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J. Biol. Chem., Vol. 276, Issue 31, 28873-28880, August 3, 2001
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§,§,,
From the Institut für Physiologie,
Ruhr-Universität Bochum, D-4480 Bochum, Germany and the
¶ Department of Pharmacology II, Graduate School and Faculty of
Medicine, Osaka University, Osaka 565-0871, Japan
Received for publication, March 15, 2001, and in revised form, May 17, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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K+ channels composed of
G-protein-coupled inwardly rectifying K+ channel
(GIRK) (Kir3.0) subunits are expressed in cardiac, neuronal, and
various endocrine tissues. They are involved in inhibiting excitability
and contribute to regulating important physiological functions such as
cardiac frequency and secretion of hormones. The functional cardiac
(K(ACh)) channel activated by
Gi/Go-coupled receptors such as muscarinic
M2 or purinergic A1 receptors is supposed to be
composed of the subunits GIRK1 and GIRK4 in a heterotetrameric (2:2)
fashion. In the present study, we have manipulated the subunit
composition of the K(ACh) channels in cultured atrial
myocytes from hearts of adult rats by transient transfection of vectors
encoding for GIRK1 or GIRK4 subunits or GIRK4 concatemeric constructs
and investigated the effects on properties of macroscopic
IK(ACh). Transfection with a GIRK1 vector did not cause any
measurable effect on properties of IK(ACh), whereas transfection with a GIRK4 vector resulted in a complete loss in desensitization, a reduction of inward rectification, and a slowing of
activation. Transfection of myocytes with a construct encoding for a
concatemeric GIRK42 subunit had similar effects on
desensitization and inward rectification. Following transfection of a
tetrameric construct (GIRK44), these changes in properties
of IK(ACh) were still observed but were less pronounced.
Heterologous expression in Chinese hamster ovary cells and human
embryonic kidney 293 cells of monomeric, dimeric, and tetrameric GIRK4
resulted in robust currents activated by co-expressed A1
and M2 receptors, respectively. These data provide strong
evidence that homomeric GIRK4 complexes form functional
G GIRK1 channels
contribute to parasympathetic reduction of cardiac frequency and reduce
excitability of central neurons and various endocrine cells (for
reviews, see Refs. 1-4). The cardiac channel complex is supposed to be
composed of GIRK1 (Kir3.1) and GIRK4 (Kir3.4) subunits in a
heterotetrameric (2:2) fashion (5), whereas neuronal channels contain,
apart from GIRK1, the subunits GIRK2 or GIRK3. Recent evidence
suggests, however, that GIRK4 is also expressed in the brain (6).
According to the initial concept, GIRK1 subunits without co-expressed
GIRK2, GIRK3, or GIRK4 subunits do not co-assemble and are not
translocated to the membrane, whereas GIRK2, GIRK3, and GIRK4 are
necessary for subunit assembly and translocation but do not form
functional homomeric channel without GIRK1 (7). However, more recently, it has been shown that in atrial myocytes, a large fraction of GIRK4
subunits exist as homomultimers (8). Moreover, in neurons, GIRK2 and
GIRK3 subunits have been localized without GIRK1 protein (9, 10),
suggesting that monomeric complexes devoid of GIRK1 may form functional channels.
Cardiac GIRK channels are activated by various heptahelical receptors
coupled to heterotrimeric G-proteins of the pertussis toxin-sensitive
class (Gi/Go), of which M2AChR is
the paradigmatic example. Receptor activation results in dissociation
of the heterotrimeric G-protein complex into its Following activation by exposure to ACh, atrial IK(ACh)
shows a peculiar type of desensitization, i.e. a partial
decay in current with a half-time of a few seconds (11-13), usually
referred to as "acute" or "fast" desensitization. This
component of desensitization is assumed to be localized downstream of
the receptor. The mechanism(s) underlying this acute desensitization, however, so far has not been resolved.
In the present study, GIRK4 subunits and GIRK4 concatemeric constructs
were overexpressed in cultured adult rat atrial myocytes by transient
transfection. Overexpression of GIRK4 resulted in ACh-activated
currents that completely lacked fast desensitization. Strong inward
rectification, a key property of GIRK currents, resulting from a block
of outward current flow by intracellular cations, particularly
polyamines, was reduced in GIRK4-transfected myocytes as compared with
native cells. Qualitatively, this was confirmed by expressing GIRK4
subunits in CHO cells and HEK293 cells, which are assumed to be devoid
of intrinsic GIRK1 subunits. These findings support the notion that
important physiological properties, such as inward rectification and
desensitization, depend on the subunit composition of the channel
complex. Moreover, in atrial myocytes GIRK channel complexes with
subunit compositions different from the
GIRK42-GIRK12 stoichiometry might contribute to
macroscopic IK(ACh).
