A Switch Mechanism for Gβγ Activation of IKACh
2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês
10.1074/jbc.m004989200
ISSN1083-351X
AutoresIgor Medina, Grigory Krapivinsky, Susanne Arnold, Pramesh Kovoor, Luba Krapivinsky, David E. Clapham,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoG protein-gated inwardly rectifying potassium (GIRK) channels are a family of K+-selective ion channels that slow the firing rate of neurons and cardiac myocytes. GIRK channels are directly bound and activated by the G protein Gβγ subunit. As heterotetramers, they comprise the GIRK1 and the GIRK2, -3, or -4 subunits. Here we show that GIRK1 but not the GIRK4 subunit is phosphorylated when heterologously expressed. We found also that phosphatase PP2A dephosphorylation of a protein in the excised patch abrogates channel activation by Gβγ. Experiments with the truncated molecule demonstrated that the GIRK1 C-terminal is critical for both channel phosphorylation and channel regulation by protein phosphorylation, but the critical phosphorylation sites were not located on the C terminus. These data provide evidence for a novel switch mechanism in which protein phosphorylation enables Gβγ gating of the channel complex. G protein-gated inwardly rectifying potassium (GIRK) channels are a family of K+-selective ion channels that slow the firing rate of neurons and cardiac myocytes. GIRK channels are directly bound and activated by the G protein Gβγ subunit. As heterotetramers, they comprise the GIRK1 and the GIRK2, -3, or -4 subunits. Here we show that GIRK1 but not the GIRK4 subunit is phosphorylated when heterologously expressed. We found also that phosphatase PP2A dephosphorylation of a protein in the excised patch abrogates channel activation by Gβγ. Experiments with the truncated molecule demonstrated that the GIRK1 C-terminal is critical for both channel phosphorylation and channel regulation by protein phosphorylation, but the critical phosphorylation sites were not located on the C terminus. These data provide evidence for a novel switch mechanism in which protein phosphorylation enables Gβγ gating of the channel complex. GTP binding protein G protein-linked inwardly rectifying K+ channels 1–4 acetylcholine l-α-phosphatidyl-d-myo-inositol-4,5-bisphosphate phosphoprotein phosphatase 2A green fluorescence protein calmodulin kinases I and II protein kinase A protein kinase C adenosine 5′-(β,γ-imino)triphosphate 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid phosphatidyl inositol At least eight transmitters activate a class of inward rectifier K+ channels via an apparently identical GTP binding protein (G protein)1-linked signal transduction mechanism. The G protein-linked receptor subtypes that activate these channels include muscarinic (m2), γ-aminobutyric acid (GABAB), serotonin (5HT1A), adenosine (P1), somatostatin, enkephalin (μ, κ, δ), α2-adrenergic, and dopamine (D2) receptors (1Wickman K. Nemec J. Gendler S.J. Clapham D.E. Neuron. 1998; 20: 103-114Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). G protein-linked receptors couple to a heterotrimeric protein complex of Gα and Gβγ subunits. After these receptors catalyze the transfer of GTP to replace GDP on the Gα subunit, the freed Gβγ subunit directly binds and activates the GIRK channel (2Krapivinsky G. Gordon E. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar, 3Logothetis D.E. Kurachi Y. Galper J. Neer E. Clapham D.E. Nature. 1987; 325: 321-326Crossref PubMed Scopus (854) Google Scholar, 4Wickman K. Iniguez-Lluhi J. Davenport P. Taussig R.A. Krapivinsky G.B. Linder M.E. Gilman A. Clapham D.E. Nature. 1994; 368: 255-257Crossref PubMed Scopus (377) Google Scholar). These G protein-linked Inwardly Rectifying K+(GIRK) channels play a role predominantly in the pacing range of cardiac cells and in the regenerative firing of neuronal cells where they oppose the slow depolarization of such currents as Ifin heart or Ih in neurons (HCN channel class) and can compensate for inactivation of the M current in neurons. In the classic example, acetylcholine (ACh) secreted from the vagus nerve binds cardiac muscarinic receptors, initiating a sequence of events leading to slowing of heart rate (1Wickman K. Nemec J. Gendler S.J. Clapham D.E. Neuron. 1998; 20: 103-114Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 5Hartzell H.C. Kuffler S.W. Stickgold R. Yoshikami D. J. Physiol. ( Lond. ). 1977; 271: 817-846Crossref PubMed Scopus (130) Google Scholar, 6Loewi O. Navratil E. Pfluegers Arch. 1926; 214: 678-688Crossref Scopus (116) Google Scholar). The GIRK channel class has four members, GIRK1, GIRK2, GIRK3, and GIRK4. GIRK1 is unique among the four in having a long C-terminal tail, whereas GIRK2, -3, and -4 are quite similar. Cardiac IKAChis composed of two homologous inward rectifier K+ channel subunits, GIRK1 and GIRK4 (2Krapivinsky G. Gordon E. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar), which form a heterotetramer consisting of two GIRK1 and two GIRK4 subunits (7Corey S. