Báo cáo khoa học: Identification of the structural determinant responsible for the phosphorylation of G-protein activated potassium channel 1 by cAMP-dependent protein kinase pdf

Identification of the structural determinant responsible
for the phosphorylation of G-protein activated potassium
channel 1 by cAMP-dependent protein kinase
Carmen Mu
¨
llner, Bibiane Steinecker, Astrid Gorischek and Wolfgang Schreibmayer
Department of Biophysics, Center for Physiological Medicine, Medical University of Graz, Austria
Introduction
G-protein activated inwardly rectifying K
+
channels
(GIRKs) link membrane potential to the presence of
extracellular signalling molecules via G-protein cou-
pled receptors and pertussis toxin sensitive, heterotri-
meric, G-proteins in a membrane delimited manner
[1,2]. Their role in brain function, as well as in the
regulation of the heartbeat, is now well established
and, increasingly, their importance in other organs is
being acknowledged [3–7]. Generally, the G-protein
b ⁄ c dimer is considered to be the primary opener of
GIRKs, although the G-protein a-subunit is also
involved in gating [8,9]. Besides this regulation via het-
erotrimeric G-proteins, a huge body of work demon-
strates that GIRK channel activity and trafficking is
regulated by various signalling molecules, comprising
nucleotides, PIP
2
, protein kinases and protein phos-
phatases [10–16]. Most remarkably, the concerted
action of protein phosphatase 2A (PP2A) [17] and
cAMP-dependent protein kinase A (PKA) [18]

provides an ‘on ⁄ off’ switch for G-protein activation.
PKA-dependent activation of I
K+ACh
itself was
observed in rat atrial cardiomyocytes [18,19] and the
Keywords
GIRK; IK+Ach; PKA
Correspondence
W. Schreibmayer, Department of Biophysics,
Center for Physiological Medicine, Medical
University of Graz, Harrachgasse 21 ⁄ 4,
A-8010 Graz, Austria
Tel: +43 316 380 4155
Fax: +43 316 380 9660
E-mail: wolfgang.schreibmayer@
medunigraz.at
(Received 17 June 2009, revised 17 August
2009, accepted 24 August 2009)
doi:10.1111/j.1742-4658.2009.07325.x
Besides being activated by G-protein b ⁄ c subunits, G-protein activated
potassium channels (GIRKs) are regulated by cAMP-dependent protein
kinase. Back-phosphorylation experiments have revealed that the GIRK1
subunit is phosphorylated in vivo upon protein kinase A activation in Xeno-
pus oocytes, whereas phosphorylation was eliminated when protein kinase
A was blocked. In vitro phosphorylation experiments using truncated ver-
sions of GIRK1 revealed that the structural determinant is located within
the distant, unique cytosolic C-terminus of GIRK1. Serine 385, serine 401
and threonine 407 were identified to be responsible for the incorporation of
radioactive
32

P into the protein. Furthermore, the functional effects of
cAMP injections into oocytes on currents produced by GIRK1 homo-
oligomers were significantly reduced when these three amino acids
were mutated. The data obtained in the present study provide information
about the structural determinants that are responsible for protein kinase A
phosphorylation and the regulation of GIRK channels.
Structured digital abstract
l
MINT-7260296, MINT-7260317, MINT-7260333, MINT-7260347, MINT-7260361, MINT-
7260270: PKA-cs (uniprotkb:P00517) phosphorylates (MI:0217) Girk1 (uniprotkb:P63251)by
protein kinase assay (
MI:0424)
Abbreviations
GIRK, G-protein activated potassium channel; GST, glutathione S-transferase; PhB, phosphorylation buffer; PKA, protein kinase A; PKA-cs,
catalytic subunit of PKA; PP2A, protein phosphatase 2A.
6218 FEBS Journal 276 (2009) 6218–6226 ª 2009 The Authors Journal compilation ª 2009 FEBS
stimulatory effect of PKA on GIRK1 ⁄ GIRK4 hete-
rooligomeric channels had been attributed to a marked
increase in the affinity of the phosphorylated channel
protein to G
b ⁄ c
[20]. A signalling complex comprising
(amongst other signalling molecules) GIRK1, GIRK4,
G
b ⁄ c
, PKA, PP2A and protein phosphatase 1 was
identified to exist in rat atrial membranes in situ [21],
supporting the physiological relevance of this regula-
tion. Several heterooligomeric combinations, including
not only GIRK1 ⁄ GIRK4, but also the homooligomeric

