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Biochemical and Biophysical Research Communications 364 (2007) 889895

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Characterizations of a loss-of-function mutation in the Kir3.4

channel subunit q
Kirstine Calloe a,*, Lasse Steen Ravn c, Nicole Schmitt a, Jin Liang Sui b, Morten Duno e,
Stig Haunso c, Morten Grunnet d, Jesper Hastrup Svendsen c, Soren-Peter Olesen a
a
The Danish National Research Foundation Centre for Cardiac Arrhythmia, Department of Biomedical Sciences, 12.5.10,
University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark
b
CombinatoRx Inc., Cambridge, MA, USA
c
Department of Cardiology, The Heart Center, Copenhagen University Hospital, Denmark
d
NeuroSearch, Ballerup, Denmark
e
Department of Clinical Genetics, Copenhagen University Hospital, Denmark

Received 10 October 2007

Available online 29 October 2007

Abstract

Kir3.4 and Kir3.1 potassium channel subunits mediate the acetylcholine induced inwardly rectifying current IKACh in the heart. We
found a glycine to arginine substitution in codon 247 of Kir3.4 in a patient with a single episode of atrial brillation (AF). Expression in
Xenopus laevis oocytes and two-electrode voltage-clamp revealed that Kir3.4-G247R basal current was reduced compared to wild-type
Kir3.4 and co-expression with the muscarinic acetylcholine receptor type 2 showed that also the acetylcholine induced current was
severely reduced in Kir3.4-G247R, indicating that the mutation interfered with activation by the stimulatory Gbc-subunits. Co-expres-
sion of Kir3.4-G247R with wild-type Kir3.4 or Kir3.1 had a compensating eect on both basal current levels and the response to mus-
carinic stimulation suggesting the function of Kir3.4-G247R is compensated in vivo. This may explain the lack of clear clinical
manifestations and further studies are necessary to elucidate if mutations in Kir3.4 are predisposing AF.
2007 Elsevier Inc. All rights reserved.

Keywords: Kir3.4; GIRK; IKACh; Gbc; Atrial brillation; Arrhythmia

The Kir3.1 and Kir3.4 potassium channel subunits
form heterotetrameric channels that mediate the acetyl-
choline (ACh) induced potassium current, IKACh [1].
IKACh hyperpolarizes the cell membrane and is important
for the negative chronotropic eect of parasympathetic
activity. ACh is released from the vagus nerve and stim-
ulates a variety of cardiac receptors and channels, includ-
ing the muscarinic ACh G-protein coupled receptor type
q
Funding: This work was supported by a grant from the Carlsberg
Foundation (K.C.), the Danish National Research Foundation (N.S.), the
John and Birthe Meyer Foundation and the Danish Academy of
Cardiovascular Research (L.S.).
*
Corresponding author. Fax: +45 35327555.
E-mail address: Kirstinec@m.ku.dk (K. Calloe).
2 (M2). Stimulation of the M2 receptor by ACh causes
dissociation of the coupled G-protein and the Gbc-sub-
units activate the Kir3.1/3.4 channel by direct binding
[2]. Despite extensive eorts, the binding site for the
Gbc-subunit on the channel is still debated and intracel-
lular domains facing the cytoplasm in both the N- and
C-termini have been suggested [38]. The mechanism
underlying Gbc activation of the channel is not well
understood but binding of Gbc to the channel complex
has been shown to stabilize the interaction between the
Kir3 channel and phosphatidylinositol (4,5)-bisphosphate
(PIP2) [9]. This interaction with PIP2 is a requirement for
Kir channels activity and a decrease in bound PIP2
strongly decreases the open probability of the Kir3.1/
3.4 channels [9,10].

