From the University of Würzburg, Department of Pharmacology & Toxicology, Versbacherstrasse 9, 97078 Würzburg, Germany
Received for publication, May 30, 2002, and in revised form, October 15, 2002
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ABSTRACT |
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G G-protein-activated inwardly rectifying K+ channels
(GIRKs)1 are expressed in
many areas of the brain and in supraventricular myocytes of the heart
(1, 2). Activation of G-protein-coupled receptors that couple to
Gi-proteins such as the M2 muscarinic acetylcholine receptors (M2-mAChRs) lead to a dissociation
of heterotrimeric G-proteins into activated GIRK channels belong to the family of strong inwardly rectifying
K+ channels, which are characterized by their strong
inwardly rectifying current-voltage relationships. The inward
rectification has been linked to the presence of intracellular
Mg2+ and polyamines (10-12). These positively charged
cytoplasmic ions are thought to block outward K+ currents
by blocking the pore of channels from the inside (10-13); however, for
a related inwardly rectifying channel Kir2.1 this hypothesis has
recently been questioned (14). Inward rectification of
K+ channels is not only voltage-dependent but
also dependent on the extracellular K+ concentration (11).
The inward rectification of these K+ channels is closely
related to their function in the heart as well as in many neuronal
tissues. In cardiac myocytes activation of inwardly rectifying
K+ channels such as IKACh causes the cell
membrane to hyperpolarize between action potentials because the
conductivity for K+ generated by these channels is high at
membrane potentials close to EK. This
hyperpolarization induced by IKACh appears to be at least
partially responsible for the negative chronotropic effect induced by
vagal activity (1, 2, 15). During action potentials, however, the
conductivity of IKACh for K+ declines
several-fold with the rise of voltage enabling the myocyte to generate
prolonged action potentials, which are critically important for cardiac
function (16).
The initial observation that led to the study presented here was the
discovery that the agonist-induced IKACh in cardiac
myocytes were quite variable in their degree of inward
rectification,2 indicating
that the modulation of the open probability of these channels (15) by
ACh may not be the only property of these channels that is regulated by
ACh. It seemed possible that, in addition, inward rectification of
these channels may be modulated as well by ACh. The present experiments
have tested this possibility.
Preparation of Feline Atrial Myocytes--
Isolation of feline
atrial myocytes was performed as described (17). Animal procedures used
were in accordance with guidelines of the Animal Care and Use Committee
of Northwestern University. Briefly, adult cats were first anesthetized
with pentobarbital sodium (70 mg/kg body weight,
intraperitoneally). The heart was quickly removed and retrograde
perfused with Krebs-Henseleit buffer. It was digested by perfusion with
collagenase-containing solution. After 10-15 min of digestion the
atria were collected and cut into small pieces, followed by a 5-min
incubation with fresh enzyme solution. Isolated atrial myocytes
were collected, placed in M199 (Invitrogen), and plated in cell culture
dishes. The cells were kept at 37 °C under 7% CO2 until
further use.
Cell Culture and Transfection--
Chinese hamster ovary
(CHO-K1) cells were grown in Ham's F-12 medium (Invitrogen).
The media were supplemented with 10% fetal bovine serum and
streptomycin/penicillin (100 units each). Cells were grown under 7%
CO2 at 37 °C. In all transfections for
electrophysiological studies the CD8 reporter gene system was used to
visualize transfected cells (18). Dynabeads coated with
anti-CD8-antibodies were purchased from Dynal. CHO-K1 cells were
transfected using adenovirus-mediated gene transfer (19) using the
following amounts of endotoxin-free cDNAs (Qiagen)/6 cm dish: human
CD8 (in
HEK 293 cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, streptomycin/penicillin (100 units each), and 1% glutamine. Cells were grown under 7% CO2 at 37 °C. In cells stably expressing GIRK1/4, the
media was supplemented with 200 mg/l G418. To visualize
transfected cells the CD8 reporter gene system was used as described
before. Transfection was performed using the Effectene transfection kit
(Qiagen) according to the manufacturer's protocol using the following
amounts of endotoxin-free cDNAs (Qiagen)/6 cm dish.
Experiments described in Fig. 4: 0-1.25 µg empty pcDNA3, 0.05 µg CD8, 0.5 µg human Solutions--
For the measurement of K+ currents an
extracellular solution of the following composition was used
(mM): NaCl, 120; KCl, 20; CaCl2, 2;
MgCl2, 1; Hepes-NaOH, 10, pH 7.3. The internal (pipette) solution contained (mM): potassium aspartate, 100; KCl, 40;
MgATP, 5; Hepes-KOH, 10; NaCl, 5; EGTA, 2; MgCl2, 1; GTP,
0.01; pH 7.3. All standard salts as well as ACh and adenosine (Ado)
were purchased either from Sigma or from Merck.