GIRK1 subunit alone, have been identified to be under
PKA regulation [18]. In addition, the GIRK1 protein
was also demonstrated to be a direct target for PKA-
catalysed phosphorylation [17,22,23]. Despite several
efforts undertaken to identify the responsible structural
determinant on the GIRK1 subunit [17,23], the exact
location still remains unknown. The present study
aimed to obtain information about PKA phosphoryla-
tion of GIRK1 in vivo and the structural motif(s) on
the GIRK1 subunit serving as PKA substrate(s), as
well as to assess its possible role in G-protein activa-
tion of the channel.
Results
Back-phosphorylation of GIRK1
Previous studies had shown that the GIRK1 subunit,
but not the GIRK4 subunit, isolated from bovine
atrium, represented a prominent target for several S ⁄ T
protein kinases in vitro, including cAMP-activated
PKA [17]. To investigate whether GIRK1 represents a
target for PKA also in vivo, back-phosphorylation
experiments were performed. Using an antibody direc-
ted against the entire C-terminus, GIRK1 was immu-
nopreciptated from oocytes, expressing rat GIRK1 and
subsequently submitted to back-phosphorylation using
the catalytic subunit of PKA (PKA-cs) and
[
32
P]ATP[cP]. Autoradiograms of subsequent SDS gels
revealed GIRK1 migrating in two bands, indicating a
glycosylated and a nonglycosylated form, as reported

previously [24]. Interestingly, prominent in vitro back-
phosphorylation of immunoprecipitated GIRK1 was
observed when RpCAMPS, a PKA inhibitor, was
injected into the oocytes prior to immunoprecipitation,
comparable to the control oocytes. This indicates that
the heterologously expressed GIRK1 subunit was a
prominent target for PKA in vitro, after the in vivo
treatment of the oocytes with a PKA inhibitor and
also in untreated oocytes. On the other hand, the
in vitro phosphorylation signal was markedly dimin-
ished when SpCAMPS, a PKA activator, was injected,
indicating that the relevant PKA site(s) had been
phosporylated already in the oocytes before the immu-
nopreciptation (Fig. 1). Clearly, this indicates that
GIRK1 is reversibly phosphorylated by native PKA
in vivo in the oocytes and that the extent of basal PKA
phosphorylation is low.
Structural determinant responsible for PKA
phosphorylation in vitro
To identify the structural determinants that are respon-
sible for phosphorylation of GIRK1 by PKA, fusion
proteins comprising truncated forms of the cytosolic
parts of GIRK1 and the glutathione S-transferase
(GST) were generated and isolated from bacterial
cultures. Recombinant proteins were submitted to
PKA-cs-catalysed phosphorylation in vitro, using
[
32
P]ATP[cP] as a co-substrate. Whereas the entire
C-terminus (amino acids 183–501) was found to be a

prominent target for PKA phosphorylation in vitro,
the N-terminus (amino acids 1–84) was only weakly
phosphorylated. Further truncation of the C-terminus
into two parts, a proximal one (G1 pC-T, amino acids
183–363) and a distal one (G1 dC-T, amino acids 365–
501), was performed to localize in more detail the
phosphorylation sites within the cytosolic C-terminal
part. The proximal part that is implicated in G
bc
bind-
ing and activation [25] was not found to be prone to
PKA phosphorylation, whereas the distal part was
extensively involved (Fig. 2A). Interestingly, this distal
part is unique for GIRK1 among the Kir3.x isoforms.
Further truncations of the C-terminus revealed a 49
amino acid stretch (amino acids 362–411) that was the
most prominent target for phosphorylation in vitro
amongst the peptides tested (Fig. 2B). The incorpo-
rated radioactivity relative to the amount of protein
Fig. 1. Back-phosphorylation of GIRK1, expressed in Xenopus
oocytes. Autoradiogram of SDS gel derived from immunoprecipi-
tated GIRK1 showing the incorporation of radioactive
32
P into
GIRK1. Before cell lysis and immunoprecipitation, SpCAMPS,
RpCAMPS or nothing (control) was injected into the oocytes the
oocytes. ), native oocytes; +, oocytes injected with RNA encoding
GIRK1 and GIRK4.
C. Mu
¨

llner et al. PKA phosphorylation of GIRK1
FEBS Journal 276 (2009) 6218–6226 ª 2009 The Authors Journal compilation ª 2009 FEBS 6219
Fig. 2. In vitro phosphorylation of recombinant, truncated, GIRK1 by the catalytic subunit of PKA. In the lower panels, the mean values and
standard error of mean values of relative specific radioactivity of the different constructs are plotted. The number of experiments is given in
parenthesis above each bar. The mean value differs statistically significant at the P < 0.01 (**) and P < 0.001 (***) levels compared to GST
alone. (A) Upper: autoradiogram of the different cytosolic regions of GIRK1. G1 pC-T, proximal C-terminus; G1 N-T, entire N-terminus; G1
dC-T, distal C-terminus. PKA-cs was present (+) or absent ()) from the reaction mixture. Lower: statistics of relative specific radioactivity
(radioactivity incorporated ⁄ amount of protein) in the different cytosolic regions of the GIRK1. G1 C-T, C-terminus. (B) Upper: Autoradiogram
of GIRK1 C-terminal fragments. Lower: statistics of relative specific radioactivity in the different regions of the GIRK1 C-terminus. (C) Upper:
autoradiogram of the entire GIRK1 C-terminus (wild-type and mutated). Lower: statistics of relative specific radioactivity incorporation into
the entire GIRK1 C-terminus (wild-type, single mutations, triple mutation and S385 + last 100 amino acids deleted). (D) Protein alignment of
the four different GIRK isoforms from rat (for GIRK3, the human sequence is shown). Transmembrane regions (TM1, TM2) and pore helix
(P) are marked. The N-terminal part that was used for G1 N-T, the region that was used for G1 pC-T and region that was used for G1 dC-T
are marked in different shades of gray. S385, S401 and T407 are marked in black (bold and underlined). Arrows indicate the peptides tested
for in vitro phosphorylation corresponding to Fig. 2B.
PKA phosphorylation of GIRK1 C. Mu
¨
llner et al.
6220 FEBS Journal 276 (2009) 6218–6226 ª 2009 The Authors Journal compilation ª 2009 FEBS
increases in the order G1 C-T > G1 dC-T > G1
362)410
(Fig. 2A, B) and is inversely related to the molecular
mass of the constructs, indicating the highest enrichment
of phosphorylation sites in G1
362)410
. Three canonical
PKA phosphorylation sites are located within this
region of GIRK1, namely serine 385, serine 401 and
threonine 407 [26]. Single mutations of these S ⁄ Ts to
cysteine (an amino acid with physicochemical properties