Isolation and Culture of Atrial Myocytes--
Experiments were
performed with local ethics committee approval. Wistar Kyoto rats of
either sex (around 200 g) were anesthetized by i.v injection of
urethan (1 g/kg). The chest was opened, and the heart was removed and
mounted on the cannula of a sterile Langendorff apparatus for coronary
perfusion at constant flow. The method of enzymatic isolation of atrial
myocytes has been described elsewhere (e.g. Ref. 12). The
culture medium was fetal calf serum-free bicarbonate-buffered M199
(Life Technologies, Inc., Karlsruhe, Germany) containing
gentamycin (25 µg/ml, Sigma Deisenhofen, Germany) and kanamycin (25 µg/ml, Sigma). Cells were plated at a low density (several thousand
cells per dish) on 36-mm culture dishes. Medium was changed 24 h
after plating and then every second day. Myocytes were used
experimentally from day 0 until day 5 after isolation. No effects of
time in culture were found for the key experiments.
Solutions and Chemicals--
For the patch clamp measurements,
an extracellular solution of the following composition was used: 120 mM NaCl, 20 mM KCl, 0.5 mM
CaCl2, 1.0 mM MgCl2, 10.0 mM Hepes/NaOH, pH 7.4. The solution for filling the
patch-clamp pipettes for whole cell voltage clamp experiments contained
110 mM potassium-aspartate, 20 mM KCl,
5.0 mM NaCl, 1.0 mM MgCl2, 2.0 mM Na2ATP, 2.0 mM EGTA, 0.01 mM GTP, 10.0 mM Hepes/KOH, pH 7.4. Standard
chemicals were from Merck (Darmstadt, Germany). EGTA, Hepes, MgATP, Ado
GTP, and ACh-chloride were from Sigma.
Current Measurement--
Membrane currents were measured using
whole-cell patch clamp. Pipettes were fabricated from borosilicate
glass and were filled with the solution listed above (direct
current resistance, 4-6 M Rat GIRK4 Constructs Encoding for Dimeric and Tetrameric
Subunits--
To obtain the different GIRK4 constructs, we amplified
the rat cDNA using different polymerase chain reaction primers to
attach restriction sites for further coupling.
The following constructs were amplified:
A,
KpnI-rGIRK4-(Met1-Met419)-(CAA)x5-EcoRV;
B,
EcoRV-(CAG)x5-rGIRK4-(Met1-Met419)-(CAA)x5-XhoI;
C,
XhoI-(CAG)x5-rGIRK4-(Met1-Met419)-(CAA)x5-XbaI;
D,
EcoRV-(CAG)x5-rGIRK4-(Met1-Met419)-(CAA)x5-XbaI;
E,
XbaI-(CAG)x5-RGIRK4-(Met1-Met419)-TGA-ApaI;
and F,
EcoRV-(CAG)x5-rGIRK4-(Met1-Met419)-TGA-ApaI.
To construct the GIRK4 dimer, GIRK4-A was
cut using the restriction enzymes KpnI and EcoRV
to ligate in the vector pcDNA3 (Invitrogen) using the same sites
and opened after ligation with EcoRV and ApaI.
F was ligated into this vector using the corresponding
restriction sites. The resulting tandem clone has the following amino
acid sequence:
kir3.41-419-QQQQQ-DI-QQQQQ-kir3.41-419,
(pGIRK4)2. To obtain the GIRK4 tetramer,
pcDNA3-GIRK4-A-E was digested using EcoRV
and XhoI, and GIRK4-B was inserted.
pcDNA3-GIRK4-A-B-E was further digested with
XhoI and XbaI, and GIRK4-C was
ligated using the corresponding restriction sites. The amino acid
sequence of the tetramer pcDNA3-GIRK4-A-B-C-E was
GIRK41-419-QQQQQ-DI-QQQQQ-GIRK41-419-QQQQQ-LE-QQQQQ-kir3.41-419-QQQQQ-SR-QQQQQ-GIRK41-419,
(pcDNA-GIRK44). All constructs were sequenced to verify
the nucleotide sequence.
Transfection of CHO and HEK293 Cells--
One day after the
inoculation of HEK293 cells or CHO cells, 3 µg of each GIRK clone was
transfected into the cells on a Petri dish (9-cm diameter) with
LipofectAMINE Plus reagent (Life Technologies, Inc.) according
to the manufacturer's protocol. CHO cells were co-transfected with
a pSV-SPORT1-A1R vector encoding for a rat brain
A1AdoR (kindly provided by Dr. A. Karschin,
Göttingen, Germany). HEK293 cells were co-transfected with a
pcDNA3-M2AChR vector, encoding a human
M2AChR. For identification of transfected cells the
reporter pIRES-EGFP vector (CLONTECH, 1.0 µg/plate) was co-transfected. Electrophysiological recordings were
made on days 3 and 4 posttransfection. Time-matched EGFP-positive cells expressing the A1AdoR or M2AChR receptor
without GIRK subunits or with GIRK1 only served as controls.