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 1998; 273: 5271-5278Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Similar complexes comprised of GIRK1 and GIRK2 (8Kofuji P. Davidson N. Lester H.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6542-6546Crossref PubMed Scopus (266) Google Scholar, 9Lesage F. Duprat F. Fink M. Guillemare E. Coppola T. Lazdunski M. Hugnot J.P. FEBS Lett. 1994; 353: 37-42Crossref PubMed Scopus (267) Google Scholar, 10Duprat F. Lesage F. Guillemare E. Fink M. Hugnot J.P. Bigay J. Lazdunski M. Romey G. Barhanin J. Biochem. Biophys. Res. Commun. 1995; 212: 657-663Crossref PubMed Scopus (132) Google Scholar), or GIRK1 and GIRK3 (11Dissmann E. Wischmeyer E. Spauschus A. Von P.D. Karschin C. Karschin A. Biochem. Biophys. Res. Commun. 1996; 223: 474-479Crossref PubMed Scopus (27) Google Scholar, 12Jelacic T.M. Sims S.M. Clapham D.E. J. Membr. Biol. 1999; 169: 123-129Crossref PubMed Scopus (47) Google Scholar) form primarily neuronal G protein-gated K+ channels. IKACh channel activity is also modulated by levels of intracellular Na+ (13Sui J.L. Chan K.W. Logothetis D.E. J. Gen. Physiol. 1996; 108: 381-391Crossref PubMed Scopus (102) Google Scholar), ATP (2Krapivinsky G. Gordon E. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar, 13Sui J.L. Chan K.W. Logothetis D.E. J. Gen. Physiol. 1996; 108: 381-391Crossref PubMed Scopus (102) Google Scholar, 14Kim D. Watson M. Indyk V. Am. J. Physiol. 1997; 272: H195-H206PubMed Google Scholar, 15Kim D. J. Physiol. ( Lond. ). 1991; 437: 133-155Crossref PubMed Scopus (60) Google Scholar), phosphatidylinositol bisphosphate (16Huang C.L. Feng S.Y. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (755) Google Scholar, 17Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (211) Google Scholar), and fatty acids (18Kim D. Pleumsamran A. J. Gen. Physiol. 2000; 115: 287-304Crossref PubMed Scopus (29) Google Scholar). These agents are not required for Gβγ activation of the channel. Two mechanisms are plausible for ATP regulation of channel activity. The first mechanism is phosphorylation at one or more sites on the channel or an associated regulatory protein that changes the open probability of the channel. The second proposed mechanism is one in which the level of PIP2, a critical cofactor for activation, is regulated by ATP (16Huang C.L. Feng S.Y. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (755) Google Scholar, 17Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (211) Google Scholar). Levels of phosphorylation of many proteins are regulated by the coordinated activities of protein kinases and phosphatases. We investigated whether IKACh activity was also regulated by such a mechanism. Here we show that the GIRK1 subunit of IKACh is phosphorylated both in vitro andin vivo. Furthermore, the treatment of inside-out patches with phosphoprotein phosphatase 2A (PP2A) prevents channel activation by Gβγ. Site-directed mutagenesis experiments demonstrate that the GIRK1 C terminus is critical for both channel phosphorylation and channel activity regulation by protein phosphorylation. Atrial auricles from neonatal rat (postnatal day 2) were microdissected from total heart tissue and digested using a myocyte isolation kit according to the manufacturer's specifications (Worthington, Lakewood, NJ). Dispersed cells were plated onto fibronectin-coated glass coverslips in Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 5% fetal bovine serum, and cultured in 10% CO2 at 37 °C for 2 days. CHO-K1 and HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal calf serum (Sigma Chemical Co., St. Louis, MO) at 37 °C, 5% CO2. Cells were plated at 3 × 106 per 100-mm dish or 4 × 105 cells per 35-mm dish, respectively, 1 day prior to transfection. Both cell lines were transfected using TransIT LT-2 (PanVera Corp.). Each 100-mm or 35-mm plate was transfected with 5 and 1.2 μg of plasmid DNA, respectively (40% of each plasmid containing either wild type or mutant GIRK1 or GIRK4 cDNA and 20% of pGREEN Lantern-GFP; Life Technologies, Inc.). Bovine atria plasma membranes were isolated as described (2Krapivinsky G. Gordon E. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar). Membranes were solubilized in immunoprecipitation (IP) buffer containing (in mm): 10 HEPES, 1 EDTA, 1 dithiothreitol, 100 NaCl, and 1.0% Triton X-100 supplemented with protease inhibitors (0.5 mmphenylmethylsulfonyl fluoride and 2 μg/ml each of leupeptin, aprotinin, and pepstatin). Native IKACh was immunoprecipitated with anti-GIRK4 (aCIRN2) and anti-GIRK1 (aCsh) (2Krapivinsky G. Gordon E. