Gαq Inhibits Cardiac L-type Ca2+ Channels through Phosphatidylinositol 3-Kinase
2005; Elsevier BV; Volume: 280; Issue: 48 Linguagem: Inglês
10.1074/jbc.m508441200
ISSN1083-351X
AutoresZhongju Lu, Yaping Jiang, Lisa M. Ballou, Ira S. Cohen, Richard Z. Lin,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoCardiac myocyte contractility is initiated by Ca2+ entry through the voltage-dependent L-type Ca2+ channel (LTCC). To study the effect of Gαq on the cardiac LTCC, we utilized two transgenic mouse lines that selectively express inducible Gαq-estrogen receptor hormone-binding domain fusion proteins (GαqQ209L-hbER or GαqQ209L-AA-hbER) in cardiac myocytes. Both of these proteins inhibit phosphatidylinositol (PI) 3-kinase (PI3K) signaling, but GαqQ209L-AA-hbER cannot activate the canonical Gαq effector phospholipase Cβ (PLCβ). L-type Ca2+ current (ICa,L) density measured by whole-cell patch clamping was reduced by more than 50% in myocytes from both Gαq animals as compared with wild-type cells, suggesting that inhibition of the LTCC by Gαq does not require PLCβ. To investigate the role of PI3K in this inhibitory effect, ICa,L was measured in the presence of various phosphoinositides infused through the patch pipette. Infusion of PI 3,4,5-trisphosphate (PI(3,4,5)P3) into wild-type myocytes did not affect ICa,L, but it fully restored ICa,L density in both Gαq transgenic myocytes to wild-type levels. By contrast, PI 4,5-bisphosphate (PI(4,5)P2) or PI 3,5-bisphosphate had no effect. Infusion with p110β/p85α or p110γ PI3K in the presence of PI(4,5)P2 also restored ICa,L density to wild-type levels. Last, infusion of either PTEN, a PI(3,4,5)P3 phosphatase, or the pleckstrin homology domain of Grp1, which Cardiac myocyte contractility is initiated by Ca2+ entry through the voltage-dependent L-type Ca2+ channel (LTCC). To study the effect of Gαq on the cardiac LTCC, we utilized two transgenic mouse lines that selectively express inducible Gαq-estrogen receptor hormone-binding domain fusion proteins (GαqQ209L-hbER or GαqQ209L-AA-hbER) in cardiac myocytes. Both of these proteins inhibit phosphatidylinositol (PI) 3-kinase (PI3K) signaling, but GαqQ209L-AA-hbER cannot activate the canonical Gαq effector phospholipase Cβ (PLCβ). L-type Ca2+ current (ICa,L) density measured by whole-cell patch clamping was reduced by more than 50% in myocytes from both Gαq animals as compared with wild-type cells, suggesting that inhibition of the LTCC by Gαq does not require PLCβ. To investigate the role of PI3K in this inhibitory effect, ICa,L was measured in the presence of various phosphoinositides infused through the patch pipette. Infusion of PI 3,4,5-trisphosphate (PI(3,4,5)P3) into wild-type myocytes did not affect ICa,L, but it fully restored ICa,L density in both Gαq transgenic myocytes to wild-type levels. By contrast, PI 4,5-bisphosphate (PI(4,5)P2) or PI 3,5-bisphosphate had no effect. Infusion with p110β/p85α or p110γ PI3K in the presence of PI(4,5)P2 also restored ICa,L density to wild-type levels. Last, infusion of either PTEN, a PI(3,4,5)P3 phosphatase, or the pleckstrin homology domain of Grp1, which Cardiac myocyte contraction is initiated by cell membrane depolarization elicited by action potentials, resulting in a small Ca2+ influx through the voltage-dependent L-type Ca2+ channel (LTCC). 2The abbreviations used are: LTCCL-type Ca2+ channelPIphosphatidylinositolPI3KPI 3-kinasePI(3,4,5)P3PI 3,4,5-trisphosphatePI(4,5)P2PI 4,5-bisphosphatePI(3,5)P2PI 3,5-bisphosphatePLCβphospholipase Cβ4-HT4-hydroxytamoxifenPH-Grp1glutathione S-transferase fused to the pleckstrin homology domain of Grp1WTwild-typeFfarad. 