similar to serine and ⁄ or threonine but nonphosphoryla-
ble) in the corresponding peptide (G1
362)411
) signifi-
cantly reduced the amount of incorporated radioactive
32
P. The most effective result was obtained by simulta-
neous mutation of all three S ⁄ Ts (subsequently denoted
3*), resulting in an almost complete absence of PKA-cs-
catalysed incorporation of
32
P (data not shown). A simi-
lar pattern was observed for the entire C-terminus of
GIRK1, when the same mutations were introduced
alone or in combination (Fig. 2C). Mutation of S385C
in combination with a deletion of the last 100 amino
acids (DC
100
) was slightly more effective than the 3*
combination, indicating that an additional, but weak
determinant may be located distal to amino acid 411.
Functional aspects of mutation of S385, S401 and
T407
The effects of PKA-catalysed phosphorylation on
rat atrial I
K+ACh
as well as on basal and agonist
induced GIRK1 ⁄ GIRK4 (and also homooligomeric
GIRK1
F137S

) currents had been described previously
[18]. To assess the role of the S ⁄ Ts in the regulation of
GIRK1 via PKA in a manner that is unbiased by the
eventual contributions of the other subunits in a het-
erooligomer, the corresponding mutations were intro-
duced into GIRK1
F137S
, a mutant capable of forming
functional, homooligomeric, channels in Xenopus oo-
cytes [27,28]. The effects of cAMP injections on basal
currents recorded from wild-type GIRK1⁄ GIRK4 het-
erooligomers and GIRK1
F137S
homooligomers were
comparable in size, with the cAMP effect amounting to
0.31 ± 0.03 (mean ± SEM) in GIRK1 ⁄ GIRK4
heterooligomers and 0.35 ± 0.04 in GIRK1
F137S
homooligomers (Fig. 3). Because cAMP injections
had been shown to be effective on basal as well as on
agonist-induced currents [18], cAMP injections were
only occasionally performed during agonist application
(data not shown) and the systematic analysis in this
study was restricted to cAMP injections in the absence
an agonist. The effects observed in the single amino acid
mutant channels were generally reduced, with the
reduction being statistically significant only for the
S385C mutation (GIRK1
F137SS385C
: 0.21 ± 0.04;

GIRK1
F137SS401C
: 0.26 ± 0.05; GIRK1
F137ST407C
:
Fig. 3. Effect of cAMP injections on homooligomeric GIRK1 wild-
type and mutated channels. (A) Effect of cAMP injection on basal
current of homooligomeric GIRK1
F137SWT
channels. (B) As in (A),
but with currents originating from the triple-mutated GIRK1
F137S***
protein. (C) Statistics of the effects of cAMP on basal currents of
heterooligomeric GIRK1 ⁄ GIRK4, homooligomeric GIRK1 and
homooligomeric, mutated GIRK1 (the cAMP effect was assessed
as I
cAMP
⁄ I
HK
of a given oocyte). Data are the mean ± SEM. The
number of experiments is given in parenthesis above each bar.
The mean value differs statistically significantly at the P < 0.05 (*)
and P < 0.01 (**) levels compared to GIRK1
F137SWT
.
C. Mu
¨
llner et al. PKA phosphorylation of GIRK1
FEBS Journal 276 (2009) 6218–6226 ª 2009 The Authors Journal compilation ª 2009 FEBS 6221
0.25 ± 0.05). The 3* mutant channel exerted a 50.8%

reduction of the cAMP effect compared to
GIRK1
F137SWT
. This reduction was statistically signifi-
cant at P < 0.005 (the cAMP effect for GIRK1
F137S3*
in absolute numbers was 0.17 ± 0.05). It must be noted,
however, that the remaining effects observed in all the
mutations tested were still statistically significant com-
pared to control values (= no injection). This indicates
that direct PKA phosphorylation of the GIRK1 protein
strongly contributes to the effects of cAMP injection,
but that other, indirect, mediators of PKA action may
also exist.
Discussion
The results obtained in the present study demonstrate
that the GIRK1 subunit serves as PKA substrate both
in vitro and in vivo. This is in line with observations
obtained in another study demonstrating in vitro PKA
phosphorylation of the GIRK1 subunit after immuno-
precipitation of GIRK from bovine atrial plasma
membranes [17]. In the present study, we report for
the first time that the entire GIRK1 subunit serves as
a substrate for PKA-catalysed phosphorylation in vivo,
when coexpressed with GIRK4 in Xenopus oocytes.
Furthermore, this phosphorylation was regulated by
cytosolic injections of PKA activators and inhibitors,
suggesting it to be the basis for the functional effects
of cAMP injections on GIRK currents. PKA-induced
phosphorylation of the recombinant entire GIRK1