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar). Proteins were immunoprecipitated for 1.5 h at 4 °C with the corresponding antibody and Protein G-Sepharose (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Immunoprecipitates were washed four times with RIPA buffer (15 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris-Cl, pH 8.0), followed by two washes with an appropriate phosphorylation buffer.32P-Labeled cells expressing recombinant GIRK1-AU5/GIRK4 were lysed in IP buffer supplemented with phosphatase inhibitor mixture (in mm); 50 NaF, 1 Na3VO4, 5 EDTA, and 0.1 μm okadaic acid. Expressed channel hetero-oligomers or individual subunits were immunoprecipitated with AU5 (Babco, Richmond, CA), FLAG (Sigma), or anti-CIRN2 antibody. Immunoprecipitated atrial IKAChwas phosphorylated for 30 min at 30 °C in 50 μl of the appropriate kinase buffer containing 10 μm [γ-32P]ATP (5 Ci/mmol), immunoprecipitate from 0.35 mg of the atria membranes, and 1 unit of protein kinase. The reaction was stopped by boiling in SDS buffer. For the dephosphorylation reaction, the in vitrophosphorylated immunoprecipitate was washed in phosphatase buffer and incubated with 0.5 unit of appropriate phosphatase for 20 min at room temperature. Reaction buffers were formulated according to the manufacturer's protocols. For in vivo 32P labeling, cells were transferred to the phosphate-free media supplemented with 10% dialyzed fetal bovine serum for 2 h followed by 3 h in the same media containing 1 mCi/ml [32P]orthophosphate (NEN Life Science Products, Boston, MA). For dephosphorylation of in vivo phosphorylated GIRK1, microsomal fractions of HEK-293 cells co-expressing GIRK1and GIRK4 and metabolically labeled with 32P were isolated as described (19Nelson D.A. Aguilar-Bryan L. Bryan J. J. Biol. Chem. 1992; 267: 14928-14933Abstract Full Text PDF PubMed Google Scholar). Membranes were resuspended in the solution used for patch-clamp recording and split into two samples, and one of the samples was treated with PP2A (1 unit/μl) for 15 min at room temperature followed by solubilization and immunoprecipitation as described above. Ptd[2- 3H]Ins(4,5)P2(22,200 cpm, NEN Life Science Products) was mixed with unlabeled PIP2 (Calbiochem), dried, resuspended in solution used for electrophysiological recording (in mm: 140 KCl, 5 EGTA, 10 K-HEPES, 2.0 MgCl2, pH 7.2) at a final concentration of 5 μm, and sonicated for 10 min on ice. Sonicated Ptd[2-3H]Ins(4,5)P2 was then incubated with protein phosphatase 2A (0.01 unit/μl) or protein phosphatase 2A catalytic subunit (0.02 unit/μl) for 30 min at 37 °C. Lipids were extracted with chloroform/methanol/1m HCl (1:1:0.1), and PIP2, PIP, and PI were separated by thin layer chromatography (20Nishikawa K. Toker A. Wong K. Marignani P.A. Johannes F.J. Cantley L.C. J. Biol. Chem. 1998; 273: 23126-23133Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Briefly, lipids were spotted onto a thin layer chromatography plate precoated with 1% potassium oxalate. The separation of the lipids was performed in 2m acetic acid/n-propanol (35/65) overnight, and plates were sprayed with EN3Hance (NEN Life Science Products). Samples were visualized by autoradiography at −80 °C for 24 h. The deletion mutant Δ373-GIRK1 was described previously (21Kennedy M.E. Nemec J. Corey S. Wickman K. Clapham D.E. J. Biol. Chem. 1999; 274: 2571-2578Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The other GIRK1 mutants were generated using the QuikChange (Stratagene) site-directed method. pCDNA3.1 vector carrying the coding region of C-terminally tagged GIRK1(GIRK1-AU5) or N-terminally tagged GIRK1(GIRK1-FLAG) was used as a template for mutagenesis. Mutations were verified by DNA sequencing. Electrophysiological recordings were performed from rat atrial cardiomyocytes, CHO-K1, and HEK-293 cells using the patch-clamp technique in the inside-out configuration. GFP-expressing cells were visualized using an inverted fluorescence microscope. Pipette and bath solutions were identical and contained (in mm): 140 KCl, 5 EGTA, 10 K-HEPES, 2.0 MgCl2, pH 7.2. The resistance of the recording borosilicate glass pipettes was 6–10 MΩ. The relatively high resistance of the patch pipette reflected a small tip diameter. Patches incorporated three to six channels and permitted us to resolve single channel openings after channel activation with Gβγ (see Figs. 2 and 4). Experiments were performed only on patches showing basal activity of IKAChin cell-attached and inside-out configurations (channel opening probability (NPo) ranging from 10−4to 10−2) to ensure the presence of a channel in the patch and to standardize the recording conditions. Single-channel currents were recorded at a holding potential of −80 mV using an Axopatch 200B amplifier (Axon Instruments, Inc.). Data were filtered at 5 kHz and recorded on compact discs using a CDR-400 recorder (Biologic) for off-line analysis. The opening kinetics of single channels were analyzed by digitizing 5- to 10-s stretches of records at 20 kHz (fo = 5 kHz) through a DigiData 1200 analog-to-digital converter using pClamp 7 software (Axon Instruments, Inc.). To calculate the NPo, records of whole experiments were digitized at 2 kHz and analyzed using pClamp 7 software. A specially designed low volume (30 μl) perfusion system was used to apply and wash out drugs during inside-out recordings. All experiments were performed at room temperature (22 ± 2 °C). All results are presented as the mean ± S.E. The Student's t test was used to compare groups of data with a significant