2The abbreviations used are: LTCCL-type Ca2+ channelPIphosphatidylinositolPI3KPI 3-kinasePI(3,4,5)P3PI 3,4,5-trisphosphatePI(4,5)P2PI 4,5-bisphosphatePI(3,5)P2PI 3,5-bisphosphatePLCβphospholipase Cβ4-HT4-hydroxytamoxifenPH-Grp1glutathione S-transferase fused to the pleckstrin homology domain of Grp1WTwild-typeFfarad. This inward Ca2+ current (ICa,L) then triggers a larger Ca2+ release from the sarcoplasmic reticulum through the ryanodine receptor that induces myofilament contraction. It is not clear if the phosphatidyinositol (PI) 3-kinase (PI3K) signaling pathway modulates LTCC function in cardiac myocytes. However, studies in neurons and vascular myocytes indicate that the LTCC in these cell types is regulated by PI3K (1Blair L.A. Marshall J. Neuron. 1997; 19: 421-429Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 2Macrez N. Mironneau C. Carricaburu V. Quignard J.F. Babich A. Czupalla C. Nurnberg B. Mironneau J. Circ. Res. 2001; 89: 692-699Crossref PubMed Scopus (87) Google Scholar). Infusion of PI3K or its lipid second messenger PI 3,4,5-trisphosphate (PI(3,4,5)P3) into rat portal vein myocytes increased the ICa,L amplitude through the LTCC (2Macrez N. Mironneau C. Carricaburu V. Quignard J.F. Babich A. Czupalla C. Nurnberg B. Mironneau J. Circ. Res. 2001; 89: 692-699Crossref PubMed Scopus (87) Google Scholar, 3Le Blanc C. Mironneau C. Barbot C. Henaff M. Bondeva T. Wetzker R. Macrez N. Circ. Res. 2004; 95: 300-307Crossref PubMed Scopus (75) Google Scholar). Increased LTCC activity in response to activation of PI3K might be due to increased trafficking of the channel to the plasma membrane, as shown using exogenous LTCC proteins expressed in human embryonic kidney 293 cells (4Viard P. Butcher A.J. Halet G. Davies A. Nurnberg B. Heblich F. Dolphin A.C. Nat. Neurosci. 2004; 7: 939-946Crossref PubMed Scopus (207) Google Scholar). L-type Ca2+ channel phosphatidylinositol PI 3-kinase PI 3,4,5-trisphosphate PI 4,5-bisphosphate PI 3,5-bisphosphate phospholipase Cβ 4-hydroxytamoxifen glutathione S-transferase fused to the pleckstrin homology domain of Grp1 wild-type farad. L-type Ca2+ channel phosphatidylinositol PI 3-kinase PI 3,4,5-trisphosphate PI 4,5-bisphosphate PI 3,5-bisphosphate phospholipase Cβ 4-hydroxytamoxifen glutathione S-transferase fused to the pleckstrin homology domain of Grp1 wild-type farad. Class I PI3K enzymes preferentially phosphorylate PI 4,5-bisphosphate (PI(4,5)P2) to form PI(3,4,5)P3 in vivo and exhibit substantial activation in response to growth factor and hormone stimulation. Class IA PI3Ks are heterodimers consisting of a catalytic subunit (p110α, p110β, or p110δ) tightly bound to a regulatory subunit (p85α, p85β, p55γ, or variants produced by alternative splicing). The class IB catalytic subunit p110γ forms a heterodimer with a p101 regulatory subunit (5Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1347) Google Scholar). Cardiac myocytes contain p85α and p85β regulatory subunits bound to either p110α or p110β catalytic subunits in unknown stoichiometric proportions (6Oudit G.Y. Sun H. Kerfant B.G. Crackower M.A. Penninger J.M. Backx P.H. J. Mol. Cell. Cardiol. 2004; 37: 449-471Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Cardiac myocytes also express p110γ (6Oudit G.Y. Sun H. Kerfant B.G. Crackower M.A. Penninger J.M. Backx P.H. J. Mol. Cell. Cardiol. 2004; 37: 449-471Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). In general, p110α is activated upon stimulation of receptor tyrosine kinases, and p110γ is activated in response to stimulation of G protein-coupled receptors. p110β is thought to be activated by both G protein-coupled and tyrosine kinase receptors (7Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1997; 272: 24252-24256Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Stimulation of G protein-coupled receptors leads to activation of the heterotrimeric G proteins that consist of α and βγ subunits. Gα and