Measurement of Membrane Currents--
Membrane currents
were recorded under voltage-clamp conditions, using conventional whole
cell patch clamp techniques (20). Patch-pipettes were fabricated from
borosilicate glass capillaries, (GF-150-10, Warner Instrument Corp.)
using a horizontal puller (P-95, Fleming & Poulsen). The DC
resistance of the filled pipettes ranged from 3-6 M
All measurements were performed at room temperature. Summarized results
are presented as mean values ± S.E. Student's t tests (two population) were performed to test for significance of differences between groups of data.
The atrial muscarinic K+ current (IKACh)
is regulated by muscarinic receptors, and the underlying pathway has
been studied in detail by many groups (15). The inwardly rectifying
properties of this channel have been the topic of many detailed studies
that provided interesting insights into the mechanisms of inward
rectification (10, 11). However, so far no physiological modulation of
the inward rectification of this current has been reported. The
following study was based on the surprising observation that the inward rectification of ACh-evoked K+ currents in feline atrial
myocytes varied as a function of the agonist concentration.
The Inward Rectification of Feline Atrial IKACh Was
Modulated by Stimulus Strength--
IKACh in isolated
feline atrial myocytes was measured in response to two different
concentrations of ACh either in the inward or outward direction using
the whole cell patch technique. The membrane potential was clamped to
either Heterologously Expressed GIRK Currents Exhibited Strong
Inward Rectification When Activated via Endogenous
G-proteins--
Whole cell currents were measured in CHO-K1 or HEK 293 cells transfected with GIRK 1 and 4 as described before to prove that GIRK channels are responsible for the observed currents in feline atrial myocytes (21, 24). Gi-coupled receptors such as the M2-mAChR or A1 adenosine receptors activated
GIRK currents in response to agonist (Fig.
2, A and B).
Although the size of the currents increased as a function of agonist
concentration (Fig. 2A), no stimulus-dependent
change in inward rectification of GIRK currents was observed (Fig.
2B). Indeed, agonist-induced currents all exhibited strong
inward rectification very similar to atrial IKACh activated
with low doses of agonist (compare Figs. 2B and 1B). Similar results were observed in transiently
transfected HEK 293 cells using M2-mAChR, A1
adenosine receptors, or Heterologously Expressed GIRK Currents Exhibited Weakened Inward
Rectification When Activated via Co-expressed
G
In a minority of cells transfected with G The Ratio of G The Weakened Inward Rectification Was Not Accompanied by Changes in
Slow Blocking Kinetics of Outward GIRK Currents Attributed to
Polyamine-induced Inward Rectification--
For further analysis
experimental conditions were chosen to consistently induce either
strong inward rectifying currents (control) or weak inward rectifying
currents (G Affinity for Ba2+ Block Was Reduced under Weak Inward
Rectifying Conditions--
A hallmark for strong inward rectifier
potassium channels is a high affinity block by external
Ba2+. Studies using crystal structures of the bacterial
KcsA channel complexed with Ba2+ have located a single
Ba2+-binding site on the cytosolic side of the selectivity
filter (29, 30). In close proximity to this site are some of the residues that have been implicated to be critical for strong inward rectification (11, 28). To test if G Cs+-induced Block of GIRK Channels Was Attenuated under
Weak Inward Rectifying Conditions--
Inwardly rectifying
K+ channels can be blocked efficiently by external
Cs+. This block is highly voltage-dependent and
most prominent at negative potentials (11, 31). Binding sites for
Cs+ in the channel have been mapped to pore-lining residues
of transmembrane domain 2 (M2) (32) and to a site close to selectivity
filter (32). Therefore, possible G Weakening of Inward Rectification of IKAch in Feline
Atrial Myocytes Is Due to Binding of G
The maximal activation of GIRK channels expressed in HEK 293 or CHO
cells was clearly limited by the availability of endogenous G The Physiological Role of Weakened Inward Rectification--
The
inward rectification of IKACh channels is important for
their physiological function to stabilize the membrane potential at
negative voltages but not for blocking the generation of the plateau
phase of action potentials (16). Because under physiological conditions
net-potassium flux through this channel will always be in outward
direction, one would predict that 2-3-fold increases in potassium
outward currents, due to weakening of the inward rectification as
observed in this study, will have a great impact on the shape and
duration of supraventricular action potentials. It seems likely that
the local in vivo concentration of ACh in the synaptic cleft
can reach levels high enough, at least for very short periods, to cause
weakening of inward rectification of atrial IKACh, because
high frequency stimulation of the vagal nerves can induce a
hyperpolarization in atrial tissue similar in amplitude as if directly
evoked by ACh in the low µM range (37).