carboxy terminus in vitro had been reported by us
previously [22] and was recently confirmed by Lopes
et al. [23].
Previously, attempts were made to identify the struc-
tural determinant that is responsible for PKA phos-
phorylation of GIRK1. In a detailed investigation,
Medina et al. [17] coexpressed GIRK1 with GIRK4 in
HEK-293 cells. Truncation of GIRK1 after amino acid
373 resulted in a complete loss of spontaneous in vivo
phosphorous incorporation into the protein, whereas
truncation to amino acid 419 had no effect. However,
when all seven S ⁄ Ts located in this 46 amino acid
stretch, including S385, S401 and T407 described in
the present study, were mutated to alanines, incorpora-
tion of radioactive phosphorus was still observed to a
considerable extent. In this experiment, however, Med-
ina et al. [17] had measured total
32
P incorporation in
cell culture rather than phosphorylation directly cataly-
sed by PKA (as performed in the present study). We
conclude that the failure of these seven mutations to
abolish protein phosphorylation was a result of other
protein kinases masking the PKA-catalysed part.
Indeed, protein kinases other than PKA, including
tyrosine kinases, have been demonstrated to directly
phosphorylate GIRK1 [12]. The observation that
PP2A was unable to dephosphorylate the constitutively
phosphorylated GIRK1 protein to a considerable
extent but the 373–418 amino acid region was essential

for functional regulation by PP2A [17] further fosters
the hypothesis that GIRK1 serves as a substrate to a
manifold of protein kinases, whereas S385, S401 and
T407 are essential for specific PKA phosphorylation
and likely also for the dephosphorylation by PP2A.
Indeed, there is a broad overlap in substrate specificity
between PKA and PP2A and both enzymes are known
to colocalize in cellular microdomains, as well as in
atrial cardiomyocytes, together with GIRK1 [21,29]. In
another study, S221 and S315 were identified to be
involved in the inhibitory action of H89, a PKA inhib-
itor, on GIRK1 ⁄ GIRK4 currents in Xenopus oocytes
[23]. In the present study, we were unable to observe
PKA-catalysed incorporation of phosphate into the
peptides comprising these residues. Hence, we conclude
that both amino acids are indirectly involved in the
PKA regulation of the GIRK1 subunit but do not
serve directly as a substrate for PKA itself. Recently,
using a different experimental approach employing
mass spectroscopic methods, Rusinova et al. [30] has
identified S385 as a prominent and specific target for
PKA-catalysed phosphorylation in vitro, greatly sup-
porting the result obtained in the present study.
Amongst the three determinants identified by us, S385
had the greatest impact on PKA phosphorylation
in vitro, being almost as effective in abolishing the
phosphorylation of GIRK1 C-T as the triple mutation.
This is in excellent agreement with theoretical predic-
tions because: (i) arginines both at positions )2 and
)3 (viewed from S385) exist; (ii) a hydrophobic valine

is located at position +1; and (iii) a serine represents a
stronger determinant than threonine does [31]. In com-
parison, S401 and T407 have only a single specificity
determinant in their surroundings, a lysine at )3
(S401) and at )2 (T407), with lysine representing a
weaker determinant than arginine. Accordingly, their
contribution to PKA-catalysed in vitro phosphoryla-
tion is substantial, but smaller, compared to that of
S385. On the other hand S ⁄ T protein phosphatases,
especially PP2A, display a striking preference for phos-
phothreonyl residues over phosphoseryl residues and
hence T407 may represent an excellent target for this
enzyme [31].
Activation of heterooligomeric GIRK1 ⁄ GIRK4 and
homooligomeric GIRK1
F137S
by PKA is well estab-
lished [18,23]. The data obtained in the present study
show that S385, S401 and T407 contribute substan-
tially to this functional effect via the GIRK1 subunit
PKA phosphorylation of GIRK1 C. Mu
¨
llner et al.
6222 FEBS Journal 276 (2009) 6218–6226 ª 2009 The Authors Journal compilation ª 2009 FEBS
but PKA activation was still observed to some extent,
demonstrating that other, indirect, effects also contrib-
ute. Furthermore, Medina et al. [17] were unable to
completely eliminate dephosphorylation-mediated
effects on heterooligomeric GIRK1 ⁄ GIRK4 channels
by mutating seven S ⁄ Ts in GIRK1, including the struc-

tural determinants identified in the present study. We
suggest that also in this case the contribution of
GIRK1 was masked by the indirect actions of PKA.
For example, such an indirect mediator of PKA action
was identified recently as RGS10 in rat atrial cells [32].
Another possibility explaining why we were unable to
completely eliminate the effects of cAMP by mutating
S385, S401 and T407 may be that isoforms other than
GIRK1 may be also targets for protein phosphoryla-
tion in the protein complex: for example, GIRK2 had
been shown to exert dramatic PKA effects upon
cAMP injection [18]. In our specific case, residual, end-
ogeneous, GIRK5 subunits that were resistant to anti-
sense oligonucleotide treatment may have contributed
to the remaining PKA effect. The distal C-T of
GIRK1, which is unique amongst GIRK isoforms, has
given rise to various proposals about its peculiar func-
tion in the past. In the present study, we identified
three S⁄ Ts within this region that play an important
role in direct phosphorylation by PKA and in mediat-
ing PKA actions on the functional, homooligomeric
complex. The next objective in this field will be to
assess the possible contribution of GIRK1 isoforms to
PKA-mediated effects on heterooligomeric channels,
with the aim of understanding in more detail this
important regulation concerning G-protein activation
of K
+
channels.
Experimental procedures