difference taken to be at the 5% level.Figure 4PP2A pretreatment modulates the activity of GIRK1/GIRK4 channels expressed in mammalian cell lines. A, 50 nm Gβγ increased GIRK1/GIRK4 channel activity in HEK-293 cells, similar to its action in atrial myocytes. Neither PP2A (0.01 unit/μl), nor addition of ATP (4 mm), affected the level of channel activity. B, pretreatment of the patch with PP2A blocked activation by Gβγ. C andD, activity of IKACh channel expressed in CHO-K1 and HEK-293 (mean ± S.E.). Channel activity (NPo) was calculated for 0.5- to 2-min stretches of records during steady-state channel activity (usually 1–2 min after drug applications). Asterisks indicate significant differences in D as compared with C(p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gβγ was isolated as described previously (2Krapivinsky G. Gordon E. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar). Calmodulin kinase (CaMKI, CaMKII), protein phosphatases (PP1, PP2A, PP2B), and alkaline phosphatase were purchased from Upstate Biotechnology (Lake Placid, NY). Glycogen synthase kinase 3 was purchased from New England BioLabs (Beverly, MA). Protein kinases A catalytic subunit (PKA), protein kinase C (PKC), and the catalytic subunit of PP2A were supplied by Promega (Madison, WI). For electrophysiological recordings, both Gβγ and PP2A were diluted with the recording solution a few minutes before the experiment by 100- and 10-fold to 50 nm and 0.01 unit/μl, respectively, and kept on ice. PIP2(l-α-phosphatidyl-d-myo-inositol-4,5-bisphosphate) was obtained from Calbiochem (catalog no. 524644; San Diego, CA). PIP2 was diluted 30 min before the experiment and continuously sonicated at 4 °C. All other reagents, unless specified, were from Sigma. Cardiac IKAChimmunoprecipitated with anti-GIRK4 antibody was phosphorylated by the catalytic subunit of PKA and other kinases (Fig.1). Two phosphorylated protein bands with electrophoretic mobilities corresponding to the core and glycosylated forms of GIRK1 (2Krapivinsky G. Gordon E. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar) were revealed after kinase exposure. We had previously shown that the immunoaffinity-purified IKAChchannel protein contained only GIRK1 and GIRK4 polypeptides (7Corey S. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 1998; 273: 5271-5278Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). To confirm that the phosphorylated proteins belonged to the IKACh complex and were not associated with impurities of unrelated strongly phosphorylated proteins in the aCIRN2 immunoprecipitate, we immunoprecipitated IKACh with anti-GIRK1 antibody. The same phosphorylation pattern was obtained for the immunopurified protein, and, in a negative control, neutral rabbit immunoglobulins did not immunoprecipitate the phosphorylated proteins. Incubation of the immunoprecipitated channel with [32P]ATP, but without additional kinases, yielded no phosphorylated proteins and demonstrated that the kinase activity was not strongly associated with the channel. Several protein kinases, including PKA, PKC, and calmodulin kinase (CaMKI and CaMKII), phosphorylated GIRK1 (Fig. 1 A). Only CaMKII phosphorylated (albeit weakly) a protein with the electrophoretic mobility of the GIRK4 subunit. To determine candidates for the channel phosphatase, in vitro phosphorylated cardiac IKACh was treated with a number of phosphoprotein phosphatases (Fig. 1 B). Among the phosphatases tested (PP1, PP2A, PP2B, and alkaline phosphatase), only PP2A effectively dephosphorylated the channel. Many attempts to modify cardiac IKAChactivity by application of ATP to the intracellular surface of excised patches either simply decreased channel run-down or produced relatively small changes in IKACh activity (see Ref. 22Wickman K. Clapham D.E. Physiol. Rev. 1995; 75: 865-885Crossref PubMed Scopus (341) Google Scholar for review). These results indicate that IKACh may have already been in a phosphorylated state before these experiments were initiated. If native IKACh is phosphorylated under normal recording conditions, then a protein phosphatase rather than ATP (or a kinase) should produce a significant functional effect. Because PP2A was most potent in dephosphorylation of the GIRK1 subunit of IKACh, we tested the ability of this phosphatase to modify IKAChchannel activity in inside-out patches. As described previously (e.g. Ref. 23Logothetis D.E. Kurachi Y. Galper J. Neer E.J. Clapham D.E. Nature. 