The Weakening of Inward Rectification Is Not Due to a Reduced
Polyamine Affinity--
It has been shown that open channel block by
polyamines and Mg2+ ions contributes to inward
rectification in GIRK channels. Therefore, changing the inward
rectification in the observed way may be related to polyamine and/or
Mg2+-binding properties to the channel. Mg2+ is
known to block instantaneously, whereas polyamine block exhibits slow
voltage-dependent blocking and unblocking kinetics (13, 27,
28). In whole cell patch clamp experiments the polyamine block is found
to be responsible for the slow inactivation/activation of GIRK currents
measured resulting from voltage steps (28). Our investigation of
polyamine block revealed no striking alteration of the
blocking/unblocking time constants in the presence of G The Weakening of Inward Rectification Is Associated with a
Reduction of Ba2+ and Cs+ Affinity--
In
Kir2.1 channels there exists an overlap between sites important for
inward rectification and blocking by external cations such as
Cs+ and Ba2+ (11, 38). Therefore,
G G
The G-protein-mediated regulation of inward rectification of atrial and
heterologously expressed GIRK channels described in this study
represents to our knowledge the first description of a regulatory
mechanism that alters the inward rectifying properties of an ion
channel. Furthermore, we demonstrate that binding of G subunits are known to bind to and
activate G-protein-activated inwardly rectifying K+
channels (GIRK) by regulating their open probability and bursting behavior. Studying G-protein regulation of either native GIRK (IKACh) channels in feline atrial myocytes or
heterologously expressed GIRK1/4 channels in Chinese hamster ovary
cells and HEK 293 cells uncovered a novel G
subunit
mediated regulation of the inwardly rectifying properties of these
channels. IKACh activated by submaximal concentrations of
acetylcholine exhibited a ~2.5-fold stronger inward
rectification than IKACh activated by saturating
concentrations of acetylcholine. Similarly, the inward rectification of
currents through GIRK1/4 channels expressed in HEK cells was
substantially weakened upon maximal stimulation with co-expressed
G
subunits. Analysis of the outward current block underlying
inward rectification demonstrated that the fraction of instantaneously
blocked channels was reduced when G
was over-expressed. The
G
induced weakening of inward rectification was associated with
reduced potencies for Ba2+ and Cs+ to block
channels from the extracellular side. Based on these results we
propose that saturation of the channel with G
leads to a
conformational change within the pore of the channel that reduced the
potency of extracellular cations to block the pore and increased the
fraction of channels inert to a pore block in outward direction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits and
dimers. G
subunits are known to bind to GIRK channels and
increase the open probability of these channels (3, 4). Cardiac
IKACh channels are formed by heteromultimers of GIRK1 and
GIRK4 subunits (4). The binding site of G
subunits to GIRK
channels was mapped primarily to the C terminus of GIRK1 and GIRK4
(4-8). Cross-linking studies have demonstrated that the
heterotetrameric channel can bind up to 4 G
subunits (9).
However, despite much experimental effort the mechanism by which
G
activates these channels is not well understood.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3; 0.15 µg; gift from Dr. G. Yellen); mouse GIRK1 (in pC1,
0.3 µg) and mouse GIRK4 (in pCDNA1, 0.3 µg; gifts from Drs. F. Lesage and M. Laszdunski); human A1-adenosine receptors (in
CLDN 10B, 0.2 µg; gift from Dr. J. Linden); human
M2-mAChR (0.8 µg, in pcDNA3; gift from Dr. E. Peralta); human G
1 (in pCMV5, 0.3 µg) and human
G
2 (in pcDNA1, 0.3 µg; gifts from Dr. H. A. Bourne). Empty pcDNA3 was used to balance the total amount of
cDNA used for transfection to 2-2.35 mg/6 cm. All assays were
performed 48-72 h post transfection if not otherwise mentioned.
2a-adrenergic receptor
(AR)-G
i1 fusion protein (in pcDNA3, kindly provided by Dr. G. Milligan), and GIRK1, GIRK4, G
1, and
G
2 as indicated in the figure. For experiments shown in
Figs. 5-7: HEK cells stably expressing GIRK1 and GIRK4 channels were
transfected with 0.7 µg
2a-AR-G
i1, 1.4 µg each of human G
1 and G
2 (in
pCDNA3), and CD8 (in
3, 0.2 µg). Experiments were performed
40-50 h post transfection.
. Membrane
currents were recorded using either a patch-clamp amplifier (Axopatch
200, Axon Instruments) or an EPC 9 (HEKA Instruments) as described
previously (21, 22). Signals were analog-filtered using a lowpass
Bessel filter (1-3 kHz corner frequency). Data were digitally stored
using either a Mac (Centrion 640 with pulse software) or an IBM
compatible PC equipped with a hardware/software package (ISO2 by MFK,
Frankfurt/Main, Germany) for voltage control, data acquisition, and
data evaluation. IKACh was measured as an inward current
using a holding potential of
90 mV as described (23). Voltage ramps
(from
120 mV to +60 mV in 500 ms, every 10 s) were used to
determine current-voltage (I-V) relationships.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
90 mV or +60 mV in the presence of 20 mM
extracellular K+. When the holding potential was negative
(
90 mV) to the potassium equilibrium potential
(EK) (Fig.
1A), superfusion of the cell with 0.1 µM ACh gave rise to inward currents that were
about 70% in amplitude compared with currents activated by 10 µM ACh. In contrast, at +60 mV outward currents induced
by 0.1 µM ACh were barely detectable and were only about
10% in amplitude compared with currents activated by 10 µM ACh. I-V curves of ACh-induced currents (Fig.