0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2007.10.106
890 K. Calloe et al. / Biochemical and Biophysical Research Communications 364 (2007) 889895
Mice decient of Kir3.1 or 3.4 exhibit mild resting
tachycardia [11] and impaired beat-to-beat control [12].
Remarkably, the Kir3.4 decient mice were resistant to
atrial brillation (AF) caused by vagal stimulation [13].
Increased activity of Kir3.4 has been linked to AF in sev-
eral animal models [1315] and constitutively active IKACh
has been demonstrated in patients with persistent AF
where it may contribute to sustaining the arrhythmia [16].
This signies that a reduction of IKACh could be benecial
in AF prevention and is concordant with the observation
that AF can be induced by gain-of-function mutation in
cardiac potassium channels (http://www.fsm.it/cardmoc/).
Conversely, loss-of-function of atrial potassium channels
causing a diminished repolarization reserve and a concom-
itant prolongation of the atrial action potential duration
has also been suggested to promote atrial tachyarrhythmias
[17,18].
This prompted us to screen AF patients for mutations in
the KCNJ5 gene that encodes the Kir3.4 potassium channel
subunit.
Methods
Clinical analysis. The investigation conforms to the principles outlined
in the Declaration of Helsinki. Patients initially participated in the
Copenhagen SAFIR investigation [19] and were included consecutively
from the Department of Cardiology, Copenhagen University Hospital
Hvidovre, Denmark. The basic inclusion criterion was AF documented on
electrocardiogram (ECG) followed by restored sinus rhythm (SR). Three
years later 158 patients attended a follow-up visit with ECG and Holter
recording), echocardiogram, registration of disease history and blood
samples.
DNA isolation and mutation analysis. Genomic DNA was isolated from
blood samples from the 158 AF patients and from 96 ethnically matched
controls using QIAamp DNA Blood Mini Kit (Qiagen, Germany). The
coding and exon anking regions of the KCNJ2 and KCNJ5 genes were
PCR amplied and directly sequenced. Other candidate genes associated
with AF, KCNQ1, KCNH2, and KCNE1-5 were amplied by PCR and
single-strand conformation polymorphism of the fragments was investi-
gated using GeneGel Excel 12.5/24 kits (Amersham Biosciences AB,
Sweden). Aberrant conformers were directly sequenced on a 3100-Avant
Genetic analyzer (Applied Biosystems, CA) using Big Dye chemistry.
Molecular biology. cDNAs encoding human Kir3.4 (Accession No.
NM_000890), Kir3.1 (NM_002239), Kir3.4-S143T (in the following
referred to as Kir3.4*, based on Accession No. NM_000890), the musca-
rinic ACh receptor type 1 (M1, NM_000738) and the muscarinic ACh
receptor type 2 (M2, NM_001006630) were kind gifts from D.E. Logo-
thetis. The cDNAs were subcloned into the oocyte expression vector
pGEM and the mutation G247R was introduced into Kir3.4 and Kir3.4*
by mutated oligonucleotide extension (Pfu Turbo Polymerase, Stratagene,
La Jolla, CA), digested with DpnI (Fermentas, Germany), and trans-
formed into Escherichia coli XL1 Blue cells. All constructs were veried by
DNA sequencing. cRNA was synthesized by standard in vitro run-o
transcription using the T7 mMessage Machine kit according to manu-
facturers instructions (Ambion, Austin, TX).
Expression in Xenopus laevis oocytes. Oocytes were surgically removed
from anaesthetized (2 g/L tricaine, Sigma) X. laevis frogs in accordance
with the guidelines from the Danish National Committee for Animal
Studies and defolliculated enzymatically as described earlier [20] before
injection with 50 nL cRNA (0.52 ng/oocyte Kir cRNA and 0.5 ng/oocyte
M1/M2 cRNA). Kir3.1 and Kir3.4 cRNAs were mixed in equal molar
ratios. In some experiments M1 or M2 encoding cRNA were injected 48 h
prior to injection of Kir3 cRNA to allow sucient expression. Oocytes
were kept in a low K solution (in mM; 90 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2,
5 Hepes, pH 7.4) at 19 C.
Electrophysiology. Whole-cell currents were measured 2472 h after
Kir cRNA injection by two-electrode voltage-clamp (TEVC) amplier
(Clampator 1, Dagan, Chicago, IL). Pipettes were pulled from borosilicate
glass and had a nal tip resistance of 0.52.5 MX when lled with 2 M
KCl and submerged in low K solution. Currents were measured in a high
K solution (in mM; 96 KCl, 10 Hepes, 1 MgCl2, 1.8 CaCl2, pH 7.4). In
some experiments 10 lM ACh (Sigma) was dissolved directly in the high K
solution. Data were sampled with Pulse (HEKA electronics, Germany)
and analyzed using the Igor software (WaveMetrics, Lake Oswego, OR)
and GraphPad Prism (GraphPad Software, San Diego, CA). All data are
presented as mean standard error of the mean. For statistical analyses
paired and unpaired T-tests as well as one way ANOVA combined with
Tukeys multiple comparison test were used. p < 0.05 was considered
signicant.
Results and discussion
The Caucasian female proband presented at age 69 years
with a single episode of AF that spontaneously reverted
after 3 days. The clinical evaluation included examination,
12-lead ECG, exercise and ambulatory ECG monitoring.
The 12-lead ECG during sinus rhythm revealed normal
PR intervals and neither signs of ventricular hypertrophy
nor bundle branch block. The patient had a normal echo-
cardiogram, the left atria had a diameter of 3.5 cm and
the ejection fraction was larger than 55 mL. The proband
showed no concomitant risk factors like diabetes, hyper-
tension or thromboembolic disease apart from age. Genetic
screening revealed that the proband was heterozygote for a
novel variant in the KCNJ5 gene, in nucleotide 739, a G
was replaced by A, resulting in a glycine to arginine substi-
tution in codon 247 of the Kir3.4 subunit. No aberrations
were found in other candidate genes associated with AF
(KCNQ1, KCNH2, KCNE1-5, and KCNJ2). The Kir3.4-
G247R substitution was absent in 96 normal control sam-
ples as well as in the remaining 157 AF patients investi-
gated and it was not registered as a Caucasian variant in
PharmGKB (www.pharmgkb.com) or in the single nucleo-
tide polymorphisms (SNP) database of National Center for
Biotechnology Information (NCBI) (http://
www.ncbi.nlm.nih.gov). The 247G residue is conserved
across species and between other members of the Kir fam-
ily suggesting that G247R either represents a de novo muta-
tion or a rare variation. Besides the G247R substitution,
three synonymous known SNPs were found in Kir3.4 from
the patient (rs6590357, rs7118824, and rs118833) and one
known non-synonymous SNP (rs7102584), resulting in a
glutamine to glutamate substitution in codon 282,
Q282E. According to the SNP database of NCBI, a Q at
position 282 is normal, but only 7 out of 158 patients were
heterozygote for 282Q and the remaining patients all were
homozygote for 282E. Alignment of Kir3.4 shows that the
residue E is conserved across species suggesting that 282Q
rather represents a common polymorphism. The proband
was homozygote for Kir3.4-Q282E. As there is increasing
evidence that the presence of a polymorphism either on
the same or a separate gene allel can aect the phenotype
 K. Calloe et al. / Biochemical and Biophysical Research Communications 364 (2007) 889895 891