Immunoblot Detection of GIRK4 Monomers and Oligomers in HEK293
Cells--
After 2 days, the cells were rinsed twice with 10 ml of
phosphate-buffered saline and collected with 1 ml of a preparation buffer (20 mM Hepes/NaOH (pH 7.4), 1 mM EDTA,
0.5 mM EGTA, 1 mM dithiothreitol, 150 mM NaCl, 2% (w/v) Triton X-100, 1% (w/v) cholate, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml each of
pepstatin, leupeptin, and chymostatin). The cell suspension was
sonicated using a TOMY ultrasonic disruptor (UD-201, Tokyo, Japan) and
centrifuged at 1000 × g for 5 min. The supernatant (3 µl) was loaded onto SDS-polyacrylamide (11%) gels and transferred to
a polyvinylidene difluoride membrane. Immunoblotting was carried out as
described previously (9). Briefly, the membrane was incubated with a primary antibody against GIRK4 (aG4N-10) raised in rabbit against a
synthetic peptide, DSRNAMNQDMEIGV, corresponding to the amino acids
4-17 of GIRK4 (14) at a concentration of 0.5 µg/ml at 4 °C
overnight. After extensive washing, the membranes were incubated with a
horseradish peroxidase-conjugated anti-rabbit antibody (1:1000) for
1 h at room temperature. The immunoreactive signals were developed
with a SuperSignal chemiluminescent substrate (Pierce) and exposed to
Hyperfilm ECL for 5 s (Amersham Pharmacia Biotech).
Transfection of Atrial Myocytes--
Following isolation,
myocytes were cultured overnight to allow for attachment. For
transfection of atrial myocytes the following vectors were used: the
reporter pIRES-EGFP vector (CLONTECH, 1.0 µg/plate), pcDNA-GIRK1, pcDNA-GIRK4,
pcDNA-GIRK42, pcDNA-GIRK44, and
pSV-SPORT1-A 1R (0.4 µg/plate). Transfection was
performed by means of LipofectAMINE Plus reagent (Life Technologies,
Inc.) according to the manufacturer's instructions.
Electrophysiological recordings were made on days 3 and 4 after
transfection. Transfected cells were identified using epifluorescence
of EGFP (excitation wavelength, 470 nm). Time-matched EGFP-positive
cells transfected with the reporter vector only served as controls.
Statistical Analysis--
Student's t test was
applied for the analysis of the results; differences at
p < 0.05 were considered statistically significant.
Immunoblot Detection of GIRK4 Tandem Constructs in HEK293
Cells--
Lysates of transfected HEK293 cells were analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotted. Antibodies against amino acids 4-17 of rat GIRK4 recognized proteins of ~45, ~90, and ~180 kDa in cells transfected with
pcDNA-GIRK44, pcDNA-GIRK42, and
pcDNA-GIRK4, respectively (Fig.
1). Because transfection rates using
LipofectAMINE methodology in atrial myocytes in terms of EGFP-positive
cells were usually less than 5%, corresponding blots to verify
expression of these proteins in myocyte cultures could not be
produced.
Transfection of Atrial Myocytes with GIRK4 Removes Rapid
Desensitization of IK(ACh)--
In native atrial myocytes,
IK(ACh), upon activation by rapid exposure to ACh at
concentrations
Fig. 2 compares representative sample
traces of ACh-induced (10 µM) inward currents recorded
from a control (EGFP-positive) myocyte (Fig. 2A), a myocyte
transfected with pcDNA-GIRK1 (Fig. 2B), a myocyte
co-transfected with pcDNA-GIRK1/pcDNA-GIRK4 (Fig. 2C), and a myocyte transfected with the pcDNA-GIRK4
vector (Fig. 2D). Whereas GIRK1 and GIRK1/GIRK4 expression
did not seem to affect the kinetic properties of IK(ACh), in the GIRK4-transfected cell, the current throughout exposure to ACh
remained constant, with no sign of desensitization. To qualitatively
assess the amount of fast desensitization, quasi-steady-state currents
(at 30 s after changing to ACh-containing solution) normalized to
peak inward current (for control myocytes) or current level at
t = 1 s (for GIRK4-transfected cells) have been
compared. The summarized data in Fig. 2E demonstrate that
fast desensitization of IK(ACh) was completely abolished in
myocytes transfected with the GIRK4 vector, whereas no significant difference was found between controls and myocytes overexpressing GIRK1
or GIRK1/GIRK4, respectively. Surprisingly, current densities of
IK(ACh) were not significantly different in the groups of
myocytes subject to the different transfection protocols.