1987; 325: 321-326Crossref PubMed Scopus (827) Google Scholar), the application of Gβγ (50 nm) to inside-out patches excised from cultured atrial cardiomyocytes induced a significant increase in the open channel probability (NPo), in this case from 0.01 ± 0.005 to 0.22 ± 0.02 (3 min after Gβγ application;n = 14; Fig. 2,A and C). Channel activity was stable and did not run down during the 10–15 min of recording (n = 5, no inhibitors of phosphatase or ATP were added). The application of PP2A phosphatase (0.01 unit/μl) to an IKACh channel-containing patch already activated by Gβγ did not significantly change the channel's open probability (Fig. 2, A and C) or mean open time (not shown). In contrast, treatment of the patch with phosphatase PP2A prior to application of Gβγ completely prevented channel activation by Gβγ in 18 of 20 experiments (Fig. 2,B and D). Heat-inactivated PP2A did not abrogate activation by Gβγ. In only two experiments under these conditions did Gβγ increase channel activity (NPo), and then only by a relatively small amount (∼2-fold). Similar results were obtained using the catalytic subunit of PP2A as well as PP1 phosphatase (5 of 5 experiments). Alkaline phosphatase did not prevent IKACh activation. Patches preincubated with PP2A and Gβγ were effectively activated by Mg-ATP (4 mm) but not by the non-hydrolyzable analog of ATP, AMP-PNP (4 mm; Fig. 2, B and D). The increase of channel activity after exposure to ATP was mainly due to an increase in the frequency of channel opening (50- to 500-fold in different patches; n = 20), because the channel mean open time was not affected significantly (1.04 ± 0.04 and 1.07 ± 0.03 ms, respectively; n = 20). Activation by ATP required Gβγ, because application of the ATP before Gβγ did not activate channels in the patch either under control conditions or after pretreatment with PP2A. Activation by ATP was not modified in the presence of protein kinase inhibitors such as staurosporine, KN-62, or lavendustin C. Phosphatidylinositol bisphosphate (PIP2) appears to be a co-factor in IKAChactivation (16Huang C.L. Feng S.Y. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (755) Google Scholar, 17Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (211) Google Scholar). ATP might have enhanced channel activity by increasing PIP2 content in the plasma membrane (16Huang C.L. Feng S.Y. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (755) Google Scholar, 17Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (211) Google Scholar). Therefore, one explanation for the effects of phosphoprotein phosphatase PP2A on IKACh channel activity could be that PP2A decreased the level of PIP2 and thus inactivated the channel. Although protein phosphatase PP2A is not known to dephosphorylate PIP2, we tested this hypothesis by incubating PIP2 in the presence of PP2A under conditions identical to those in the patch-clamp experiments (see "Materials and Methods"). No hydrolysis of PIP2 by PP2A was detected in these experiments (data not shown). The effect of PP2A might also be explained by dephosphorylation of one or more membrane-associated proteins, which regulate PIP2levels in the membrane. However, this suggestion contradicts the results shown in Fig. 2; phosphatase 2A was effective only when added before Gβγ activation of the channel, and it failed to modify activated channels. In contrast, the hydrolysis of PIP2resulted in the inhibition of the active channel (16Huang C.L. Feng S.Y. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (755) Google Scholar, 17Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (211) Google Scholar). The application of phospholipase C or PIP2 antibodies to IKACh already activated by Gβγ significantly decreased the channel open probability in rat atrial myocytes (n= 8 out of 8 patches) as has been previously demonstrated (16Huang C.L. Feng S.Y. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (755) Google Scholar, 17Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (211) Google Scholar). Finally, if PP2A decreased the level of PIP2 in the membrane, then application of a saturating concentration of PIP2 to the PP2A-treated patch should completely restore channel activity, and subsequent application of ATP should not further activate the channel. However, Fig. 3shows that the application of a saturating concentration of bovine brain PIP2 only partially restored activity of the channel pretreated with PP2A and Gβγ. Subsequent application of ATP induced an additional significant increase in channel activity (NPo) in all experiments (p = 0.002, n = 6; paired t test). Thus the effect of phosphoprotein phosphatase 2A on GIRK channel activity cannot be explained by PIP2 degradation in the membrane. To further delineate the molecular mechanism of IKACh inactivation by protein phosphatases, we expressed GIRK1 and GIRK4 subunits in two mammalian cell lines, CHO-K1 and HEK-293 (Fig. 4). In both cell lines expressing the GIRK1/GIRK4 channel, Gβγ induced a robust and highly reproducible activation of the multimeric channel. Channel activity was sustained and did not run down during 10–15 min of recording. As in atrial cardiomyocytes, neither PP2A nor ATP significantly modified channel activity after Gβγ application in HEK-293 or CHO-K1 cells (Fig. 4 A). Patch pretreatment with PP2A prior to application of Gβγ completely blocked Gβγ activation in CHO-K1 cells and significantly decreased the extent of channel activation in HEK-293 cells (Fig. 4, B and D). Subsequent ATP application completely restored channel activity in HEK-293 cells but not substantially in CHO-K1 cells (Fig. 4 D). Consecutive