of a mutation constructs containing either an E or a Q at
position 282. No dierence in current levels, kinetics or rec-
tication was found. Only data using 282E constructs are
presented in the following as the proband was homozygote
for 282E.
Family history was obtained (Fig. 1A). One son was het-
erozygote for Kir3.4-G247R but was asymptomatic. The
four other sons did not carry the substitution, one of
which, however, had uncharacteristic chest complaint that
could be caused by arrhythmia. Despite thorough investi-
gation no arrhythmia was documented or signs of struc-
tural heart disease observed.
Electrophysiological characterization of Kir3.4-G247R
To investigate if the electrophysiological phenotype of
Kir3.4-G247R deviated from WT, both were co-expressed
with the Kir3.1 subunit in oocytes. To mimic the heterozy-
gote state of the patient, Kir3.1/3.4 and Kir3.1/3.4-G247R
cRNAs were mixed in a 1:1 ratio. Whole-cell currents were
measured by TEVC and large, inward rectifying currents
were observed in both WT and mutant expressing oocytes
(Fig. 1B). The currentvoltage relationship is shown in
Fig. 1C. There was no apparent dierence in the reversal
potential, rectication or the kinetic parameters of the
Kir3.1/3.4-G247R current compared to WT but a clear
eect on the current levels was observed. At 100 mV,
the average current of Kir3.1/3.4-G247R was approxi-
mately 40% less than that of Kir3.1/3.4 expressing oocytes.
Oocytes co-expressing Kir3.1/3.4 with Kir3.1/3.4-G247R
gave 10% less current than Kir3.1/3.4 WT expressing
oocytes. These experiments clearly demonstrated that
G247R substitution resulted in a loss-of-function and there
was no dominant negative eect of Kir3.4-G247R on co-
expressed Kir3.1 or 3.4 subunits.
As co-expression with Kir3.1 could have a compensating
eect on the phenotype of the Kir3.4-G247R mutant, we
attempted to measure currents from oocytes expressing
homomeric Kir3.4 channels, but the currents were very
low as described earlier [21]. To circumvent this obstacle,
we used the Kir3.4-S143T construct, referred to as
Kir3.4*. Kir3.4* channels yield large currents in the absence
of Kir3.1 due to a higher open probability [22] and have
been demonstrated to interact in a qualitative similar way
with Gbc as Kir3.1/3.4 WT channels [21]. Hence, we intro-
duced the G247R substitution in Kir3.4*. Kir3.4* or