Fig. 3 illustrates that apart from the
removal of acute desensitization, GIRK4 overexpression resulted in a
slowing of activation upon fast agonist application. The mean time
constant of activation in this series of experiments was increased from
about 300 ms to 750 ms. Because a slowing of the rise time of
IK(ACh) per se results in a decrease or blunting
of the fast desensitizing component (13, 16), it is conceivable
that the absence of desensitization in GIRK4-overexpressing cells
reflects a consequence of the slower rise time. Although the
mechanism(s) underlying fast desensitization in the system under study
is not understood, there is strong evidence that it reflects a
phenomenon related to a signaling element downstream of the receptor,
rather than the receptor itself. The major arguments against receptor
desensitization come from the heterologous nature of fast
desensitization. Two experimental protocols demonstrating the
independence of desensitization and its removal by GIRK4 overexpression
on the species of the activating receptor are illustrated in Figs.
4 and
5.
Fig. 4A shows membrane currents recorded from a
representative (control) myocyte. After a reference current had been
elicited by ACh (10 µM, which yields the maximum
IK(ACh) available in a given cell, thus reflecting the
total population of available channels), a saturating concentration of
Ado (100 µM) was applied. In line with previous reports,
the maximum current that could be activated by Ado via
A1AdoR amounted to about 30% of peak IK(ACh)
elicited by a saturating concentration of ACh due to a lower membrane
density of A1R as compared with M2AChR (13). In
the presence of Ado, superimposed pulses of ACh resulted in inward
currents, the total amplitude of which was smaller than the amplitude
of IK(ACh) in the absence of Ado. This occlusive,
subadditive behavior, first described by Kurachi et al. (11)
and confirmed for other receptor combinations (17, 18), results from
fast desensitization. The Ado-induced current itself does not show
desensitization in terms of a distinct relaxation subsequent to
activation. However, the current desensitizes during its slow onset,
reflecting the heterologous nature of fast desensitization. A
representative result from a GIRK4-transfected myocyte is illustrated
in Fig. 4B. The current in the presence of ACh and Ado
matches the current amplitude of the current evoked by the saturating
[ACh], i.e. exposure to Ado did not cause heterologous
desensitization. The difference between GIRK4-transfected myocytes and
controls was highly significant, as confirmed by the summarized data
(Fig. 4C; p < 0.02;
n = 6). Both fast desensitization and its removal by
GIRK4 overexpression are not limited to currents activated by
stimulation of the M2AChR. Fig. 5 illustrates the effect of overexpressing the A1AdoR. In Fig. 5A (control),
in line with Fig. 4, the maximum current induced by a saturating
concentration of Ado (100 µM) was about 30% of peak
IK(ACh) elicited by 20 µM ACh and never
showed a fast desensitizing component. In Fig. 5B, the same
protocol was applied to a myocyte transfected with a vector encoding
for the A1AdoR. As shown previously, in about 70%
of these cells, Ado-induced IK(ACh) was larger than
ACh-induced IK(ACh). Moreover, the Ado-induced current
showed a prominent desensitizing component that was never seen in
native myocytes (13) If, as shown in Fig. 5C, myocytes were
co-transfected with the vectors encoding for A1AdoR and
GIRK4, the majority of measurements yielded Ado-induced currents that
were larger than ACh-induced currents. However, desensitization was
absent, underscoring independence of desensitization and its removal
by GIRK4 overexpression on receptor species.
Previously, it has been shown that the speed and amount of acute
desensitization are increased at positive membrane potentials (19),
which can be considered as additional evidence in support of the notion
that it represents a phenomenon related to the channel. As shown in
Fig. 6A, in a control myocyte,
desensitization is more pronounced for outward as compared with inward
IK(ACh) (see legend for experimental details), whereas in
GIRK4-transfected myocytes, desensitization was lacking at both
membrane potentials (Fig. 6B). This observation, which is
representative of five time-matched GIRK4-transfected and control
myocytes, demonstrates that desensitization is genuinely removed rather
than altered in its voltage dependence.
Inward Rectification of Atrial IK(ACh) Is Reduced by
GIRK4 Transfection--
GIRK channels are characterized by their
strong inward-rectifying properties. Inward rectification of these
channels reflects a block by endogenous intracellular cations, in
particular by polyamines (spermine and spermidine) (20, 21). A
comparison of current-voltage relations obtained by voltage ramps from
-100 to +60 mV reveals a reduction in inward rectification in
GIRK4-transfected as compared with control myocytes (Fig.
7, A and B). To
statistically compare inward rectification in the two groups of
myocytes, ratios of current at 0 and -100 mV were calculated from I/V
curves of individual cells and summarized in Fig. 7C. This
qualitative assessment yields a highly significant difference in
inward-rectifying properties between the two groups. No difference was
found if data from native (i.e. nontransfected) myocytes and
myocytes transfected with the EGFP vector only were compared (not
shown).