Gβγ then signal independently to downstream effectors. While the p110γ and p110β PI3Ks are activated by Gβγ subunits (7Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1997; 272: 24252-24256Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 8Kerchner K.R. Clay R.L. McCleery G. Watson N. McIntire W.E. Myung C.S. Garrison J.C. J. Biol. Chem. 2004; 279: 44554-44562Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), the role of Gα subunits in regulating PI3K is less clear. Recently, we reported that Gαq coprecipitates with and inhibits the lipid kinase activity of the p110α/p85α PI3K complex (9Ballou L.M. Lin H.Y. Fan G. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2003; 278: 23472-23479Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Using purified recombinant proteins, we demonstrated that Gαq binds directly to the enzyme to inhibit its activity. 3L. M. Ballou, M. Chattopadhyay, Y. Li, S. Scarlata, and R. Z. Lin, submitted for publication. 3L. M. Ballou, M. Chattopadhyay, Y. Li, S. Scarlata, and R. Z. Lin, submitted for publication. We also found that the GαqQ209L mutant, which signals constitutively to its effectors, inhibits p110α but not p110β (9Ballou L.M. Lin H.Y. Fan G. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2003; 278: 23472-23479Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Furthermore, Gαq can inhibit PI3K without activating its canonical effector phospholipase Cβ (PLCβ), as shown by the use of a mutant (GαqQ209L-AA) that cannot activate PLCβ (9Ballou L.M. Lin H.Y. Fan G. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2003; 278: 23472-23479Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10Venkatakrishnan G. Exton J.H. J. Biol. Chem. 1996; 271: 5066-5072Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 11Fan G. Ballou L.M. Lin R.Z. J. Biol. Chem. 2003; 278: 52432-52436Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Using transgenic mice that selectively express an inducible GαqQ209L protein in cardiac myocytes, we have demonstrated that activation of Gαq leads to inhibition of the cardiac ICa,L. 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. A second line of transgenic mice expressing an inducible GαqQ209L-AA protein showed a similar inhibition of the ICa,L, suggesting a role for PI3K but not PLCβ in this response. 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. In this study, we used whole-cell patch clamping to further investigate the role of PI3K in mediating Gαq inhibition of the ICa,L in myocytes isolated from these transgenic animals. We found that PI(3,4,5)P3 and some PI3K isoforms can reverse the inhibitory effect of Gαq on the cardiac LTCC. Materials—Tamoxifen and 4-hydroxytamoxifen (4-HT) were from Sigma. Recombinant p110β/p85α, p110γ, and PTEN were from Upstate Biotechnology, Inc. (Lake Placid, NY). PI(4,5)P2 di-C8, PI(3,5)P2 di-C8, PI(3,4,5)P3 di-C8 and glutathione S-transferase fused to the pleckstrin homology domain of Grp1 (PH-Grp1) were from Echelon Biosciences, Inc. (Salt Lake City, UT). Akt1/2 antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-Erk1/2 antibody was from Cell Signaling Technology, Inc. (Beverly, MA). Recombinant p110α/p85α purified from baculovirus-infected Sf9 cells was described previously (9Ballou L.M. Lin H.Y. Fan G. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2003; 278: 23472-23479Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). GαqQ209L-hbER Transgenic Mice—C57BL/6 transgenic mice expressing either GαqQ209L-hbER or GαqQ209L-AA-hbER in cardiac myocytes under the control of the α myosin heavy chain promoter were described previously. 