A C Vm (mV)
AF, 69
-100 -50 50
-10
I (A)
*** -20

48 50 52 53 56 ** -30

-40

B Kir3.1/3.4 D

3.4*-G247R
I (A)

-10 0mV 50mV

Kir3.1/3.4-G247R
20 A

-100mV
3.4*
-20
500 ms 0.0 0.2 0.4 0.6 0.8
Time (s)

Fig. 1. Pedigree and electrophysiological characterization of Kir3.4-G247. (A) Pedigree for the family (circle = female; squares = males). The age is
indicated. The proband and one son, age 48, were heterozygote for Kir3.4-G247R as indicated by half-lled circle/square. (B) Kir3.1/3.4 and Kir3.1/3.4-
G247R currents elicited by a step protocol, from 100 mV to 60 mV in 20 mV increments. (C) Currentvoltage relationship Kir3.1/3.4 (squares), Kir3.1/
3.4-G247R (triangles) and Kir3.1/3.4+Kir3.1/3.4-G247R (open triangles). At 100 mV, the average current for Kir3.1/3.4 was 37.80 2.5 lA (n = 7),
for Kir3.1/3.4+Kir3.1/3.4-G247R it was 33.7 1.7 lA (n = 7) and for Kir3.1/3.4-G247R, 22.70 1.522 lA (n = 7). The Kir3.1/3.4 was signicantly
dierent from Kir3.1/3.4-G247. Kir3.1/3.4+Kir3.1/3.4-G247R was signicantly dierent from Kir3.1/3.4-G247R but not signicantly dierent from
Kir3.1/3.4. (D) Kir3.4* or Kir3.4*-G247R or Kir3.4* and Kir3.4*-G247R currents elicited by the depicted ramp protocol. At 100 mV the average current
was 20.5 2.5 lA (n = 5) for Kir3.4*, 8.83 1.2 lA (n = 6) for Kir3.4*/Kir3.4*-G247R and for Kir3.4*-G247R it was 4.32 0.97 lA (n = 5). The
Kir3.4* current was signicantly dierent than Kir3.4*/Kir3.4*-G247 (p < 0.001) and from Kir3.4*-G247R (p < 0.001). The dierence between Kir3.4*/
Kir3.4*-G247R and Kir3.4*-G247R was not signicantly dierent.
892 K. Calloe et al. / Biochemical and Biophysical Research Communications 364 (2007) 889895