application of protein kinase A, protein kinase C, glycogen synthase kinase-3, CaMKI, and CaMKII did not significantly enhance the effect of ATP in CHO-K1 cells. Channel inactivation by protein phosphatase 2A suggests that protein phosphorylation by the specific protein kinase could be an important condition of channel activation by Gβγ. The different sensitivity of the channel to ATP in the two expression systems may be the result of a difference in the specific protein kinase activity in CHO-K1 and HEK-293 cells. To determine if atrial IKACh was phosphorylated in native cells, we attempted to immunoprecipitate IKACh from primary cultures of neonatal rat atrial cardiomyocytes metabolically labeled with32P. Unfortunately, we could not obtain sufficient numbers of cells for detection of IKACh. To circumvent this shortcoming, we expressed the functionally active GIRK1/GIRK4 heteromeric channel in HEK-293 and CHO-K1 cell lines, metabolically labeled the cells with 32P, and immunoprecipitated the channel. We have previously established that the properties of GIRK1/GIRK4 in HEK-293 cells, CHO-K1, and native cardiomyocytes are functionally indistinguishable (2Krapivinsky G. Gordon E. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar) and that the mode of channel regulation by PP2A is similar in native cardiomyocytes and expression systems (see previous section). In the steady state, only the GIRK1 subunit of the channel complex was phosphorylated when expressed in HEK-293 cells, as detected by 32P labeling and immunoprecipitation of the heteromeric GIRK1/GIRK4 channel (Fig.5). Identical results were obtained for channels expressed in CHO-K1 cells (data not shown). In some experiments, we observed a weakly phosphorylated band with a mobility corresponding to the GIRK4 subunit, but phosphorylation of this protein was not consistently observed. The same result was obtained when the heteromeric channel was immunoprecipitated with anti-GIRK4 antibody. Interestingly, GIRK4 was phosphorylated when expressed alone without GIRK1 (Fig. 5), a condition whose physiological relevance has not been established. Assuming that PP2A inactivation of IKACh is due to GIRK1 serine/threonine residue dephosphorylation, then mutation of the phosphorylated amino acids should produce an inactive channel. GIRK1 has a wealth of putative serine/threonine phosphorylation sites located in its long carboxyl tail region (Fig. 6). Most of these sites are present in the GIRK1 but not the GIRK4 subunit. Based on the results presented so far, we expected that a point mutation at one of these serine/threonine residues would prevent phosphorylation and result in a GIRK1/GIRK4 channel that could not be activated by Gβγ. Thus, most of the putative serine/threonine phosphorylation sites on GIRK1 were mutated to alanine (Fig. 6). None of the 18 single point, or 8 paired, amino acid mutations disrupted the ability of Gβγ to activate the expressed mutant GIRK1/GIRK4 channel. However, two point mutations, T193A and S221A, resulted in expression of channel with a much shorter mean open time (less than 0.3 ms) and one mutation (S278A) displayed lower sensitivity to Gβγ. When these three mutant proteins were expressed in HEK-293 cells, none exhibited decreased levels of GIRK1 phosphorylation. More complex combinations of mutations were also tried. The GIRK1 subunit with a mutation of 3 residues (193, 221, and 278) to alanines did not express in either HEK-293 or CHO-K1 cells as determined by Western blot (not shown). The failure of the point mutations to disrupt channel activation by Gβγ indicated that multiple phosphorylation sites were relevant to GIRK1 function or that non-consensus serine/threonine residues might be involved. Tyrosine phosphorylation of GIRK1 is unlikely to be pertinent, because the phosphorylated and immunoprecipitated channel was not recognized with an anti-phosphotyrosine antibody (data not shown). Several truncation mutants were constructed to determine the regions of the GIRK1 protein that were phosphorylated in vivo. We found that truncation of GIRK1 after residue 373 resulted in complete disappearance of the phosphorylation signal (Fig.7). The absence of the phosphorylation was not due to decreased Δ373-GIRK1 subunit expression, because its expression level was even higher than that of the wild type GIRK1 subunit (Fig. 7). Furthermore, the ability of Δ373-GIRK1 to associate with the GIRK4 subunits was not decreased compared with wild type GIRK1 (data not shown, see also Fig. 8 where the functional heterochannel is shown). These results suggested that the phosphorylation site(s) are located on the carboxyl tail of the GIRK1 subunit. To further define the region of the relevant phosphorylation site, we truncated the GIRK1 molecule after amino acid 419. The level of phosphorylation of the Δ419-GIRK1 mutant protein was indistinguishable from that of the wild type subunit (Fig. 7). This suggests that the relevant phosphorylation sites were located between amino acids 373 and 419 of GIRK1. Surprisingly, the