1B) were determined by subtracting currents measured in the
absence of agonist from currents measured in the presence of agonist in
response to linear voltage ramps from
120 mV to +60 mV. I-V curves of
the currents elicited by 0.1 µM ACh or by 10 µM ACh exhibited inward rectification and identical
reversal potentials close to the EK (Fig.
1B), suggesting that the currents generated by either
concentration of ACh were attributable to activation of
IKACh. However, inward rectification of the current activated by 0.1 µM ACh was found to be considerably
stronger than that of the current activated by 10 µM
(Fig. 1B). Plotting GIRK conductance (normalized to the
amplitude measured at
90 mV) against voltage shows a 15-20 mV shift
to more positive potentials when currents were activated by 10 µM compared with 0.1 µM ACh (Fig.
1C).
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Fig. 1.
The inward rectification of feline atrial
IKACh is dependent on stimulus strength. ACh-induced
changes in whole cell currents of freshly isolated feline atrial
myocytes were measured in the presence of 20 mM
extracellular K+ (EK ~ 50 mV).
Currents were activated in response to either submaximal (0.1 µM) or saturating (10 µM) concentrations of
ACh at
90 mV and +60 mV as indicated (A). Current-voltage
curves of ACh induced currents as shown in B were calculated
after subtraction of background currents. The voltage
dependencies of IKACh conductance in the presence of
low or high agonist concentrations are plotted in C. These
results were representative for similar experiments performed in four
different atrial myocytes obtained from two different myocyte
preparations.
2A adrenergic receptors (data
not shown and Refs. 21 and 22).
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Fig. 2.
G
modulates inward rectification of heterologously expressed GIRK
currents. HEK 293 cells were transiently transfected with
cDNAs for GIRK1, GIRK4, and A1 adenosine receptors and
with G
1
2 (C, D,
E, F, indicated as +G
) or without
additional exogenous G-protein subunits (A, B,
E, F, indicated as agonist). GIRK
currents activated via A1 adenosine receptors or by
co-expression with G
subunits were measured using whole cell
voltage clamp recording similar as described in the legend to Fig. 1.
GIRK current-voltage curves were calculated by subtracting either
background currents in the absence of agonist (B) or
currents insensitive to 1 mM Ba2+
(D). Normalized GIRK current conductance in cells
co-transfected with or without G
subunits were plotted against
voltage (E). To quantify the degree of inward rectification,
an inward rectification factor was defined (Fir = I(Erev
50 mV)/I(Erev + 50 mV)) (F). Summarized data were compared for
adenosine-evoked currents in the absence of exogenous G
(agonist) and Ba2+-sensitive currents evoked by
heterologous expression of G
(F) (n = 9 each, the two groups were significantly different at
p < 0.05).
--
Agonist-induced GIRK currents obtained from cells
heterologously transfected with GIRK1/4 were activated via endogenous
G-proteins (Fig. 2, A and B). It seemed likely
that the pool of endogenous G-proteins might have been limiting for the
extent of maximal GIRK current activation. Therefore,
G
1
2 subunits were co-expressed with GIRK
channels. GIRK currents were constitutively active due to G
subunits. The amplitude of GIRK currents was determined via inhibition
by Ba2+ (Fig. 2C). In most cases, activation of
co-expressed A1 adenosine receptors by 10 µM
Ado induced no further stimulation of Ba2+-sensitive GIRK
currents, indicating a maximal stimulation of GIRK channels by G
.
Under these conditions, total Ba2+-sensitive GIRK currents
compared with control conditions (activation via receptor and
endogenous G-proteins) were about 2-fold larger in amplitude (147 ± 12.7 pA/pF with co-expressed
G
1
2 versus 71.2 ± 20.7 pA/pF activated via A1 adenosine receptors and endogenous G-proteins) and exhibited a weaker inward rectification (Fig. 2D versus Fig. 2B). Comparing the
conductance-voltage relationship revealed a shift to more positive
voltages for GIRK currents activated by heterologously expressed
G
subunits compared with GIRK currents activated by agonist only
(Fig. 2E). To quantify the relative inward rectification of
GIRK currents the ratio of GIRK current conductance in outward
versus inward direction (Ba2+-sensitive GIRK
currents at reversal potential (Erev) ± 50 mV) was calculated. The ratio of outward/inward currents of
Ba2+-sensitive GIRK currents activated by heterologously
expressed G
was significantly increased compared with
Ba2+-sensitive GIRK currents activated via
A1 adenosine receptors and endogenous G-proteins (0.39 ± 0.11, n = 9 versus 0.14 ± 0.05, n = 8) (Fig. 2F). The voltage-dependence of
GIRK currents maximally activated by heterologous expression of G
was comparable with atrial IKACh activated by saturating
concentrations of ACh (10 µM), whereas agonist-induced
GIRK currents activated via endogenous G-proteins exhibited similar
strong inward rectification as submaximally activated atrial
IKACh. This result indicated that the inwardly rectifying
properties of GIRK channels were modulated depending on the internal
G
concentration.
subunits addition of
adenosine to stimulate A1 adenosine receptors resulted in a
further increase in GIRK currents (Fig.