Effects of GIRK4 Transfection on Atrial IK(ACh) Are
Mimicked by Concatemeric GIRK42 and
GIRK44--
The data presented so far demonstrate that
transfection of atrial myocytes with a vector encoding for the GIRK4
subunit affects key properties of macroscopic IK(ACh), suggesting that functional channel complexes with a subunit composition different from the native GIRK channel population are formed. To obtain
further information on this issue, myocytes were transfected with
concatemeric GIRK4 constructs (GIRK42 and
GIRK44). The results are summarized in Fig.
8. Panels A and B
show representative current recordings; summarized data on fast
desensitization and inward rectification are shown in panels
B and C, respectively. Currents recorded from myocytes
transfected with pcDNA-GIRK42 were indistinguishable from currents recorded from myocytes overexpressing the GIRK4 monomer,
i.e. they lacked fast desensitization and inward
rectification was significantly reduced. In the group of myocytes
transfected with the tetrameric construct
(pcDNA-GIRK44), fast desensitization and inward
rectification of IK(ACh) showed an intermediate behavior
between control, GIRK4-transfected, and GIRK42-transfected
groups.
Expression of GIRK4 Constructs in CHO Cells--
The
data presented thus far suggest that GIRK4 homomeric complexes are
functional channels with properties different from the native channel
population that determines macroscopic IK(ACh), although
other interpretations are possible. It is conceivable, for example,
that the GIRK4 subunits overexpressed in atrial myocytes interfere in
an unknown fashion with the signaling pathway, causing the changes in
macroscopic IK(ACh) described above. We therefore studied
whether heterologous expression of GIRK4 homo- and tetramers in
combination with a Gi/o-coupled receptor in principle
results in agonist-activation of GIRK currents in two different cell
lines (CHO and HEK293) frequently used as mammalian expression systems.
Both cell lines are assumed to be devoid of intrinsic GIRK subunits. To
provide a receptor for activation of the signaling pathway, in CHO
cells an A1AdoR was co-expressed. This was preferred over
the M2AChR, because activation of the latter in CHO cells
causes a novel long lasting heterologous desensitization not present in
cardiac myocytes (22). Cells transfected with pSV-SPORT1-A1R only served as controls. As positive
controls, cells were transfected (apart from the A1
receptor) with pcDNA-GIRK1 or pcDNA-GIRK4 (see under
"Experimental Procedures"). In corresponding experiments on HEK293
cells, a rat M2AChR was used for activation of expressed
GIRK currents.
The principle question to be addressed by this series of
experiments was whether expression of monomeric or tetrameric GIRK4 results in receptor-activated GIRK currents in a cell line devoid of
intrinsic GIRK subunits. Current amplitudes in this expression system
for a given set of transfection variables were more variable than in
atrial myocytes, and fast desensitization was intrinsically weak.
Therefore, reliable information on this issue was not available from
this series of experiments.
Fig. 9 compares representative responses
of CHO cells to A1 receptor stimulation by 100 µM Ado. Whereas cells transfected with vectors encoding
for GIRK1 and A1AdoR did not respond to Ado with a
measurable change in whole cell current (Fig. 9A), in cells
co-expressing the A1-receptor and GIRK1/GIRK4, exposure to
Ado, as expected, resulted in activation of sizable inward rectifying
currents (B). Robust Ado-induced currents were also routinely recorded from cells expressing GIRK4 monomers (Fig. 9C) and tetramers (D). Inward rectification of
GIRK4 currents was significantly less pronounced as compared with
currents recorded from GIRK1/GIRK4-transfected cells. This is
illustrated by the sample recordings of current voltage relations and
the summarized data (F). Qualitatively similar data were
obtained in HEK293 cells expressing the GIRK4 constructs plus the
M2AChR (data not shown). Thus, at least with regard to
inward rectification, receptor-activated GIRK currents in the two cell
lines mimic those currents observed in myocytes transfected with the
same subunits. This supports the idea that the changes in properties of
IK(ACh) caused by GIRK4 overexpression are due to formation
of homomeric channels rather than an indirect effect at some stage of
the signaling pathway.
The cardiac K(ACh) channel that is expressed
predominantly in supraventricular tissue of the heart but also, at a
lower level, in ventricular myocytes (23) is supposed to represent a
heterotetrameric complex of GIRK1 and GIRK4 (24). Evidence has been
provided that binding of G Initially, it was assumed that homomeric GIRK1 channels represented the
atrial K(ACh) channel (31). This resulted from an intrinsic
GIRK subunit (XIR), homologous to GIRK4 of the oocyte expression system
(32). According to the current concept, GIRK1 subunits do not assemble
to form functional channels (7). Assembly and membrane translocation
require the expression of GIRK4 or a related neuronal type of subunit.