simultaneous mutation of all seven serine/threonine residues located between these amino acids did not eliminate GIRK1 subunit phosphorylation (Fig. 7). This suggests that residues between amino acids 373 and 419 are critical for channel phosphorylation, but that the actual phosphorylated sites must be located proximal to amino acid 373. Membranes isolated from HEK-293 cells and expressing the GIRK1/GIRK4 heterotetramer were treated with PP2A under conditions similar to those in patch-clamp experiments. GIRK1 phosphorylation was not changed under these conditions. But PP2A completely dephosphorylated in vitro phosphorylated IKACh, suggesting that the sites phosphorylated in IKACh in vitro are different from those phosphorylated in vivo. These results could be explained if PP2A had dephosphorylated a regulatory membrane-associated protein or if it had dephosphorylated residues that were critical for channel activity but that were only a small fraction of the total GIRK1 phosphorylated residues.Figure 8Truncation of the C-tail of GIRK1 prevents channel regulation by PP2A. A, single channel currents from inside-out patches excised from HEK-293 cells expressing GIRK4 and wild type or mutant subunits of GIRK1. Currents were recorded in the presence of phosphatase PP2A (left traces) or Gβγ applied after phosphatase wash out (right traces).fo = 5 kHz. B, mean ion channel activity (quantified as NPo) of channels comprised of GIRK4 and mutants of GIRK1 subunits (HEK-293 cells). Experiments were performed as illustrated in Figs. 4 A and4 C. Asterisks show values that were significantly different from those recorded from the wild type channel.NPo was calculated for 0.5–2 min during steady-state channel activity (usually 1–2 min after drug application). seven mutants, GIRK1 incorporating all of the following mutations: T377A, S379A, S385A, S396A, T397A, S401A, S407A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We suggested above that expression of a dephosphorylated GIRK1 subunit should yield an inactive channel. Surprisingly, co-expression of Δ373-GIRK1 with GIRK4 subunits produced single channel currents that had the identical conductance and channel kinetics of those expressed by wild type GIRK1 and GIRK4 subunits (Fig. 8 A, see also Ref. 21Kennedy M.E. Nemec J. Corey S. Wickman K. Clapham D.E. J. Biol. Chem. 1999; 274: 2571-2578Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). However, unlike the wild type channel, Δ373-GIRK1/GIRK4 was not sensitive to PP2A pretreatment (Fig. 8). The removal of the more distal portion () of the GIRK1 C terminus did not affect channel phosphorylation but (like the Δ373-GIRK1 channel mutant) was not sensitive to PP2A pretreatment (Fig. 8 A). The mutation of all GIRK1 serine and threonine residues between amino acids 373 and 419, and co-expressed with GIRK4, yielded a channel that was sensitive to the phosphatase and biophysically indistinguishable from the wild type channel (Fig. 8 B). Taken together, these results support the conclusion that the GIRK1 C terminus is critical not only for GIRK1 phosphorylation but that it also mediates the inhibitory effect of the protein phosphatase. This study provides direct evidence that both the native IKACh and expressed GIRK1/GIRK4 channel are phosphorylated. Only the GIRK1 subunit of the heteromeric channel is phosphorylated when co-expressed with GIRK4. We have also shown that protein phosphorylation is an important switch mechanism for IKAChchannel activation; treatment of the inside-out patch with protein phosphatases completely prevented the channel activation by Gβγ. Finally, we have shown also that the GIRK1 C terminus is critical for both GIRK1 phosphorylation and GIRK1/GIRK4 heteromeric channel regulation by the phosphatase. This discussion addresses the possible mechanisms involved in phosphorylation-dependent regulation of IKAChactivity. We have shown that the GIRK1 subunit of the IKACh channel protein is phosphorylated and that protein phosphatase prevents IKAChgating by Gβγ. Several previous papers have shown that ATP enhanced the activity of IKACh and suggested that phosphorylation was a potential mechanism of channel modulation (13Sui J.L. Chan K.W. Logothetis D.E. J. Gen. Physiol. 1996; 108: 381-391Crossref PubMed Scopus (102) Google Scholar, 14Kim D. Watson M. Indyk V. Am. J. Physiol. 1997; 272: H195-H206PubMed Google Scholar, 15Kim D. J. Physiol. ( Lond. ). 1991; 437: 133-155Crossref PubMed Scopus (60) Google Scholar, 24Mullner C. Vorobiov D. Bera A.K. Uezono Y. Yakubovich D. Frohnwieser-Steinecker B. Dascal N. Schreibmayer W. J. Gen. Physiol. 2000; 115: 547-558Crossref PubMed Scopus (50) Google Scholar). One potential phosphorylation mechanism suggested by Mullner et al. (24Mullner C. Vorobiov D. Bera A.K. Uezono Y. Yakubovich D. Frohnwieser-Steinecker B. Dascal N. Schreibmayer W. J. Gen. Physiol. 2000; 115: 547-558Crossref PubMed Scopus (50) Google Scholar) is through β-adrenergic receptor stimulation of PKA-dependent protein phosphorylation. Another hypothesis suggests that IKACh could be activated through phosphorylation of phosphoinositides (16Huang C.L. Feng S.Y. