ACh was comparable to that in heart cells, but
short-term desensitization was lost if the cells were not transfected
with GRK2 or were transfected with a mutant m2 muscarinic receptor
lacking the third intracellular loop containing the phosphorylation
sites. The present study confirms that if CHO cells are not expressed
with GRK2 (with or without
-arrestin 2) then desensitization is
reduced (Fig. 1E).
The present study has shown that transfection of CHO cells with both
GRK2 and
-arrestin 2 rather than GRK2 alone enhances desensitization
of iK,ACh (Fig. 1E). This effect of
-arrestin 2 is dependent on the presence of GRK2, if the cells were not transfected with GRK2, transfection with
-arrestin 2 had no
significant effect (Fig. 1E). The requirement for GRK2 is
consistent with the known property of
-arrestin 2 that it
preferentially binds to receptor kinase-phosphorylated receptor.
-Arrestin 2 is expected to enhance desensitization by uncoupling the
receptor from the G protein and by promoting internalization of the
receptor (see Introduction). The results of Kobrinsky et al.
(32) are not necessarily in disagreement with our hypothesis that the
desensitization is the result of a modification of the receptor rather
than the channel, because U-73122 is known to inhibit m2 muscarinic
receptor internalization (33). It is also possible that in native
atrial cells
-arrestin 2 acts in concert with other mechanisms,
which act directly on the channel, to affect
desensitization. Although we favor the hypothesis that
-arrestin 2 affects desensitization by acting on the receptor, the
possibility that it acts on another component in the pathway cannot be
excluded. Recently, Appleyard et al. (34) have shown that,
in Xenopus oocytes transfected with the rat
opioid
receptor and the muscarinic K+ channel, co-transfection
with a receptor kinase (GRK3 or GRK5) and
-arrestin 2 increased the
desensitization of iK,ACh in the presence of the agonist,
U69,593.
Resensitization of iK,ACh--
This study has shown
that
-arrestin 2 expression slows recovery from short-term
desensitization (resensitization). Resensitization presumably involves
unbinding of receptor kinase and the arrestin from the receptor and
dephosphorylation of the receptor. There are at least two explanations
for the slowing of resensitization with
-arrestin 2 overexpression
(for arguments in favor of overexpression see below): resensitization
may be rate-limited by the dissociation of the arrestin and
overexpression of
-arrestin 2 might slow this dissociation. Another
possibility is that the overexpression of
-arrestin 2 pushes
receptors to a more slowly resensitizing state: for example, in the
absence of
-arrestin 2 overexpression, desensitization-resensitization may involve receptor-G protein uncoupling-recoupling only (possibly fast processes), whereas with
-arrestin 2 overexpression it may involve
internalization-recycling to the membrane (possibly slower processes).
Activation and Deactivation of
iK,ACh--
Overexpression with
-arrestin 2 slowed both
the activation and deactivation of iK,ACh on application
and wash-off, respectively, of ACh (Figs. 2 and 3). Activation of
iK,ACh involves ACh-receptor binding, G protein activation,
and the subsequent activation of the channel by the G protein 
subunits. It is known that binding of a
-arrestin to the receptor
inhibits receptor-G protein interaction (5). Although arrestins
preferentially bind to the agonist-occupied receptor
kinase-phosphorylated receptor, arrestins still bind but with reduced
efficacy to the agonist-unoccupied, dephosphorylated receptor (29).
Perhaps with
-arrestin 2 overexpression, as in the present study,
there is increased interaction of
-arrestin 2 with the
agonist-unoccupied, dephosphorylated receptor and this is manifested as
impaired coupling with the G protein and a consequent slowing of the
activation of iK,ACh on application of ACh. Alternatively, it is possible that
-arrestin 2 has a direct effect on the
muscarinic K+ channel to slow activation of
iK,ACh.
On wash-off of ACh, deactivation of iK,ACh involves the
splitting of GTP by the G protein
subunit, unbinding of the G
protein 
subunits from the channel and reformation of the G
protein trimer. It is possible that interaction of the G protein trimer with the receptor is involved in the deactivation process, perhaps by
stabilizing the trimer. In this case, with
-arrestin 2 overexpression as in the present study, there may be increased
interaction of
-arrestin 2 with the agonist-unoccupied,
dephosphorylated receptor as argued above and this may impair coupling
of the receptor with the G protein and this may slow deactivation of
iK,ACh. Alternatively, it is possible that
-arrestin 2 has a direct effect on the G protein or channel.