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. Starting at 8 weeks of age, GαqQ209L-hbER animals were injected intraperitoneally with 1 mg of tamoxifen daily for 14 days, and GαqQ209L-AA-hbER mice were injected for 28 days. Matching wild-type (WT) littermates that were injected with tamoxifen for an equivalent number of days serve as controls; the WT data shown are pooled from these animals, except where otherwise indicated. All animal-related experimental protocols were approved by the Institutional Animal Care and Use Committee. Ventricular Myocyte Isolation—Mice were euthanized by intraperitoneal injection of 100 mg/kg sodium pentobarbital, and ventricular myocytes were isolated as previously described. 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. Electrophysiology—Only clearly rod-shaped myocytes were studied. Whole-cell patch clamp recordings used 2-3 mΩ borosilicate glass pipettes measured prior to sealing (Sutter Instrument), pCLAMP 8 software, the DigiData 1350 interface, and the Axopatch 1D amplifier (Axon Instruments). For the recording of ICa,L, pipettes contained internal solution (111 mm CsCl, 20 mm tetraethylammonium chloride, 10 mm glucose, 14 mm EGTA, 10 mm HEPES, and 5 mm MgATP, pH 7.2, adjusted with CsOH) and cells were perfused in a Na+-free bath solution (137 mm tetraethylammonium chloride, 1 mm MgCl2, 2 mm CaCl2, 10 mm HEPES, and 10 mm glucose, pH 7.4, adjusted with tetraethylammonium hydroxide). Except where otherwise noted, the bath solution also contained 1 μm 4-HT. The membrane capacity was measured in response to a voltage step from the holding potential, and the current amplitude was divided by cell capacitance to give ICa,L density in pA/pF. Membrane capacitance is an indirect measure of cell membrane area. This commonly used approach assumes a specific capacitance of 1 μF/cm2, which could vary slightly depending on physiologic conditions. This variation is expected to be extremely small and well below the detection limit of this technique. Phosphoinositides, PI3K isozymes, PH-Grp1, and PTEN were diluted 100-500-fold in the internal solution and infused through the patch pipette. PTEN buffer contained 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 81 mm imidazole, 1 mm EDTA, 5 mm glutathione, 2 mm dithiothreitol, 267 mm sucrose, and 10% glycerol. Akt Kinase Assays and Western Blots—Frozen mouse organs were homogenized with a PRO250 (Pro Scientific, Inc., Monroe, CT) in ice-cold lysis buffer (50 mm HEPES, 1% Triton X-100, 50 mm NaCl, 5 mm EDTA, 50 mm NaF, 10 mm sodium pyrophosphate, 1 mm sodium orthovandate, 0.5 mm phenylmethylsulfonyl fluoride, and 10 μg/ml each of aprotinin and leupeptin, pH 7.5). Homogenates were centrifuged at 15,000 × g for 30 min at 4 °C. Protein concentrations of the supernatants were determined using the Bradford assay (Bio-Rad). Akt activity was assayed in immunoprecipitates starting with 0.5 mg of tissue lysate protein following a method described earlier (12Ballou L.M. Cross M.E. Huang S. McReynolds E.M. Zhang B.X. Lin R.Z. J. Biol. Chem. 2000; 275: 4803-4809Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Western blot signals were visualized and quantitated using the Odyssey Infrared Imaging System with version 1.2 software (LI-COR Biosciences, Lincoln, NE). IRDye800-conjugated second antibodies were from Rockland Immunochemicals (Gilbertsville, PA). Data Analysis—Values are means ± S.E., and Student's t tests were performed to estimate the significance of the differences between mean values. A value of p < 0.05 was considered significant. Effects of PI(3,4,5)P3 on ICa,L in Cardiac Myocytes—In this study we employed myocytes isolated from two transgenic mouse lines that selectively express silent Gαq proteins in the heart. These proteins, GαqQ209L-hbER and GαqQ209L-AA-hbER, are inactive until they bind to 4-HT. Both of them can inhibit PI3K signaling, but the latter cannot activate PLCβ. Activation of these Gαq proteins in response to injection with tamoxifen, which is converted to 4-HT in animals, causes a large reduction in ICa,L density in cardiac myocytes from both transgenic animals. 