Moreover, in GIRK4 knockout mice, a concomitant loss of GIRK1 protein
has been observed (7), suggesting a role of GIRK4 in controlling expression of GIRK1. Only small currents were measured in
Xenopus oocytes expressing wild type GIRK4 alone, whereas
large currents could be recorded if the GIRK4 subunit contained a point
mutation (S143F) (33). Other authors described robust macroscopic
G In bovine atria, a substantial fraction of GIRK4 protein
exists as homotetrameric complex (8). The physiological significance of
this finding, however, remained unknown. Because the present data
clearly demonstrate sizable whole cell currents in CHO cells expressing
monomeric, dimeric, and tetrameric GIRK4 and a suitable receptor,
intrinsic GIRK4 homotetramers are likely to contribute to macroscopic
IK(ACh) in atrial myocytes.
Fast desensitization is a key property of atrial
IK(ACh). Its heterologous nature between various receptors
(11, 12, 17) provided the major argument that this component of
agonist-dependent decay in current is unlikely to reflect
receptor desensitization or down-regulation, which is common to
many, if not all, G-protein-coupled receptors (36-38). Desensitization
of the M2AChR in the system studied here requires much
longer periods of exposure to an agonist than were used in the present
study. Moreover, reversibility is much slower (16). Various mechanisms
underlying fast desensitization have been proposed so far, such as a
dephosphorylation of the channel (39) or a nonidentified component of
the signaling pathway (40). More recently, it was proposed to reflect
depletion of phosphatidylinositol 4,5-bisphosphate due to simultaneous
activation of a Gq-coupled M3 receptor
activating phospholipase C (41). This is contradictory to the
heterologous nature of fast desensitization. Moreover, recent evidence
suggested that depletion of phosphatidylinositol 4,5-bisphosphate
following stimulation of intrinsic PLC-coupled receptors results in
inhibition of IK(ACh) that is slower than fast
desensitization by at least one order of magnitude (15). Some of the
properties of fast desensitization can be accounted for by the kinetics
of the G-protein cycle (42). The present data, however, support the
notion that fast desensitization reflects a property of the channel
complex. Either it is genuinely absent in GIRK4 homomeric channel
complexes or, alternatively, desensitization of the current carried by
these complexes proceeds during the activation phase, which is
significantly slowed as compared with the native current (compare Fig.
3). This can be formally modeled by a scheme in which desensitization
is linked to activation of the channel complex by
G
gated ion channels and that kinetic
properties of GIRK channels, such as activation rate,
desensitization, and inward rectification, depend on subunit composition.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and 
subunits. In turn, the subunits interact with the GIRK subunits
in a membrane-delimited fashion, causing an increase in open-state
probability of the channel complex.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
). Currents were measured by means
of a patch clamp amplifier (List LM/EPC 7, Darmstadt, Germany). Signals
were analog filtered (corner frequency, 1-3 KHz), digitally sampled at
5 KHz and stored on a computer equipped with a hardware/software
package (ISO2, MFK, Frankfurt/Main, Germany) for voltage control and
data acquisition. Experiments were performed at ambient temperature
(22-24 °C). Cells were voltage-clamped at -90 mV, i.e.
negative to EK, resulting in inward K+
currents. Current-voltage relations were determined by means of voltage
ramps between -120 and +60 mV. Rapid superfusion of the cells for
application and withdrawal of different solutions was performed by
means of a solenoid-operated flow system that permitted switching
between up to six different solutions (t1/2 
100 ms). Performance of this system was dependent on the positioning of the
outlet tube in relation to the cell studied. This was routinely optimized by measuring the time course of the blocking action of
Ba2+ on IK(ACh).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Detection of GIRK4 concatemers expressed in
HEK293 cells. Immunoblots were produced from transfected HEK293
cells as described under "Experimental Procedures." Nontransfected
cultures served as controls.
1 µM, shows various components of
desensitization (12). The acute component, not related to the
activating receptor has a half-time on the order of magnitude of 5 s and is heterologous (15). Its magnitude varies in individual cells,
and it is affected by the experimental conditions, such as rise time of
the agonist concentration, which depends on the superfusion device,
temperature, or density of functional receptors (12;13;16). In order to
separate fast desensitization from receptor desensitization, exposures to ACh were usually limited to <60 s. For simplicity, in the following experiments, the current level reached after 30 s was
considered as quasi-steady-state current (15). As shown in that study, complete recovery from fast desensitization following washout of ACh
takes less than 30 s. Thus, apart from its fast onset, acute
desensitization is defined by its rapid reversibility and by its
heterologous nature (cf. Fig. 4)
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Fig. 2.