3A), indicating submaximal stimulation of GIRK channels by heterologously expressed G
. Under
these circumstances, basal G
-induced GIRK currents exhibited strong inward rectification, whereas addition of adenosine resulted in
a pronounced weakening of inward rectification (Fig. 3B),
demonstrating that inward rectification of heterologously expressed
GIRK currents can be modulated via stimulation of G-protein-coupled
receptors similar to atrial myocytes. Taken together, these results
suggested that G
at submaximal concentrations induces strong
inwardly rectifying GIRK currents, whereas at maximal concentrations,
G
-evoked GIRK currents exhibited weakened inward
rectification.
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Fig. 3.
Agonist-mediated modulation of inward
rectification in cells submaximally stimulated with transfected
G . In a minority of cells transfected
with G
, stimulation of co-expressed A1 adenosine
receptors caused, in addition to the constitutively active GIRK
currents, a further increase in GIRK currents (A). I-V
curves for the Ba2+-sensitive currents in the absence
(a-c) and presence of 1 µM adenosine
(Ado, b-c) as well as the currents that were
stimulated by Ado in addition to the constitutively active currents
(b-a) were determined (B). Note a substantial
weakening of GIRK current inward rectification in response to
adenosine.
to GIRK Channel Expression Is Critical for
Regulation of the Inward Rectification of GIRK Currents--
If the
ratio of GIRK channels versus available G
in the cells
is important for the degree of inward rectification as suggested by
these results, it should be possible to achieve a high ratio of
endogenous G
to GIRK channels by lowering the GIRK channel expression. Contrarily, a strengthening of the inward rectification should occur when GIRK channel expression is increased relative to the
G
expression. We attempted to counteract the G
mediated weakening of inward rectification by transfecting HEK 293 cells with steady amounts of G
subunits but increasing amounts of GIRK1 and GIRK4 subunits as illustrated in Fig.
4. G
expression in the presence of
co-expressed GIRK channels decreased cell survival. Therefore, we
choose to co-express an
2A-adrenergic receptor fused to
a Gi
1-protein (26) to reduce constituitive
G
signals. GIRK channels were stimulated using saturating
concentrations of norepinephrine (10 µM), and
subsequently GIRK currents were blocked by superfusion of the cells
with 1 mM BaCl2 to determine background
currents. The ratio of outward to inward GIRK currents significantly
declined with increasing amounts of GIRK channels transfected (Fig. 4,
upper panel). The GIRK current density measured at
90 mV
increased with increasing amounts of GIRK1/4 channel expression (Fig.
4, lower panel), suggesting that G
expression was not
limiting for maximal inward GIRK currents in cells transfected with 0.1 µg cDNA/5 cm dish of GIRK1/4. These results supported the
hypothesis that inward rectification of GIRK channels is
modulated dependent on the ratio of G
subunits relative to GIRK
channels. In addition, we tried to lower GIRK expression relative to
endogenous G-proteins by prolonging the time after transfection and
found a significant increase in the ratio of outward to inward currents from day 3 to 4 post-transfection in transiently transfected CHO cells
(Iout/Iin: 0.22 ± 0.06 d.4
versus 0.095 ± 0.025 d.3) accompanied by a small
reduction in GIRK current density determined at
90 mV (43 ± 13 pA/pF, d.4 n = 12 compared with 64 ± 10 pA/pF,
d.3 n = 6). This weakening of inward rectification of
GIRK currents reflected most likely a decrease in GIRK channel
expression in the individual cells, resulting in an increase of the
ratio of G-proteins versus GIRK channels.
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Fig. 4.
The ratio of
G subunits to GIRK channel is
important for regulation of inward rectification. HEK 293 cells
were transiently transfected with indicated amounts of cDNAs
encoding for GIRK1, GIRK4, G
1, and G
2 as
well as constant amounts of cDNA of a
2A-AR-G
i1-fusion protein and the
CD8-reporter gene. 40-48 h post transfection whole cell currents were
recorded in response to voltage ramps either in the presence of 10 µM norepinephrine (maximal GIRK activation) or 1 mM Ba2+ (to specifically block GIRK currents).
Summarized data for the degree of GIRK current inward rectification
(Fir = I(Erev + 50 mV)/I(Erev
50 mV)) obtained under the indicated
conditions is illustrated in the upper panel
(n = 5-9, of 2-3 transfections). Corresponding
maximal GIRK current densities measured in inward direction (
90 mV
holding potential) are shown in the lower panel. Differences
from the results shown in the first column (0.1 µg
cDNA of GIRK1/4; 0.5 µg cDNA for
G
1
2) that reached significance are
indicated (*, p < 0.05; **, p < 0.01).