Kir3.4*-G247R constructs were expressed in oocytes. Rep-
resentative currents for both WT and mutant Kir3.4* are
shown in Fig. 1D. At 100 mV, Kir3.4*-G247R expressing
oocytes had approximately 80% less current than Kir3.4*
expressing oocytes. In experiments where Kir3.4* was co-
expressed with Kir3.4*-G247R in a 1:1 ratio, the current
was reduced by 57% compared to Kir3.4* current. This
could suggest that if homomeric Kir3.4 channels were pres-
ent in the patient, the current through these channels would
be severely reduced. However, the absence of attributable
cardiac or non-cardiac phenotype in the heterozygote car-
riers suggest reduced current through Kir3.4 homomeric
channels is either well compensated in vivo or alternatively
that homomeric Kir3.4 channels only play a small role
physiologically, in agreement with the ndings in Kir3.4
decient mice [11].
Kir3.4-G247 and muscarinic stimulation
The G247R substitution is located in the cytoplasmic
region of the Kir3.4 C-terminus and alignment with the
published structure of the cytoplasmic region of mouse
Kir3.1 proteins (PDB: 1U4F [23] and 1N9P [8]) it suggests
that the G247R residue is facing the cytoplasm proximal to
the interaction site between two Kir subunits and near a
turn in the secondary structure. The interaction between
the Kir3.1/3.4 protein and Gbc-subunits is presumed to
take place within a part of the intracellular region but the
exact domain(s) of the interaction is a matter of debate.
The region just upstream the G247R mutation has been
suggested as a putative Gbc-subunit binding site in
Kir3.4 [6]. Replacing a small glycin with a bulky arginine
residue may disturb the secondary protein structure adja-
cent to the mutation and this could aect the Gbc mediated
activation. Interestingly, the same sequence stretch has also
been demonstrated to aect the PIP2 anity of the channel
[24] and we therefore addressed the eect of muscarinic
stimulation on mutant and WT channels.
To test if the interaction between the Gbc-subunits and
the Kir3.1/3.4 channel was aected by the G247R substitu-
tion, Kir3.1/3.4 and Kir3.1/3.4-G247R were co-expressed
with the M2 receptor and currents measured before and
during exposure to 10 lM ACh (Fig. 2A). For both WT
and mutant, the current was signicantly increased during
ACh stimulation. On average, WT current was increased
by almost 90% during ACh exposure and the Kir3.1/3.4-
G247R/M2 current was increased by 80% (Fig. 2B and
C) during ACh exposure.
The experiments were repeated with Kir3.4*. As Kir3.4*
-G247R had very low currents, the amount of injected
Kir3.4*-G247R cRNA was increased fourfold to yield
basal current comparable to that of Kir3.4*. Representative
currents recordings for Kir3.4*/M2 and Kir3.4*-G247R/
M2 are shown in Fig. 3A. The Kir3.4*/M2 current was

A Ach Kir3.4/3.1
B 0

PIP2
M2
-10
I (uA)

i/o
K+
***
-20
3.1/3.4/M2

-30 ***
3.1/3.4/ 3.1/3.4-G247R/
M2 M2

ACh C
100 NS
3.1/3.4-G247R/M2
% increase

50

ACh
10 A

0
3.1/3.4/ 3.1/3.4-G247R/
200 ms M2 M2

Fig. 2. Kir3.4-G247 and Gbc-stimulation. Kir3.1/3.4/M2 and Kir3.1/3.4-G247R/M2 currents before and during stimulation with 10 lM ACh. (A) The
activated pathway is shown and representative Kir3.1/3.4/M2 and Kir3.1/3.4-G247R/M2 currents. (B) Average current at 100 mV before and during
ACh stimulation (grey bar). The Kir3.1/3.4/M2 current was increased from 13.4 1.3 lA (n = 13) to 25.2 1.7 lA (n = 13) during ACh stimulation.
For Kir3.1/3.4-G247R/M2, the current increased from 6.66 0.66 lA (n = 13) to 11.8 1.35 lA (n = 13). (C) Percentage increase in current during
ACh stimulation. For Kir3.1/3.4/M2 ACh stimulation increased currents at 100 mV by 96.7 10% (n = 13) and for Kir3.1/3.4-G247R/M2, the currents
were increased by 78.4 11% (n = 13).
 K. Calloe et al. / Biochemical and Biophysical Research Communications 364 (2007) 889895 893

A B 0

3.4*/M2 -10

I (A)
-20
NS
-30 **
ACh
3.4*/ 3.4*-G247R/
M2 M2

C
3.4*-G247R/M2 40

30

% increase
20
10 A

10
***
0
200 ms 3.4* 3.4*-G247R

Fig. 3. Kir3.4*-G247 and Gbc-stimulation. Kir3.4*/M2 and Kir3.4*_G247R/M2 currents before and during stimulation with 10 lM ACh. (A)
Representative Kir3.4*/M2 and Kir3.4*-G247R/M2 currents. (B) Average currents at 100 mV before and during ACh stimulation (grey bar). Kir3.4*/
M2 had an average current of 19.5 3.0 lA (n = 9) before ACh stimulation and 25.5 4.0 lA (n = 9) during ACh stimulation. For Kir3.4*-G247R/
M2, currents were 19.7 5.2 lA (n = 7) before stimulation and 19.8 5.2 lA (n = 7) during stimulation. (C) Percentage increase in current during
ACh stimulation. For Kir3.4*/M2 ACh stimulation increased to currents at 100 mV by 30.7 4.7% (n = 9), whereas Kir3.4*-G247R/M2 currents were
increased by 1.5 0.56% (n = 7).