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. This result suggests that inhibition of the cardiac LTCC by Gαq occurs independently of PLCβ and may be due to reduced PI3K signaling. This idea is supported by studies showing that PI3Ks stimulate ICa,L in isolated portal vein myocytes (2Macrez N. Mironneau C. Carricaburu V. Quignard J.F. Babich A. Czupalla C. Nurnberg B. Mironneau J. Circ. Res. 2001; 89: 692-699Crossref PubMed Scopus (87) Google Scholar, 3Le Blanc C. Mironneau C. Barbot C. Henaff M. Bondeva T. Wetzker R. Macrez N. Circ. Res. 2004; 95: 300-307Crossref PubMed Scopus (75) Google Scholar). As a first test of this hypothesis, we asked if PI(3,4,5)P3, the lipid product of PI3K, reverses the depressed ICa,L in GαqQ209L-hbER and GαqQ209L-AA-hbER cells. Myocytes were isolated from tamoxifen-treated WT and transgenic mice, and internal solution with or without 1 μm PI(3,4,5)P3 was infused into the cells through the patch pipette. The peak ICa,L density was measured by whole-cell patch clamping at +10 mV following a single depolarizing step of 300 ms duration from a holding potential of -50 mV. PI(3,4,5)P3 did not have a significant effect on ICa,L density in WT myocytes (Fig. 1). However, PI(3,4,5)P3 significantly increased the ICa,L density by 2.4-fold in GαqQ209L-hbER myocytes and 2.2-fold in the GαqQ209L-AA-hbER cells (Fig. 1). The values for PI(3,4,5)P3-treated transgenic myocytes were statistically indistinguishable from those observed in WT myocytes treated with or without PI(3,4,5)P3 (Fig. 1). We performed additional control experiments in GαqQ209L-AA-hbER myocytes using other phosphoinositides. Infusion with PI(3,5)P2, which is generated from PI 3-phosphate by a PI 5-kinase, had no effect (Fig. 1). Similarly, PI(4,5)P2, which is converted to PI(3,4,5)P3 by PI3K, did not alter the peak ICa,L density (Fig. 1). These results are consistent with the hypothesis that decreased ICa,L density in the transgenic myocytes is due to Gαq-dependent inhibition of PI3K. We also constructed current density-voltage (I-V) relationships for WT and transgenic myocytes infused with or without PI(3,4,5)P3. Activation of ICa,L was elicited by depolarizing voltage pulses from -50 mV to +50 mV in 10 mV increments (300 ms duration) from a holding potential of -50 mV. Fig. 2A shows typical recordings of ICa,L activation from WT cells in the absence (top panel) or presence (middle panel)of PI(3,4,5)P3. The peak I-V curves for both conditions are plotted in the bottom panel (Fig. 2A). There was no significant difference in ICa,L density between the two conditions at any of the voltages tested. In contrast, infusion with PI(3,4,5)P3 resulted in a large enhancement of ICa,L activation in both the GαqQ209L-hbER (Fig. 2B) and GαqQ209L-AA-hbER (Fig. 2C) myocytes. The I-V relationships (bottom panels of Fig. 2, B and C) show that ICa,L density was increased at nearly all of the voltages tested in both groups of myocytes. To further characterize the action of PI(3,4,5)P3 in transgenic myocytes, we investigated its time-dependent effect on ICa,L activation in single cells. In this protocol, the myocyte was infused with or without PI(3,4,5)P3 and repeatedly depolarized with voltage steps to +10 mV from a holding potential of -50 mV (300 ms duration). The ICa,L densities were normalized to the value obtained from the first voltage step following opening of the patch and initiation of whole-cell recording. As shown in the left panel of Fig. 3, the typical "run-down" of ICa,L density was observed in GαqQ209L-hbER myocytes when the patch pipette contained the control internal solution