-induced) in HEK cells stably expressing GIRK1 and 4. Strong inward rectifying currents were induced via agonist stimulation
of
2A adrenergic receptors in the absence of exogenous
G
, whereas weak inward rectifying currents were evoked by
additional co-transfection of G
1
2. As described above the current model of the inward rectifying mechanism is
a voltage-dependent open channel block by internal
Mg2+ and polyamines such as spermine and spermidine. To
test whether an alteration of the polyamine- and
Mg2+-induced open channel block was the cause for the
observed weakening of inward rectification, blocking and unblocking
kinetics were determined using whole cell recording. According to Refs.
13, 27, and 28, the polyamine block is responsible for the
time-dependent (slow) activation and inactivation of
K+ currents through GIRK channels (or other inward
rectifier channels) in response to voltage steps, whereas current block
induced by internal Mg2+ occurs almost instantaneously.
Therefore, whole cell currents resulting from voltage steps (
120 mV
to 60 mV; 60 mV to
120 mV) were measured to determine the time
constants of polyamine block onset and offset. In case inward
rectification was weakened due to lowered polyamine block affinity, a
faster polyamine unbinding from the channel and/or a slower-polyamine
binding to the channel should be observed. In contrast, if
Mg2+ block was altered, the fraction of channels blocked
instantaneously in outward direction should be decreased, whereas
changes in blocking and unblocking kinetics should not be observed.
Background currents were determined by inhibiting GIRK channels via
Ba2+ and subtracted from each measured whole cell current.
A second-order exponential function was used to fit the current curves
and determine time constants. Comparison of currents measured under
control (strong inward rectification) and G
over-expressed (weak
inward rectification) conditions showed no striking alteration of the slow blocking kinetics (Fig.
5A). As expected, the
unblocking appeared to be faster (Fig. 5B), however, this
effect did not reach statistical significance (1.28 ms ± 0.1 versus 1.05 ms ± 0.46; 10 ms ±0.95 versus
9.1 ms ± 2.8). In contrast to the proposition, blocking of the
channel in the outward direction (reflecting binding of polyamines) was
faster, too (4.9 ± 0.97 ms versus 2.75 ± 0.62 ms; 58.8 ± 16 ms versus 36 ± 9.3 ms).
Normalizing to the maximum inward current revealed that the probability
of channel opening at voltages positive to EK
was increased under weak inward rectifying conditions compared with
control conditions. Normalizing to the outward maximum current
demonstrated that the same percentage of channels underwent a slow
blockade under control as well as under weak inward rectifying
conditions. Because the fraction of channels instantaneously blocked in
the outward direction was lower when G
was over-expressed the
potency of internal Mg2+ to block the channels might have
been reduced. Therefore, we increased internal Mg2+ up to
20 mM to compensate for a reduced potency of
Mg2+ to block GIRK channels, however, no change in inward
rectification was observed (data not shown).
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Fig. 5.
The slow component of outward current block
is not altered by co-expression of G
subunits. Illustrated are representative current recordings
measured in response to voltage steps (
120 mV, 60 mV, and
120 mV,
as indicated) from cells, which did (red) or did not express
exogenous G
(black). Currents were normalized
either to the maximal inward (upper and lower right
panel) or outward (lower left panel) currents, and the
time course of the onset of outward current block (lower left
panel) as well as the recovery from outward current block
(lower right panel) was fitted best by a bi-exponential
decay. Summarized data for the resulting time constants are illustrated
in the figure (n = 5-7).
mediates a conformational change of the GIRK channel that causes weakening of inward
rectification by altering structures close to the selectivity filter,
we questioned whether or not GIRK channel block by Ba2+ was
affected by G
. Whole cell currents at a holding potential of
90
mV were measured in the presence of 1 µM, 10 µM, 40 µM, 140 µM, 1 mM, and 2 mM extracellular Ba2+
under strong and weak inward rectifying conditions (Fig.
6). Ba2+ effectively
inhibited GIRK currents under both conditions, however, the potency of
Ba2+ to block GIRK currents was substantially decreased
when channels were maximally activated by G
(IC50: 73 µM versus 20 µM; Hill coefficient: n = 1.14 versus
n = 2). These results strongly suggested that
interaction with G
subunits induced conformational changes of
GIRK channel structures close to the Ba2+-binding site.
View larger version (19K):
[in a new window]
Fig. 6.
Concentration-response curve for
Ba2+-induced GIRK current inhibition. The inhibition
of steady GIRK currents (holding potential: 90 mV) in response to
extracellular Ba2+ was determined in CHO-K1 cells
transfected with the same set of cDNAs as described in the legend
to Fig. 2. Curve fitting using conventional dose-response equations
(Origin 6.1 software) determined the concentration for half-maximal
inhibition of GIRK currents to be 30 µM Ba2+
for agonist-activated (strong inward rectifying) currents and 70 µM for G
1
2-activated
currents. Hill slopes were n = 1.1 (agonist)
and 2.0 (+G
1
2), respectively
(n = 3-5).
