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 eliminat
Trang 1for 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, PIP2, 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 IK+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 32P 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.
Trang 2stimulatory 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 Gb ⁄ c [20] A signalling complex comprising
(amongst other signalling molecules) GIRK1, GIRK4,
Gb⁄ 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
[32P]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 vivoin 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 [32P]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 Gbc 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.
Trang 3Fig 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.
Trang 4increases in the order G1 C-T > G1 dC-T > G1362)410
(Fig 2A, B) and is inversely related to the molecular
mass of the constructs, indicating the highest enrichment
of phosphorylation sites in G1362)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 (G1362)411)
signifi-cantly reduced the amount of incorporated radioactive
32P 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 of32P (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 (DC100) 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 IK+ACh as well as on basal and agonist
induced GIRK1⁄ GIRK4 (and also homooligomeric
GIRK1F137S) 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 GIRK1F137S, 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 GIRK1F137S 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 GIRK1F137S
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 (GIRK1F137SS385C: 0.21 ± 0.04;
GIRK1F137SS401C: 0.26 ± 0.05; GIRK1F137ST407C:
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 GIRK1F137S*** 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 IcAMP⁄ 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
Trang 50.25 ± 0.05) The 3* mutant channel exerted a 50.8%
reduction of the cAMP effect compared to
GIRK1F137SWT This reduction was statistically
signifi-cant at P < 0.005 (the cAMP effect for GIRK1F137S3*
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 total32P 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 posipredic-tions )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 GIRK1F137S 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
Trang 6but 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); [32P]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 MgCl2; 5 mmolÆL)1 EGTA; 0.05% Tween-20
Homogenization buffer: 100 mmolÆL)1 sodium phosphate
buffer; pH 5.8; 10 mmolÆL)1EDTA; 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)1Tris⁄ Cl, pH 6.8, 20% sucrose, 4% SDS, 20% mercaptoethnole; Comassie staining solution: 40 mgÆL)1 Co-massie blue, 500 mLÆL)1methanol, 100 mLÆL)1acetic 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)1NaCl, 2 mmolÆL)1 KCl, 1 mmolÆL)1 MgCl2, 1 mmolÆL)1CaCl2, 5 mmolÆL)1Hepes, buffered with NaOH to pH 7.4; NDE: same as ND96, but CaCl2 was 1.8 mmolÆL)1and 2.5 mmolÆL)1pyruvate and 0.1% antibiot-ics (G-1397; ·1000 stock from Sigma-Aldrich) were added; HK: 96 mmolÆL)1 KCl, 2 mmolÆL)1NaCl, 1 mmolÆL)1 MgCl2, 1 mmolÆL)1 CaCl2, 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 19C, 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 4C Antibody complexes were precipitated by another addition of 10% Protein A Sepha-rose and incubation for 1 h at 4C
Back-phosphorylation
In vitro back-phosphorylation was performed after the immunoprecipitate was washed twice with ice-cold phos-phorylation buffer Then, 1 lL [32P]ATP[cP] and 1 lL of PKA-cs were added to the pellet for 5 min at 30C Subse-quently, the reaction was put on ice, washed twice with phosphorylation buffer and supplemented with 40 lL of 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,
Trang 7destained 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 m2R [18] and GIRK1F137S [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 GIRK1F137S 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 N2 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 [32P]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 lL of 4· 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 32P 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): m2R: 1500; GIRK1F137S: 37.5; GIRK1F137SS385C: 37.5; GIRK1F137SS401C: 37.5; GIRK1F137ST407C: 37.5; GIRK1F137SS385CS401CT407C:
150 Oocytes were kept in NDE at 19C 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)5molÆL)1 acetylcholine) at 21C 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 IHK 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 G1F137S and Dr T DeVaney (Medical University of Graz, Graz, Austria) for correcting the English language Support provided by
Trang 8the Austrian Research Foundation (SFB708) and the
Research Foundation of the Austrian National Bank
(OENB12575) is gratefully acknowledged
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