Desensitization of inward IK(ACh)
in myocytes subject to different transfection protocols.
A, control (EGFP-positive); B, GIRK1-transfected;
C, GIRK1/GIRK4-transfected; D, GIRK4-transfected.
ACh (20 µM) was superfused as indicated by the
horizontal lines. The rapid vertical deflections in
D represent changes in membrane current caused by voltage
ramps from -120 to +60 mV, which were superimposed in the majority of
measurements. Holding potential was -90 mV in all experiments.
E shows the summarized data. Desensitization was expressed
as ratio of quasi-steady-state current at t = 20 s
by peak current or current at t = 1 s in the case
of GIRK4-transfcted myocytes. Differences between GIRK1- and
GIRK1/4-transfected cells and controls were not significant, whereas
the differences between the GIRK4 group and the other three groups were
highly significant. The number of cells was between 12 and 20 for each
group.
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Fig. 3.
Activation of IK(ACh) is slowed
by overexpression of GIRK4. A, representative
superimposed recordings of activation of IK(ACh) from a
control and a GIRK4-transfected myocyte. The superfusion was switched
to ACh-containing solution at the point of time indicated by the
arrow. The trace labeled GIRK4 has been scaled up
vertically to match the peak of the control current. B,
summarized data from 12 time-matched myocytes each. The time constant
of activation ( 
) was approximated by means of a least square fitting
procedure. In this and subsequent figures, a star indicates
a difference at p < 0.05 compared to control
groups.
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Fig. 4.
Absence of heterologous desensitization in
GIRK4-transfected myocytes. Saturating concentrations of ACh (20 µM) and Ado (100 µM) were superfused as
indicated. Representative current recordings from a control
(A) and a GIRK4-transfected myocyte (B).
C, summarized data from six time-matched myocytes for each
group. The bars indicate the ratios of peak current induced
by Ado plus ACh (average of three consecutive responses from individual
traces, as shown in A and B) divided by peak
ACh-induced current in the absence of Ado.
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Fig. 5.
Removal of desensitization by GIRK4
overexpression is not limited to activation via
M2AChR. Representative traces showing inward
IK(ACh) activated by ACh (20 µM) and Ado (100 µM). Panel A, control myocyte transfected with
the EGFP vector only; panel B, myocyte transfected with
pSV-SPORT-A1R; panel C, myocyte transfected with
pSV-SPORT-A1R plus pcDNA-GIRK4.
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Fig. 6.
GIRK4 overexpression removes
desensitization of inward and outward IK(ACh).
A, representative recordings from a control myocyte at two
different extracellular K+ concentrations and holding
potentials: 20 mM/90 mV/EK = -49 mV
(a), and 5 mM/-40 mV/EK = -84 mV
(b). ACh (20 µM) was applied as indicated. The
trace labeled 5 K+ has been inverted and scaled
up in c to match the peak of inward IK(ACh) at
20 mM K+ and -90 mV holding potential.
B, representative recordings from a GIRK4-transfected
myocyte. Panels a-c have the same meaning as in
A.
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Fig. 7.
Inward rectification is reduced in
GIRK4-overexpressing myocytes. A and B
represent difference current-voltage relations obtained by electronic
subtraction of background current from ACh-induced current (voltage
ramps from -120 to +60 mV). A, control; B,
GIRK4-transfected myocyte. C, summarized data from 12 cells
each. Inward rectification was expressed as ratio of current at 0 mV
divided by current at -100 mV.
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Fig. 8.
Properties of IK(ACh) in myocytes
transfected with GIRK42 and GIRK44
concatemers. Representative recording of inward
IK(ACh) evoked by 20 µM ACh from a myocyte
transfected with a GIRK42 (A) and a
GIRK44 (B) construct. C, summarized
data on fast desensitization, as in Fig. 2E. The quotient
was significantly different from controls for the GIRK42
group but not different from those for the GIRK44 group
(n = 6). Summarized data on inward rectification are as
in Fig. 5. The bars representing the control and GIRK4
groups C and D are the same as in Figs.
1E and 4C, respectively.
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Fig. 9.
Current-voltage relations of GIRK currents
induced by activation of A1 receptors in CHO cells.
Difference I/V-curves of currents evoked by 100 µM
adenosine (background subtracted) representative of CHO cells
transfected with GIRK1 (A), GIRK1 plus GIRK4 (B),
GIRK4 (C), and GIRK44 (D).
E, summarized data on inward rectification (quotients of
current at 0 mV divided by current at -100 mV).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to the
carboxyl terminus of the GIRK4 subunit is essential for channel
activation, whereas the requirement for interaction of G with GIRK1 remained unclear (25-29). In a very recent study, it was demonstrated that both heterotetrameric GIRK1/GIRK4 complexes and GIRK4 homotetramers exhibit a 1:1
subunit-G. binding stoichiometry
(30).