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (755) Google Scholar, 17Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (211) Google Scholar). Because PIP phosphorylation is a powerful mechanism of IKAChregulation, we have extensively tested whether the effect of protein phosphatase on channel activity could involve PIP2hydrolysis and whether the ATP-induced recovery of the channel activity involved additional PIP phosphorylation mechanisms. Phosphatase 2A is known to be a protein phosphatase, and we demonstrated that it did not directly dephosphorylate the lipid, PIP2. It is possible that PP2A dephosphorylated and regulated the activity of an unknown protein involved in PIP2 metabolism that resulted in a decrease in PIP2 concentration in the membrane. In that case, however, PP2A should have inactivated the channel, independent of whether Gβγ was present or not, because PIP2 degradation did not depend on Gβγ (16Huang C.L. Feng S.Y. Hilgemann D.W. Nature. 1998; 391: 803-806Crossref PubMed Scopus (755) Google Scholar, 17Sui J.L. Petit-Jacques J. Logothetis D.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1307-1312Crossref PubMed Scopus (211) Google Scholar). The fact that prior activation of the channel by Gβγ completely blocked channel inactivation by PP2A rules out this possibility. Finally, ATP increases channel activity even in the presence of saturating PIP2 concentrations, strongly suggesting that PP2A-sensitive protein phosphorylation is involved in IKAChgating independent of PIP2. Our data point to a potential switch mechanism that would effectively block receptor-dependent activation of IKACh (IKG or GIRK channels) by making the channel insensitive to gating by Gβγ. But the upstream mechanism is still obscure. Is dephosphorylation of the constitutively phosphorylated GIRK1 responsible for the PP2A effect, or is another protein involved in this mechanism? If we assume that PP2A dephosphorylates GIRK1, then mutations of phosphorylation sites critical for channel activation might be expected to result in expression of a non-activatable channel protein. To identify phosphorylated serine/threonine residues, we performed multiple point mutations/deletions of the GIRK1 subunit. Deletion of the GIRK1 C terminus resulted in expression of non-phosphorylated but functionally normal channel. The fact that PP2A did not dephosphorylate the in vivo phosphorylated channel does not support a model in which dephosphorylation of the GIRK1 subunit itself is responsible for channel inactivation. Instead, these results suggest that either PP2A dephosphorylated an associated regulatory membrane protein or that it dephosphorylated only a small fraction of the phosphorylated residues in GIRK1 critical for channel activity. If there is an associating protein, it would be likely to be relatively loosely bound to IKACh, because in at least one study no proteins were found to be associated with GIRK1/GIRK4 subunits after purification of the native IKACh complex (7Corey S. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 1998; 273: 5271-5278Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The unique C-terminal tail of GIRK1 has prompted speculation over several years about its function. Several hypotheses have been advanced, including the proposal that the tail was the site of Gβγ activation (25Huang C.L. Slesinger P.A. Casey P.J. Jan Y.N. Jan L.Y. Neuron. 1995; 15: 1133-1143Abstract Full Text PDF PubMed Scopus (284) Google Scholar, 26Kunkel M.T. Peralta E.G. Cell. 1995; 83: 443-449Abstract Full Text PDF PubMed Scopus (133) Google Scholar). Subsequently, it was found that Gβγ could activate homomultimeric GIRK2, -3, or -4 channels (2Krapivinsky G. Gordon E. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (747) Google Scholar) and that the GIRK1 C terminus was not required even for GIRK1/GIRKx heteromultimer activation (21Kennedy M.E. Nemec J. Corey S. Wickman K. Clapham D.E. J. Biol. Chem. 1999; 274: 2571-2578Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The present studies have shown that phosphatase 2A treatment of the in vivo channel abrogates Gβγ activation of the channel complex and that the GIRK1 C terminus participates in this switch mechanism. The targets of the phosphatase are not the phosphorylated residues on the GIRK1 C terminus, but the C terminus is required for the action of the phosphatase. The simplest model supported by these results is one in which the GIRK1 C terminus is an inhibitor of the channel. This inhibition is removed by phosphorylation of either GIRK1 or an accessory protein. When phosphorylated, the channel complex is competent for activation by Gβγ. The channel complex is normally in the phosphorylated state and readily gated by Gβγ when any one of several neurotransmitter receptors are bound. The next objective of work in this area will be to determine if phosphatases can be physiologically induced to switch off the Gβγ sensitivity of the channel.
Referência(s)