Comparison of iK,ACh in Atrial and CHO
Cells--
While a comparison of iK,ACh in atrial and CHO
cells may be useful, such a comparison must be considered cautiously,
because of the absence/presence of additional regulatory components in the two cell types as well as the effects of protein overexpression on
the relative stoichiometries of the different components. Fig. 1E shows that the amplitude of desensitization in rat atrial
cells was comparable to that in CHO cells transfected with GRK2 alone and less than that in CHO cells transfected with GRK2 and
-arrestin 2. Comparison of Figs. 5 and 6 shows that in rat atrial cells the speed
of resensitization (time constant,
, 64 or 75 s depending on
the method of measurement) was intermediate between that in CHO cells
in the absence and presence of
-arrestin 2 (
, 34 and 232 s,
respectively). It is possible that transfection of CHO cells with
-arrestin 2 resulted in overexpression of arrestin, i.e. a level of
-arrestin 2 higher than normal,
especially as CHO cells express endogenous arrestin (35). In the
present study, activation and deactivation of iK,ACh in CHO
cells transfected with or without
-arrestin 2 were slower than in
rat atrial cells (Figs. 2 and 3). Activation and deactivation of
iK,ACh in heterologous expression systems are accelerated
upon transfection with RGS proteins, activators of the GTPase activity
of the G protein
subunit (36), and it is possible that this
difference between rat atrial cells and CHO cells is the result of a
lack of RGS protein in CHO cells.
 |
SUMMARY |
Evidence from this study as well as our previous studies (12, 13,
23) suggests that the short-term desensitization of iK,ACh
both in cardiac cells and in the reconstituted system in CHO cells is
the result of a change in the receptor and, on the basis of the present
study, it is concluded that
-arrestin 2 has the potential
to be involved in this desensitization process. In addition, it is
concluded that
-arrestin 2 has the potential to be
involved in other properties of the channel (activation, deactivation,
and resensitization). However, whether
-arrestin 2 is involved in
regulation of the muscarinic K+ channel in the heart
remains to be elucidated.
 |
FOOTNOTES |
*
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.
¶
To whom correspondence should be sent: School of Biomedical
Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom. Tel.:
44-113-2334298; Fax: 44-113-2334224; E-mail:
m.r.boyett@leeds.ac.uk.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M011007200
 |
ABBREVIATIONS |
The abbreviations used are:
iK, ACh, cardiac muscarinic K+ current;
CHO, Chinese hamster ovary;
GTP
S, guanosine
5'-3-O-(thio)- triphosphate;
Ach, acetylcholine.
 |
REFERENCES |
1.
|
Boyett, M. R.,
Kodama, I.,
Honjo, H.,
Arai, A.,
Suzuki, R.,
and Toyama, J.
(1995)
Cardiovasc. Res.
29,
867-878[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Boyett, M. R.,
Kirby, M. S.,
Orchard, C. H.,
and Roberts, A.
(1988)
J. Physiol.
404,
613-635[Abstract]
|
3.
|
Nishimura, M.,
Habuchi, Y.,
Hiromasa, S.,
and Watanabe, Y.
(1988)
Am. J. Physiol.
255,
H7-H14[Abstract/Free Full Text]
|
4.
|
Reuveny, E.,
Slesinger, P. A.,
Inglese, J.,
Morales, J. M.,
Iniguez-Lluhi, J. A.,
Lefkowitz, R. J.,
Bourne, H. R.,
Jan, Y. N.,
and Jan, L. Y.
(1994)
Nature
370,
143-146[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Lefkowitz, R. J.
(1998)
J. Biol. Chem.
273,
18677-18680[Free Full Text]
|
6.
|
Haga, T.,
Haga, K.,
and Kameyama, K.
(1994)
J. Neurochem.
63,
400-412[Medline]
[Order article via Infotrieve]
|
7.
|
Kwatra, M. M.,
Leung, E.,
Maan, A. C.,
McMahon, K. K.,
Ptasienski, J.,
Green, R. D.,
and Hosey, M. M.
(1987)
J. Biol. Chem.
262,
16314-16321[Abstract/Free Full Text]
|
8.
|
Martin, P.
(1983)
Am. J. Physiol.
245,
H584-H591[Abstract/Free Full Text]
|
9.
|
Martin, P.,
Levy, M. N.,
and Matsuda, Y.
(1982)
Am. J. Physiol.
243,
H219-H225[Medline]
[Order article via Infotrieve]
|
10.
|
Boyett, M. R.,
and Roberts, A.
(1987)
J. Physiol.
393,
171-194[Abstract]
|
11.
|
Honjo, H.,
Kodama, I.,
Zang, W.-J.,
and Boyett, M. R.
(1992)
Am. J. Physiol.
263,
H1779-H1789[Abstract/Free Full Text]
|
12.
|
Shui, Z.,
Boyett, M. R.,
Zang, W.-J.,
Haga, T.,
and Kameyama, K.
(1995)
J. Physiol.
487,
359-366[Abstract]
|
13.
|
Shui, Z.,
Khan, I. A.,
Tsuga, H.,
Haga, T.,
and Boyett, M. R.