Antibodies, reagents and solutions
GIRK1-Ab: Anti-Kir3.1 (APC-005; Alomone Labs, Ltd,
Jerusalem, Israel); Protein A Sepharose (CL-4B beads;
Pharmacia LKB Biotechnology AB, Uppsala, Sweden);
SpCAMPS, RpCAMPS (A166, A165, respectively; Sigma-
Aldrich, St Louis, MO, USA); [
32
P]ATP[cP] (25001748; GE
Healthcare Europe GmbH, Vienna, Austria); PKA-cs (1529
307; Boehringer Ingelheim GmbH, Ingelheim, Germany;
400 mU per 80 mL). All other reagents used were of
reagent grade throughout if not stated otherwise. Phosphor-
ylation buffer (PhB): 25 mmolÆL
)1
HEPES ⁄ Na; pH 7.4;
5 mmolÆL
)1
MgCl
2
; 5 mmolÆL
)1
EGTA; 0.05% Tween-20.
Homogenization buffer: 100 mmolÆL
)1
sodium phosphate
buffer; pH 5.8; 10 mmolÆL
)1
EDTA; 5 mmolÆL
)1
a-glycero-

phosphate; 5 mmolÆL
)1
BSA; 0.5 mmolÆL
)1
vanadate;
50 mmolÆL
)1
KF; 20% Triton-X-100. Seven mililiters of
buffer were supplemented with one tablet of complete mini
(Roche, Basel, Switzerland). 4· SDS-loading buffer:
400 mmolÆL
)1
Tris ⁄ Cl, pH 6.8, 20% sucrose, 4% SDS, 20%
mercaptoethnole; Comassie staining solution: 40 mgÆL
)1
Co-
massie blue, 500 mLÆL
)1
methanol, 100 mLÆL
)1
acetic acid;
Destain I: 500 mLÆL
)1
methanol, 100 mLÆL
)1
acetic acid;
Destain II: 50 mLÆL
)1
methanol, 70 mLÆL
)1

acetic acid.
ND96: 96 mmolÆL
)1
NaCl, 2 mmolÆL
)1
KCl, 1 mmolÆL
)1
MgCl
2
, 1 mmolÆL
)1
CaCl
2
, 5 mmolÆL
)1
Hepes, buffered with
NaOH to pH 7.4; NDE: same as ND96, but CaCl
2
was
1.8 mmolÆL
)1
and 2.5 mmolÆL
)1
pyruvate and 0.1% antibiot-
ics (G-1397; ·1000 stock from Sigma-Aldrich) were added;
HK: 96 mmolÆL
)1
KCl, 2 mmolÆL
)1
NaCl, 1 mmolÆL

)1
MgCl
2
, 1 mmolÆL
)1
CaCl
2
, 5 mmolÆL
)1
Hepes buffered
with KOH to pH 7.4; Glutathione buffer: 120 mmolÆL
)1
NaCl; 0.05% Tween-20; 100 mmolÆL
)1
Tris; 15 mmolÆL
)1
glutathione pH 8.0.
Immunoprecipitation
For the immunoprecipitation experiments, Xenopus laevis
oocytes were injected with cRNAs coding for GIRK1 and
GIRK4 (5 ng per oocyte for each RNA) and the KHA2
antisense oligonucleotide as described previously [33] (25 ng
per oocyte). After incubation of cells for 6 days at 19 °C,
the oocytes were checked for expression with the two elec-
trode voltage clamp technique as described below and the
cells were injected with either SpcAMPS or RpcAMPS
(2.5 mmolÆL
)1
, 20 nL per oocyte). Incubation with PKA
activator ⁄ inhibitor was allowed to take place for approxi-