-dependent
modulation of GIRK current block by external Cs+ (3 mM) was studied (Fig. 7). At
a membrane potential of
90 mV, whole cell GIRK currents were
inhibited under control (strong inward rectifying) conditions by
85 ± 2.6%, whereas whole cell currents in the presence of
heterologously expressed G
(weak inward rectifying conditions)
were inhibited only by 28 ± 4% (Fig. 7, A-C). To
verify whether or not the attenuation of the Cs+ block by
co-expression of G
was correlated to the G
-mediated weakening of inward rectification, the degree of inward rectification (defined as Fir = I(Erev
50 mV)/I(Erev + 50 mV)) was plotted against the
potency of Cs+ to block GIRK channels. We obtained a close
inverse correlation of the degree of inward rectification and the
ability of Cs+ to block GIRK currents (Fig. 7D).
This result suggested that a G
-mediated conformational change of
GIRK channels caused the reduced inward rectification and was
mechanistically coupled to a reduction of the Cs+ block. We
further analyzed the voltage-dependencies of the Cs+ block
by comparing GIRK currents activated via endogenous G-proteins and
selected GIRK currents activated via co-expressed G
subunits, but
exhibiting a different degree of inward rectification (most likely due
to different expression levels of G
subunits).
Background-subtracted, current-voltage relationships of strong inward
rectifying (Fir = 0.10; no G
co-transfected) and
medium and weakly inward rectifying currents (Fir = 0.14, Fir = 0.20; both with co-expression of G
) were
determined in the presence or absence of 3 mM external
Cs+ and fitted according to the Woodhull model (33, 34)
(Fig. 7E).
View larger version (23K):
[in a new window]
Fig. 7.
G
over-expression attenuates Cs+-induced inward current
block of GIRK channels. Current-voltage relationships of GIRK
currents were recorded in the presence or absence of 3 mM
Cs+ in the bath solution. GIRK currents were evoked either
via
2A adrenergic receptors and endogenous G-proteins
(A and C, blue-colored bar) or by
co-expression of G
(B and C,
red-colored bar). The potency of 3 mM
Cs+ to block GIRK currents at
90 mV were plotted against
the degree of inward rectification in cells expressing or not
expressing exogenous G
(D). The voltage dependence of
the current block induced by 3 mM extracellular
Cs+ was determined in dependence of the degree of inward
rectification (E). Representative experiments have been
selected for strong (Fir = 0.1, control; no
G
transfected), medium, and weak (Fir = 0.14, Fir = 0.20; both with co-transfection of G
subunits)
inwardly rectifying currents and were fitted according to the Woodhull
model (Equation 1 under "Results").
The half-blocking voltage EBlock1/2
was shifted in the negative direction by up to
(Eq. 1)
30 mV by G
(EBlock1/2 =
64 mV for Fir = 0.10; EBlock1/2 =
79 mV for
Fir = 0.14; EBlock1/2 =
93
mV for Fir = 0.20). Interestingly, the apparent voltage dependence of the Cs+-induced current block as indicated by
the electrical distance
was up to 3-fold steeper under weak
inwardly rectifying conditions (
= 2.2 for Fir = 0.10;
= 3.0 for Fir = 0.14;
= 6.9 for
Fir = 0.20), suggesting a deeper penetration of
Cs+ into the pore or a change in
voltage-dependent binding parameters for Cs+
within the pore. This result strongly suggests that G
induced a
significant conformational change within the GIRK channel pore.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
to GIRK
Channels--
This study discovered that G-proteins do not only
activate GIRK channels, but in addition also regulate the degree of
inward rectification of these channels. In isolated atrial myocytes
from adult cats, submaximally activated IKACh exhibited
strong inward rectification, whereas maximal stimulation resulted in a
2-3-fold weakening of the inward rectification of IKACh.
The fact that GIRK channels heterologously expressed in cell lines
devoid of any other measurable inward rectifying currents were
modulated in their inward rectifying properties by co-expression of
G
subunits, strongly suggested that the inward rectification of GIRK channels themselves can be modulated by G
and that the observed stimulus-dependent weakening of IKACh
inward rectification was the result of a G
-mediated modulation of
GIRK channels.
subunits as the co-expression of G
subunits boosted GIRK currents
2.5-4-fold (Figs. 2 and 4 and Refs. 21 and 35). This may explain why
in the absence of G
co-expression no agonist-mediated modulation
of GIRK inward rectification was observed (unless GIRK channel
expression was very low). In cells exhibiting strong inwardly rectifying GIRK currents despite expression of exogenous G
, additional stimulation via G
i-coupled receptors led to a
dramatic weakening of the inward rectification of these currents (Fig. 3). This observation suggested that modulation of inward rectification was not an artifact of G
over-expression. At a constant
expression level of G
subunits an increase of GIRK expression
strengthened inward rectification and increased inward current density
(Fig. 4), suggesting that the ratio of available G
subunits to
expressed GIRK channels is critical for regulating inward
rectification. Taken together these results point to a bimodal
regulation of GIRK channels by G
subunits: at submaximal
concentrations G
increased the open probability of GIRK channels
(as demonstrated before (15, 36)), whereas at saturating concentrations
G
weakened inward rectification of GIRK channels giving rise to a
substantial increase in outward K+ current conductance.
over-expression. If a decrease of the polyamine affinity had been the
cause for weakened inward rectification, a major increase in the
blocking time constants and/or a major decrease in the unblocking time
constant should have been observed. However, we found the contrary.