without PI(3,4,5)P3. Within 180 s, the normalized ICa,L density in these cells decreased by about 20%. In contrast, in the presence of PI(3,4,5)P3 we observed a "run-up" of ICa,L density in both the GαqQ209L-hbER (Fig. 3, middle panel) and GαqQ209L-AA-hbER (Fig. 3, right panel) myocytes. ICa,L density started to decrease after reaching a maximum after ∼120 s and the rate of decline was similar to that seen in cells infused with the control solution. Interestingly, even the first voltage step elicited a significantly larger ICa,L in cells infused with PI(3,4,5)P3 as compared with the control internal solution. Modulation of ICa,L by PI3K Isozymes in GαqQ209L-AA-hbER Myocytes—We next asked if infusion of purified PI3K proteins into GαqQ209L-AA-hbER myocytes has the same effect as PI(3,4,5)P3 in increasing ICa,L density. Multiple isoforms of PI3K have been identified in the adult heart of different species (6Oudit G.Y. Sun H. Kerfant B.G. Crackower M.A. Penninger J.M. Backx P.H. J. Mol. Cell. Cardiol. 2004; 37: 449-471Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar), and we tested three of them: p110α/p85α, p110β/p85α, and p110γ. The effects of these PI3K isozymes on the peak ICa,L density at +10 mV are shown in Fig. 4. In the absence of PI(4,5)P2, none of the three PI3K isozymes had an effect on ICa,L density. However, in the presence of PI(4,5)P2, both p110β/p85α and p110γ induced a significant increase in ICa,L density (2.6- and 2.2-fold, respectively). These increased ICa,L density values were similar to those observed in WT myocytes (see Fig. 1). Interestingly, infusion with p110α/p85α plus PI(4,5)P2 had no effect on ICa,L density (Fig. 4). These results suggest that ICa,L in the Gαq transgenic myocytes is modulated by specific PI3K isoforms. Fig. 5 shows typical traces of ICa,L activation in the presence of PI3K isozymes without (A) or with (B) PI(4,5)P2 that were used to construct I-V relationships (Fig. 5, C and D). The I-V curves show that infusion of the PI3K isozymes alone did not change ICa,L density across the entire voltage range examined as compared with the control cells infused with internal solution only (Fig. 5C). In the presence of PI(4,5)P2, both p110β/p85α and p110γ stimulated ICa,L at nearly all the voltages tested (Fig. 5D). The I-V curve for myocytes infused with p110α/p85α plus PI(4,5)P2 is nearly identical to the curve obtained from cells infused with PI(4,5)P2 alone (Fig. 5D). We also examined the time-dependent effect of PI3K isozymes plus PI(4,5)P2 on ICa,L in GαqQ209L-AA-hbER myocytes subjected to repeated depolarizing voltage steps. Infusion of p110α/p85α plus PI(4,5)P2 did not prevent the typical run-down (Fig. 6, left panel). In contrast, a run-up of ICa,L followed by a slow decrease was observed when either p110β/p85α or p110γ plus PI(4,5)P2 were infused into the cells (Fig. 6, middle and right panels). Reduction of ICa,L by Decreasing Endogenous PI(3,4,5)P3 in WT Myocytes—Since the reduction of ICa,L density in Gαq transgenic myocytes can be reversed by infusing exogenous PI(3,4,5)P3 or some PI3Ks, we hypothesized that depletion of endogenous PI(3,4,5)P3 in WT myocytes would lower ICa,L density. Two approaches were used to test this hypothesis. In the first approach, we infused myocytes isolated from WT mice (not treated with tamoxifen) with purified PH-Grp1 protein to sequester intracellular PI(3,4,5)P3. PH-Grp1 has been shown to bind specifically to PI(3,4,5)P3 (13Gray A. Van Der Kaay J. Downes C.P. Biochem. J. 1999; 344: 929-936Crossref PubMed Scopus (180) Google Scholar) and has been used to block PI3K signaling (4Viard P. Butcher A.J. Halet G. Davies A. Nurnberg B. Heblich F. Dolphin A.C. Nat. Neurosci. 