mately 30 min; thereafter, oocytes were homogenized in
homogenization buffer by pipetting up and down. Immuno-
precipitation of GIRK1 channels from 15 oocytes solubi-
lized in 100 lL of homogenization buffer was initiated by
adding 4 lL of non-immune IgG and incubating for 1 h at
room temperature to prevent unspecific binding. Twenty
microliters of 10% Protein A Sepharose per reaction were
added for precipitation of unspecific antibody complexes,
whereas 4 lL of GIRK1-Ab were added to the supernatant.
The immunoprecipitation reaction was incubated over night
under constant agitation at 4 °C. Antibody complexes were
precipitated by another addition of 10% Protein A Sepha-
rose and incubation for 1 h at 4 °C.
Back-phosphorylation
In vitro back-phosphorylation was performed after the
immunoprecipitate was washed twice with ice-cold phos-
phorylation buffer. Then, 1 lL[
32
P]ATP[cP] and 1 lLof
PKA-cs were added to the pellet for 5 min at 30 °C. Subse-
quently, the reaction was put on ice, washed twice with
phosphorylation buffer and supplemented with 40 lLof
SDS-loading buffer. The denatured proteins were loaded on
a 10% SDS gel [34] and run for 1 h at 150 V. Afterwards,
the gel was stained with Comassie staining solution,
C. Mu
¨
llner et al. PKA phosphorylation of GIRK1
FEBS Journal 276 (2009) 6218–6226 ª 2009 The Authors Journal compilation ª 2009 FEBS 6223
destained and dried on a slab gel dryer before exposure to

X-ray film.
Genetic engineering
Plasmid vectors were grown in bacteria, purified and linear-
ized using standard procedures [35]. Plasmids with inserts
encoding m
2
R [18] and GIRK1
F137S
[28] have been
described previously. Plasmids for the production of recom-
binant protein were constructed by a PCR, where forward
and reverse primers encoded the desired parts of the C- and
N-termini of GIRK1. These sequences were each preceded
or followed, respectively, by restriction recognition
sequences appropriate for cloning in frame with GST in the
bacterial expression vector pGEX-4T-1. Isolated PCR prod-
ucts were digested with the appropriate enzymes, ligated
into pGEX-4T-1 and the sequences were verified by
conventional sequencing. Mutants of GIRK1
F137S
and
truncated GIRK1 ⁄ GST fusion constructs for protein pro-
duction were produced by PCR using homologous primers
containing the appropriate mutation in addition to a silent
mutation creating an additional restriction site to facilitate
identification of the mutants. Before bacterial transforma-
tion, template DNA was digested with DpnI as described
previously [35].
Recombinant protein purification
Constructs were transfected into BL-21(RIL) competent

cells (Stratagene, La Jolla, CA, USA), the corresponding
proteins were overexpressed and purified as described previ-
ously [25]. Protein was quantitated by the method of Brad-
ford [36], diluted to a concentration of 1 lgÆlL
)1
, and
aliquots were shock frozen in liquid N
2
and stored at
)70 °C until use.
In vitro phosphorylation
One microgram of the appropriate protein was incubated in
300 lL of PhB containing 18.5 kBq [
32
P]ATP[cP] and
0.4 lL of PKA-cs for 30 min at room temperature (agitated
by a Labquake laboratory shaker; Cole-Parmer Instrument
Company, Vernon Hills, IL, USA). Next, 30 lL of gluta-
thione Sepharose 4B beads (washed and suspended in PhB)
were added and incubation ⁄ agitation continued for another
30 min. Samples were centrifuged (1 min; maximum g;
picofuge, Stratagene) and the supernatant discarded care-
fully. Beads were washed three times in 1 mL of PhB by
resuspension and centrifugation. Finally, the protein was
eluted by adding 30 lL of glutathione buffer to the beads
and incubating for 10 min. Thirty-two microliters of super-
natant were removed, combined with 10 lLof4· SDS
loading buffer and run on a 12% SDS gel [34]. Gels were
stained with Comassie blue, dried and scanned. Subse-
quently, autoradiograms were performed using the Storm

Phosphorimager (GE Healthcare Europe GmbH, Vienna,
Austria). Incorporation of radioactive
32
P into the protein
was quantitated and normalized to the total amount of pro-
tein as detected by the Comassie stain (= relative specific
radioactivity).
Xenopus laevis oocyte expression
Oocytes were prepared as described previously [37]. Approxi-
mately 24 h afterwards, they were injected with the KHA2
antisense oligonucleotide (25 ng per oocyte) together with the
appropriate RNA (amounts in pg per oocyte): m
2
R: 1500;
GIRK1
F137S
: 37.5; GIRK1
F137SS385C
: 37.5; GIRK1
F137SS401C
:
37.5; GIRK1
F137ST407C
: 37.5; GIRK1
F137SS385CS401CT407C
:
150. Oocytes were kept in NDE at 19 °C for 3–5 days after
injection before electrophysiological experiments were
performed.
Electrophysiology

Oocytes were placed in a recording chamber, allowing
superfusion with either ND96 or HK (with and without
10
)5
molÆL
)1
acetylcholine) at 21 °C and currents were
recorded via the two electrode voltage clamp technique
using agarose cushion electrodes [38] and the Geneclamp
500 amplifier (Axon Instruments, Foster City, CA, USA).
Membrane potential was kept at )80 mV and the medium
was changed from ND96 to HK, HK+ acetylcholine, HK
and back to ND96. Cytosolic injection of cAMP and
cAMP analogs (30–60 pmol per oocyte) was performed as
described previously [18]. The current increase, following
cAMP injection, was normalized to the basal current of
the given oocyte (where the basal current is defined as the
current induced by a change of the extracellular medium
from ND96 to HK, designated as I
HK
in Fig. 3). Current
traces were low pass filtered at 10 Hz and digitized using
the Digidata 1322A interface (Axon Instruments) con-
nected to computer running pclamp 9.2 software (Axon
Instruments).
Statistical analysis
Given experimental groups were tested for statistical signifi-
cant differences using Student’s t-test and sigmaplot 9.0
(Systat Software Inc., Chicago, IL, USA).
Acknowledgements