Ach
Kir3.4/3.1
A B
PIP2
M1

PLC NS
IP3 + DAG 11/q
30
% Decrease

3.1/3.4/M1 20

10
ACh
0
3.1/3.4/ 3.1/3.4-G247R/
M1 M1

C
30 NS
3.1/3.4-G247R/M1
% Decrease

20

ACh
10
5 A

0
3.4*/M1 3.4*-G247R/
200 ms
M1

Fig. 4. Kir3.4-G247 and PIP2 interaction. Kir3.1/3.4/M1 and Kir3.1/3.4-G247R/M1 currents before and during stimulation with 10 lM ACh. (A) The
activated pathway and representative Kir3.1/3.4/M1 and Kir3.1/3.4-G247R/M1 currents. (B) The percentage decrease in current during ACh stimulation.
For Kir3.1/3.4/M1 ACh stimulation decreased currents at 100 mV by 32.7 1.9% (n = 11). Kir3.1/3.4-G247R/M1 currents were decreased by
29.8 1.6% (n = 8). The decrease in Kir3.1/3.4/M1 and Kir3.1/3.4-G247R/M1 current were not signicantly dierent. (C) The percentage decrease in
current during ACh stimulation for Kir3.4*/M1 and Kir3.4*-G247R/M1. For Kir3.4*/M1 ACh stimulation signicantly decreased currents at 100 mV by
24.6 5.3% (n = 7) and for Kir3.4*-G247R/M1, the current was reduced by 24.0 3.19% (n = 7).
894 K. Calloe et al. / Biochemical and Biophysical Research Communications 364 (2007) 889895
increased by 31% during ACh exposure (Fig. 3B and C). In
contrast, Kir3.4*-G247R/M2 currents were increased less
than 2% by ACh, indicating that the activation of Kir3.4-
G247R by Gbc-subunit was perturbed by the substitution.
Binding of the Gbc-subunits to Kir3.4 channels has
been shown to stabilize interaction between PIP2 and the
channel complex and thereby increasing the open probabil-
ity [9,10]. The level of PIP2 in the membrane can be
reduced by stimulation of a whole range of receptors that
activate phospholipase C (PLC), such as the muscarinic
ACh receptor type 1 (M1). PLC activation results in hydro-
lysis of PIP2 into potent second messengers. The depletion
of PIP2 per se results in a decrease in current but activation
of PKC may further destabilize the interaction between the
channel complex and PIP2 and exacerbate the current
reduction [25]. To test if the G247R substitution aected
this interaction, Kir3.1/3.4 or Kir3.1/3.4-G247R were co-
expressed with the M1 receptor.
Application of ACh resulted in a prominent decrease in
currents measured in WT Kir3.1/3.4/M1 as well as in
Kir3.1/3.4-G247R/M1 expressing oocytes (Fig. 4A). The
relative decrease was not dierent between the WT and
the mutant, with 30% or 33% decreases, respectively
(Fig. 4B). The experiments were repeated using the
Kir3.4* constructs. Exposing Kir3.4*/M1 or Kir3.4*
-G247R/M1 expressing oocytes to ACh, resulted in current
reductions by 24.6 5.3% and 24.0 3.2%, respectively
(Fig. 3C). These reductions were not signicantly dierent.
Together these results indicate that the interaction between
the channels complex and PIP2 is not aected by the
G247R substitution and we speculate if a possible allosteric
change conferred by the mutation could interfere with
either Gbc binding or activation without aecting the
PIP2 anity as such.
In our experiments, we found that heteromerization of
Kir3.1 or 3.4 had a compensating eect on the phenotype
of Kir3.4-G247R, both with regard to the basal current
level and the response to ACh stimulation. This could oer
an explanation for the lack of clear clinical manifestations
and in conclusion and in conclusion further studies are nec-
essary to elucidate if mutations in Kir3.4 could be predis-
posing to or even protecting against AF.
Acknowledgments
P. Hagman, T. Soland, and P. Westermann are thanked
for expert technical assistance.
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