2004; 7: 939-946Crossref PubMed Scopus (207) Google Scholar). In the presence of 20 nm PH-Grp1, the peak ICa,L density measured after a 300 ms pulse at +10 mV from a holding potential of -50 mV was 6.6 ± 0.6 pA/pF (n = 7) as compared with 9.6 ± 0.4 pA/pF (n = 7) for cells infused with the control solution. This 31% decrease is statistically significant (t test, p < 0.01). Shown in Fig. 7A (top two panels) are representative current traces that were used to construct the I-V relationships. The I-V curves show that PH-Grp1 reduced ICa,L density at nearly all of the voltages tested (Fig. 7A, bottom panel). In the second approach, we infused myocytes from untreated WT mice with the lipid phosphatase PTEN, which specifically dephosphorylates the D3 position on the inositol ring of PI(3,4,5)P3 to form PI(4,5)P2. When 20 nm purified PTEN protein was infused through the patch pipette, the peak ICa,L density at +10 mV measured after a 300 ms pulse from a holding potential of -50 mV was reduced by 28% to 6.5 ± 0.4 pA/pF (n = 20) versus 9.0 ± 0.6 pA/pF (n = 5) for control cells infused with an equivalent volume of PTEN buffer diluted into internal solution. The difference between the two conditions is statistically significant (t test, p < 0.01). The top two panels in Fig. 7B show sample current traces that were used to construct the I-V relationships for these two conditions (Fig. 7B, bottom panel). The ICa,L densities were reduced at nearly all of the voltages tested when PTEN was infused into the cells as compared with the control solution. Taken together, these results demonstrate that reduction of endogenous PI(3,4,5)P3 levels negatively modulates ICa,L activation in mouse cardiac myocytes. Reduced PI3K/Akt Signaling in GαqQ209L-AA-hbER Hearts—We previously demonstrated using an Akt activity assay that activation of GαqQ209L-AA-hbER in human embryonic kidney 293 cells inhibits PI3K signaling. 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. The protein kinase Akt is a downstream effector of PI3K that is activated by PI(3,4,5)P3. Here, we used Akt assays to confirm that PI3K signaling is reduced in hearts of GαqQ209L-AA-hbER mice as compared with WT mice. Both groups of animals were injected with tamoxifen for 28 days. Since food intake can affect PI3K signaling in some tissues due to changes in circulating insulin levels, the animals were fasted overnight and then injected with either insulin or saline as a control. Even though cardiac Akt activities were very low in mice injected with saline, we detected a consistent decrease in basal Akt activity in the GαqQ209L-AA-hbER hearts as compared with WT (Fig. 8A). GαqQ209L-AA-hbER inhibition of PI3K signaling was more obvious in animals treated with insulin. Insulin treatment induced large increases in Akt activity in both WT and GαqQ209L-AA-hbER hearts. However, total Akt activity was 33% less in the GαqQ209L-AA-hbER hearts as compared with WT (Fig. 8A). The difference in Akt activity is statistically significant (t test, p < 0.01). In contrast, GαqQ209L-AA-hbER did not reduce insulin-induced phosphorylation of Erk1/2 as measured by Western blotting using a phospho-specific antibody (Fig. 8B, bottom panel). Insulin stimulates a modest increase in Erk1/2 phosphorylation in the heart (data not shown). Expression of Akt protein was not reduced in GαqQ209L-AA-hbER hearts, as demonstrated by Western blotting of tissue lysates (Fig. 8B, top panel). These results indicate that activation of GαqQ209L-AA-hbER results in reduced PI3K/Akt signaling in the heart. Our initial study showing that ICa,L in GαqQ209L-hbER and GαqQ209L-AA-hbER transgenic myocytes is similarly depressed as compared with WT myocytes 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. suggested that PLCβ was not involved in this response. The ability of both of these fusion proteins to inhibit PI3K signaling