We thank Dr D. Logothetis (Virginia Commonwealth
University, Richmond, VA, USA) for kindly providing
the clone encoding G1
F137S
and Dr T. DeVaney
(Medical University of Graz, Graz, Austria) for
correcting the English language. Support provided by
PKA phosphorylation of GIRK1 C. Mu
¨
llner et al.
6224 FEBS Journal 276 (2009) 6218–6226 ª 2009 The Authors Journal compilation ª 2009 FEBS
the Austrian Research Foundation (SFB708) and the
Research Foundation of the Austrian National Bank
(OENB12575) is gratefully acknowledged.
References
1 Dascal N (2001) Ion-channel regulation by G proteins.
Trends Endocrinol Metab 12, 391–398.
2 Dascal N (1997) Signalling via the G protein-activated
K+ channels. Cell Signal 9, 551–573.
3 Smith PA, Sellers LA & Humphrey PPA (2001)
Somatostatin activates two types of inwardly rectifying
K+ channels in MIN-6 cells. J Physiol (Lond) 532,
127–142.
4 Shankar H, Kahner BN, Prabhakar J, Lakhani P,
Kim S & Kunapuli SP (2006) G-protein-gated inwardly
rectifying potassium channels regulate ADP-induced
cPLA(2) activity in platelets through Src family kinases.
Blood 108, 3027–3034.
5 Perry CA, Pravetoni M, Teske JA, Aguado C, Erickson
DJ, Medrano JF, Lujan R, Kotz CM & Wickman K

(2008) Predisposition to late-onset obesity in GIRK4
knockout mice. Proc Natl Acad Sci USA 105, 8148–
8153.
6 Iwanir S & Reuveny E (2008) Adrenaline-induced
hyperpolarization of mouse pancreatic islet cells is
mediated by G protein-gated inwardly rectifying
potassium (GIRK) channels. Pflugers Arch 456, 1097–
1108.
7 Stringer BK, Cooper AG & Shepard SB (2001) Overex-
pression of the G-protein inwardly rectifying potassium
channel 1 (GIRK1) in primary breast carcinomas corre-
lates with axillary lymph node metastasis. Cancer Res
61, 582–588.
8 Schreibmayer W, Dessauer CW, Vorobiov D, Gilman
AG, Lester HA, Davidson N & Dascal N (1996) Inhibi-
tion of an inwardly rectifying K+ channel by G-protein
alpha-subunits. Nature 380, 624–627.
9 Rubinstein M, Peleg S, Berlin S, Brass D & Dascal N
(2007) G alpha(i3) primes the G protein-activated K+
channels for activation by coexpressed G beta gamma in
intact Xenopus oocytes. J Physiol (Lond) 581, 17–32.
10 Ruppersberg JP (2000) Intracellular regulation of
inward rectifier K+ channels. Pflugers Arch 441, 1–11.
11 Mao JZ, Wang XR, Chen FX, Wang RP, Rojas A,
Shi Y, Piao H & Jiang C (2004) Molecular basis for the
inhibition of G protein-coupled inward rectifier K+
channels by protein kinase C. Proc Natl Acad Sci USA
101, 1087–1092.
12 Mora SI & Escobar LI (2005) Phosphorylation of a
tyrosine at the N-terminus regulates the surface expres-

sion of GIRK5 homomultimers. FEBS Lett 579, 3019–
3023.
13 Ippolito DL, Xu M, Bruchas MR, Wickman K &
Chavkin C (2005) Tyrosine phosphorylation of K(ir)3.1
in spinal cord is induced by acute inflammation, chronic
neuropathic pain, and behavioral stress. J Biol Chem
280, 41683–41693.
14 Chung HJ, Qian X, Ehlers M, Jan YN & Jan LY
(2009) Neuronal activity regulates phosphorylation-
dependent surface delivery of G protein-activated
inwardly rectifying potassium channels. Proc Natl Acad
Sci USA 106, 629–634.
15 Huang CL, Feng SY & Hilgemann DW (1998) Direct
activation of inward rectifier potassium channels by
PIP2 and its stabilization by G beta gamma. Nature
391, 803–806.
16 Zhang HL, He C, Yan XX, Mirshahi T & Logothetis
DE (1999) Activation of inwardly rectifying K+ chan-
nels by distinct PtdIns(4,5)P-2 interactions. Nat Cell
Biol 1, 183–188.
17 Medina I, Krapivinsky G, Arnold S, Kovoor P,
Krapivinsky L & Clapham DE (2000) A switch
mechanism for G ss gamma activation of I-KACh.
J Biol Chem 275, 29709–29716.
18 Mullner C, Vorobiov D, Bera AK, Uezono Y,
Yakubovich D, Frohnwieser-Steinecker B, Dascal N &
Schreibmayer W (2000) Heterologous facilitation of
G protein-activated K+ channels by beta-adrenergic
stimulation via cAMP-dependent protein kinase. J Gen
Physiol 115, 547–557.