(1998)
J. Physiol.
507,
325-334[Abstract/Free Full Text]
|
14.
|
Pals-Rylaarsdam, R.,
Gurevich, V. V.,
Lee, K. B.,
Ptasienski, J. A.,
Benovic, J. L.,
and Hosey, M. M.
(1997)
J. Biol. Chem.
272,
23682-23689[Abstract/Free Full Text]
|
15.
|
Pals-Rylaarsdam, R.,
and Hosey, M. M.
(1997)
J. Biol. Chem.
272,
14152-14158[Abstract/Free Full Text]
|
16.
|
Feron, O.,
Smith, T. W.,
Michel, T.,
and Kelly, R. A.
(1997)
J. Biol. Chem.
272,
17744-17748[Abstract/Free Full Text]
|
17.
|
Boyett, M. R.,
Shui, Z.,
Khan, I.,
and Dobrzynski, H.
(1999)
J. Physiol.
521,
23
|
18.
|
Krupnik, J. G.,
and Benovic, J. L.
(1998)
Annu. Rev. Pharamacol. Toxicol.
38,
289-319[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Lohse, M. J.
(1995)
Trends Cardiovasc. Med.
5,
63-68[CrossRef]
|
20.
|
Kameyama, K.,
Haga, K.,
Haga, T.,
Kontani, K.,
Katada, T.,
and Fukada, Y.
(1993)
J. Biol. Chem.
268,
7753-7758[Abstract/Free Full Text]
|
21.
|
Tsuga, H.,
Kameyama, K.,
Haga, T.,
Kurose, H.,
and Nagao, T.
(1994)
J. Biol. Chem.
269,
32522-32527[Abstract/Free Full Text]
|
22.
|
Harrison, S. M.,
McCall, E.,
and Boyett, M. R.
(1992)
J. Physiol.
449,
517-550[Abstract]
|
23.
|
Zang, W.-J., Yu, X.-J.,
Honjo, H.,
Kirby, M. S.,
and Boyett, M. R.
(1993)
J. Physiol.
464,
649-679[Abstract]
|
24.
|
Shui, Z.,
Boyett, M. R.,
and Zang, W.-J.
(1997)
J. Physiol.
505,
77-93[Abstract]
|
25.
|
Kim, D.
(1993)
Circ. Res.
73,
89-97[Abstract]
|
26.
|
Kim, D.
(1991)
J. Physiol.
437,
133-155[Abstract]
|
27.
|
Hong, S.-G.,
Pleumsamran, A.,
and Kim, D.
(1996)
Am. J. Physiol.
270,
H526-H537[Abstract/Free Full Text]
|
28.
|
Chuang, H. H., Yu, M.,
Jan, Y. N.,
and Jan, L. Y.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11727-11732[Abstract/Free Full Text]
|
29.
|
Gurevich, V. V.,
Dion, S. B.,
Onorato, J. J.,
Ptasienski, J.,
Kim, C. M.,
Sterne-Marr, R.,
Hosey, M. M.,
and Benovic, J. L.
(1995)
J. Biol. Chem.
270,
720-731[Abstract/Free Full Text]
|
30.
|
Gurevich, V. V.,
and Benovic, J. L.
(1993)
J. Biol. Chem.
268,
11628-11638[Abstract/Free Full Text]
|
31.
|
Gurevich, V. V.,
Pals-Rylaarsdam, R.,
Benovic, J. L.,
Hosey, M. M.,
and Onorato, J. J.
(1997)
J. Biol. Chem.
272,
28849-28852[Abstract/Free Full Text]
|
32.
|
Kobrinsky, E.,
Mirhahi, T.,
Zhang, H.,
Jin, T.,
and Logothetis, D. E.
(2000)
Nat. Cell Biol.
2,
507-514[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Thompson, A. K.,
Mostafapour, S. P.,
Denlinger, L. C.,
Bleasdale, J. E.,
and Fisher, S.
(1991)
J. Biol. Chem.
266,
23856-23862[Abstract/Free Full Text]
|
34.
|
Appleyard, S. M.,
Celver, J.,
Pineda, V.,
Kovoor, A.,
Wayman, G. A.,
and Chavkin, C.
(1999)
J. Biol. Chem.
274,
23802-23807[Abstract/Free Full Text]
|
35.
|
Santini, F.,
Penn, R. B.,
Gagnon, A. W.,
Benovic, J. L.,
and Keen, J. H.
(2000)
J. Cell Sci.
113,
2463-2470[Abstract/Free Full Text]
|
36.
|
Saitoh, O.,
Kubo, Y.,
Miyatani, Y.,
Asano, T.,
and Nakata, H.
(1997)
Nature
390,
525-529[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.