Under weak inwardly rectifying conditions blocking time constants were
slightly decreased and no major differences in unblocking time
constants was observed. The observed weakening of inward rectification
could be attributed to a decrease of the fraction of channels that were
blocked instantaneously in outward direction (Fig. 3), pointing to
attenuation of either the Mg2+-induced channel blockade or
some yet unknown intrinsic outward current block (14). However, no
change in inward rectification was observed when increasing internal
Mg2+ up to 20 mM to compensate for a possibly
reduced potency. So far, there is no direct experimental evidence to
attribute the weakening of inward rectification to altered binding
properties of polyamines or Mg2+ to the channels. However,
we cannot exclude that Mg2+-induced outward current block
was completely impaired in weak inwardly rectifying GIRK channels.
-induced reduction of the affinity of Ba2+ to block
GIRK currents supports the assumption that weakening of inward
rectification is induced by conformational changes in the pore region
of GIRK channels. Extracellular Cs+ is known to block
strongly inwardly rectifying K+ channels in a highly
voltage-dependent manner. The Cs+-binding site
is also located within the channel pore, probably deeper in the channel
than the blocking site for Ba2+. Similarly to
Ba2+-induced GIRK channel block, Cs+-induced
block was attenuated under weak inward rectifying
conditions and the weakening of inward rectification correlated to
weakening of Cs+-induced current block. G
-induced
weakening of inward rectification was correlated as well with a
stronger voltage dependence of Cs+ block and a shift to
more negative potentials. These G
-mediated changes in the pore
blocking properties of GIRK channels compare well to the differences of
the pore blocking properties between members of the week and strong
inwardly rectifying K+ channel family. Weak inwardly
rectifying K+ channels exhibit usually a weaker affinity
for Cs+ and Ba2+ compared with strong inwardly
rectifying K+ channels (11, 28, 39). Based on these results
we propose that maximal activation of GIRK channels by G
subunits
induces a conformational change within the channel pore that tunes GIRK channels from a strong to a weak inwardly rectifying channel.
Induces a Conformational Change in the Pore of GIRK
Channels--
How does binding of G
to the channel induce a
conformational change of the channel that leads to reduced affinity for
Cs+ and Ba2+? The fact that G
binds near
the intracellular C terminus makes it unlikely that the cations and
G
subunits share common binding sites within the pore.
Considering that a tetrameric channel can bind up to four G
subunits (9) and the open probability is gradually regulated by at
least three G
-binding sites (40), we propose that binding of the
third or more likely the fourth G
subunit to the channel may
force the channel into a weak inward rectifying conformation. How could
this work? Recently L.Y. Jan and co-workers (41) presented
convincing data, which suggested that opening of GIRK channels requires
a rotation of the M2 transmembrane helix. Because the residues
important for cation pore block are located either on the M2 helix or
are in close proximity to the M2 helix, a rotation of these helices may
likely alter the position of these residues. Assuming that the model of
Jan and co-workers is correct and binding of a G
subunit to a
GIRK channel subunit causes a rotation of the M2 helix of this
particular GIRK channel subunit, it is obvious that the structures
close to the cation-binding site(s) within the channel pore of a
tetrameric GIRK channel will be different depending on how
many G
subunits are bound. According to Refs. 36 and
42, single channel characteristics in respect to open and closed times
GIRK channels are different depending on the concentration of available
G
subunits. If strong inward rectification and high affinity
Ba2+ and Cs+ block require one or two of the
four M2 helices not to be rotated, rotation of the last two helices
(induced by binding of the 3rd or 4th G
subunit to the tetrameric channel) could potentially weaken inward
rectification. This working hypothesis needs to be verified in future studies.
subunits
to the channel alter the conformation at known cation-binding sites
within the channel pore, supporting the hypothesis that G
might
gate the channel at the selectivity filter rather than at a cytoplasmic gate.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Hosey and Dr. B. TenEick for generous support of this work, which included providing lab space, equipment, and materials as well as scientific advice. Feline atrial myocytes were kindly provided by C. Hansen. The skillful assistance of M. Frank for work related to cDNA cloning and cell culture accelerated this project significantly.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Research Fellowship BU 1133/1 (to M. B.) and a Leibniz award (to M. J. L.).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 addressed: Dept. of Pharmacology
and Toxicology, University of Würzburg, Versbacherstrasse 9, 97078 Würzburg, Germany. Tel.: 49-931-201-48854; Fax:
49-931-201-48539; E-mail: m-buenemann@toxi.uni-wuerzburg.de.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M205325200
2 M. Bünemann, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GIRK, G-protein-activated inwardly rectifying K+ channels; CHO, Chinese hamster ovary; Ado, adenosine; ACh, acetylcholine; M2-mAChRs, M2 muscarinic acetylcholine receptors; AR, adrenergic receptor; M2, transmembrane domain 2.
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