19 Kim D (1990) Beta-adrenergic regulation of the
muscarinic-gated K+ channel via cyclic Amp-dependent
protein-kinase in atrial cells. Circ Res 67, 1292–1298.
20 Mullner C, Yakubovich D, Dessauer CW, Platzer D &
Schreibmayer W (2003) Single channel analysis of the
regulation of GIRK1 ⁄ GIRK4 channels by protein
phosphorylation. Biophys J 84, 1399–1409.
21 Nikolov EN & Ivanova-Nikolova TT (2004) Coordina-
tion of membrane excitability through a GIRK1 signal-
ing complex in the atria. J Biol Chem 279, 23630–
23636.
22 Muellner C, Steinecker-Frohnwieser B & Schreibmayer
WF (2002) Structural determinant responsible for the
cAMP dependent phosphorylation of the GIRK1 sub-
unit. Biophysical Society Meeting Abstracts Biophys J
Supplement, 82, 2890-Pos.
23 Lopes CMB, Remon JI, Matavel A, Sui JL, Keselman I,
Medei E, Shen YM, Rosenhouse-Dantsker A, Rohacs T
& Logothetis DE (2007) Protein kinase A modulates
PLC-dependent regulation and PIP2-sensitivity of K+
channels. Channels 1, 124–134.
24 Krapivinsky G, Gordon EA, Wickman K, Velimirovic
B, Krapivinsky L & Clapham DE (1995) The G-pro-
tein-gated atrial K+ channel I-Kach is a Heteromulti-
mer of 2 inwardly rectifying K+-channel proteins.
Nature 374, 135–141.
25 Ivanina T, Rishal I, Varon D, Mullner C, Frohnwieser-
Steinecke B, Schreibmayer W, Dessauer CW & Dascal
N (2003) Mapping the G beta gamma-binding sites in
C. Mu

¨
llner et al. PKA phosphorylation of GIRK1
FEBS Journal 276 (2009) 6218–6226 ª 2009 The Authors Journal compilation ª 2009 FEBS 6225
GIRK1 and GIRK2 subunits of the G protein-activated
K+ channel. J Biol Chem 278, 29174–29183.
26 Kennelly PJ & Krebs EG (1991) Consensus sequences
as substrate-specificity determinants for protein-kinases
and protein phosphatases. J Biol Chem 266, 15555–
15558.
27 Chan KW, Sui JL, Vivaudou M & Logothetis DE
(1996) Control of channel activity through a unique
amino acid residue of a G protein-gated inwardly recti-
fying K+ channel subunit. Proc Natl Acad Sci USA 93,
14193–14198.
28 Vivaudou M, Chan KW, Sui JL, Jan LY, Reuveny E &
Logothetis DE (1997) Probing the G-protein regulation
of GIRK1 and GIRK4, the two subunits of the K-ACh
channel, using functional homomeric mutants. J Biol
Chem 272, 31553–31560.
29 McConnachie G, Langeberg LK & Scott JD (2006)
AKAP signaling complexes: getting to the heart of the
matter. Trends Mol Med 12, 317–323.
30 Rusinova R, Shen Y-MA, Dolios G, Padovan J, Yang
H, Kirchberger M, Wang R & Logothetis DE (2009)
Mass spectrometric analysis reveals a functionally
important PKA phosphorylation site in a Kir3 channel
subunit. Pflugers Arch-Eur J Physiol 458, 303–314.
31 Pinna LA & Ruzzene M (1996) How do protein kinases
recognize their substrates? Biochim Biophys Acta 1314,
191–225.

32 Bender K, Nasrollahzadeh P, Timpert M, Liu B, Pott L
& Kienitz MC (2008) A role for RGS10 in beta-adren-
ergic modulation of G-protein-activated K+ (GIRK)
channel current in rat atrial myocytes. J Physiol (Lond)
586, 2049–2060.
33 Hedin KE, Lim NF & Clapham DE (1996) Cloning
of a Xenopus laevis inwardly rectifying K+ channel
subunit that permits GIRK1 expression of I-KACh
currents in oocytes. Neuron 16, 423–429.
34 Laemmli UK (1970) Cleavage of structural proteins
during assembly of head of bacteriophage-T4. Nature
227, 680–685.
35 Sambrook J & Russel DW (2001) Molecular Cloning,
3 edn. CSH, Cold Spring Harbour.
36 Bradford MM (1976) Rapid and sensitive method
for quantitation of microgram quantities of protein
utilizing principle of protein-dye binding. Anal Biochem
72, 248–254.
37 Dascal N & Lotan I (1992) Expression of exogenous
ion channels and neurotransmitter receptors in RNA-
injected Xenopus oocytes.InMethods in Neurobiology
(Longstaff ARP, ed), pp. 205–225. Humana Press,
Totowa, NJ.
38 Schreibmayer W, Lester HA & Dascal N (1994) Voltage
clamping of Xenopus laevis oocytes utilizing agarose-
cushion electrodes. Pflugers Arch 426, 453–458.
PKA phosphorylation of GIRK1 C. Mu
¨
llner et al.
6226 FEBS Journal 276 (2009) 6218–6226 ª 2009 The Authors Journal compilation ª 2009 FEBS