-activated currents in
Xenopus oocytes injected with GIRK4 mRNA (34, 35). In a
mammalian expression system, macroscopic currents carried by homomeric
GIRK4 channels so far have not been identified. Single channel currents
carried by GIRK4 homomers expressed in oocytes and CHO cells have
extremely short open times, which renders them inaccessible to an
analysis of their basic properties (24).
, as illustrated in Fig.
10. In this simulation, agonist-induced activation of IK(ACh) was modeled for simplicity as a
-induced activation. Inactivation or desensitization was modeled
using first order kinetics with a time constant of 500 ms (Fig.
10A). The simulated normalized current traces in Fig. 10B yield a rapidly activating current with a distinct
desensitizing component, whereas the current activating with the slower
rate apparently lacks desensitization (compare Fig. 3A).
Although this simple model does not support any particular mechanism of
GIRK channel-associated desensitization, it would be in line with a lower affinity of the carboxyl-terminal binding site of GIRK4 as
compared with GIRK1 to -subunits reported in Ref. 43.
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Fig. 10.
Simulation of desensitization properties of
receptor-activated current at two different rates of activation.
A, normalized activation with time constants of 125 ms
(a) and 400 ms (b). Trace c represents
an inactivation process with a time constant of 500 ms and a
steady-state amplitude of 50% of steady-state activation. Traces in
B represent simulated normalized currents combining
inactivation with fast activation (a) and slow activation
(b).
Our data suggest that GIRK4 homotetrameric complexes might contribute to macroscopic IK(ACh) with a nondesensitizing component, whereas GIRK1/GIRK4 heteromeric channels show desensitization. This hypothesis, however, does not explain why, in GIRK4- or GIRK42-transfected myocytes, the fast desensitizing component was always completely lost, because endogenous GIRK1 should still be able to associate with GIRK4. We assume that on the background of a high expression level of monomeric or dimeric GIRK4, the low probability of formation of heteromeric complexes results in whole cell currents that are highly dominated by the properties of homomeric GIRK4 channels. This would be in line with the observation that in myocytes transfected with the tetrameric construct, IK(ACh) had intermediate properties, because intrinsic GIRK1/GIRK4 complexes should still exist.
An alternative explanation for the loss of contribution of heteromeric channels would be a competition of the channel complexes for a limited number of putative anchoring domains required for functional membrane targeting. This would also explain why we did not find significant changes in current densities even in myocytes transfected with both the GIRK1 and GIRK4 encoding vectors. An anchoring protein interacting with GIRK4, however, so far has not been identified. Alternatively, the total current might be limited by the expression level of endogenous G-proteins.
Inward rectification of Kir channels reflects a block by intracellular cations, such as Mg2+ and spermine (see Refs. 3 and 21 for reviews). In the situation of a whole cell patch clamp experiment, also exogenous constituents of the pipette filling solution, such as organic buffers, might contribute to inward rectification (44). Evidence has been provided that this block depends on two amino acid residues located in the M2 transmembrane segment and the carboxyl-terminal of the channel subunits. These residues were initially identified in the strong inward rectifier IRK1 as Asp172 and Glu224 (45). The corresponding residues are Asp173 and Ser225 in GIRK1 and Asn179 and Glu231 in GIRK4, respectively (see Ref. 3 for review). In a study using homomeric mutants expressed in Xenopus oocytes, Vivaudou et al. (33) found an enhancement of inward rectification of GIRK4-containing tetramers by GIRK1, underscoring the importance of GIRK1 residues 173 and 179 as dominant determinants of inward rectification.
As for inward rectification, our data obtained in native transfected
myocytes are confirmed by the finding that inward rectification of
receptor-activated current is stronger in CHO and HEK293 cells transfected with GIRK1/GIRK4 as compared with GIRK4 alone or either of
the concatemeric constructs. Assuming that the cell lines are devoid of
endogenous GIRK subunits, this provides additional support for the
conclusion that functional channels can be formed by assembly of
homomeric GIRK4 complexes in mammalian cells.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Anke Galhoff, Bing Liu, and Gabriele Reimus for expert technical assistance.
| |
FOOTNOTES |
|---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant Po212/9-2.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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
49-234-3229200; Fax: 49-234-3214449; E-mail:
lutz.pott@ruhr-uni-bochum.de.
Published, JBC Papers in Press, May 30, 2001, DOI 10.1074/jbc.M102328200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: GIRK, G-protein-coupled inwardly rectifying K+ channel; ACh, acetylcholine; Ado, adenosine; HEK, human embryonic kidney; CHO, Chinese hamster ovary; M2AChR, muscarinic M2 acetylcholine receptor; A1AdoR, A1 adenosine receptor; EGFP, enhanced green fluorescent protein.
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REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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