led us to examine this pathway as a possible mediator of Gαq inhibition of the LTCC. In this study, we demonstrate that infusion of exogenous PI(3,4,5)P3 into GαqQ209L-hbER and GαqQ209L-AA-hbER myocytes completely reverses the inhibition of ICa,L. ICa,L is also fully restored in GαqQ209L-AA-hbER myocytes infused with certain PI3K isozymes in the presence of their phospholipid substrate. Together, these results support the idea that Gαq inhibits certain PI3Ks to cause a reduction in ICa,L. Furthermore, since reduction of endogenous PI(3,4,5)P3 levels in WT myocytes depresses ICa,L density, it appears that constitutive PI3K signaling is required for normal LTCC function. It is well established that activation of Gαs and subsequent activation of cAMP-dependent protein kinase stimulates cardiac LTCC function. On the other hand, activation of pertussis toxin-insensitive Gα proteins such as Gαq/11 inhibits neuronal L-type (14Shapiro M.S. Loose M.D. Hamilton S.E. Nathanson N.M. Gomeza J. Wess J. Hille B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10899-10904Crossref PubMed Scopus (98) Google Scholar) and N- and P/Q-type (15Dolphin A.C. Pharmacol. Rev. 2003; 55: 607-627Crossref PubMed Scopus (234) Google Scholar) Ca2+ channels. Gβγ subunits released from pertussis toxin-sensitive G proteins also inhibit N- and P/Q-type channels through direct protein-protein interactions, but they do not bind to or inhibit the LTCC (15Dolphin A.C. Pharmacol. Rev. 2003; 55: 607-627Crossref PubMed Scopus (234) Google Scholar). It is not known how activation of Gαq leads to inhibition of neuronal Ca2+ channels, but our results here using myocytes suggest that it could be due to inhibition of PI3K. Interestingly, a recent study indicates that inhibition of the neuronal LTCC by the M1 muscarinic receptor is mediated by Gαq/11 but does not appear to involve PLCβ (16Bannister R.A. Melliti K. Adams B.A. Biophys. J. 2002; 83: 3256-3267Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Our studies consistently support the concept that Gαq can inhibit PI3K independently of PLCβ activation. We initially showed that Gαq/ 11-coupled α1A adrenergic receptors inhibit growth factor and insulin activation of PI3K (12Ballou L.M. Cross M.E. Huang S. McReynolds E.M. Zhang B.X. Lin R.Z. J. Biol. Chem. 2000; 275: 4803-4809Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 17Ballou L.M. Tian P.Y. Lin H.Y. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2001; 276: 40910-40916Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Subsequently, we demonstrated that activated Gαq directly binds to and inhibits the p110α/p85α PI3K 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. (9Ballou L.M. Lin H.Y. Fan G. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2003; 278: 23472-23479Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). By contrast, transfected GαqQ209L did not inhibit p110β immunoprecipitated from cotransfected cells (9Ballou L.M. Lin H.Y. Fan G. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2003; 278: 23472-23479Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We have also reported that activated Gαq does not bind to p110γ, 4Fan, G., Jiang, Y.-P., Lu, D., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (October 6, 2005) J. Biol. Chem. 10.1074/jbc.M506810200. and studies are ongoing in our laboratory to determine whether GTP-bound Gαq interacts with other isoforms of PI3K. Results in this study show that p110β/p85α or p110γ, but not p110α/p85α, reversed Gαq inhibition of ICa,L (Figs. 4 and 5). One explanation for this result is that activated Gαq inhibits only p110α, and the transgenic myocytes express enough activated Gαq proteins to neutralize the infused p110α/p85α. Activation of PI3K potentiates ICa,L in rat portal vein myocytes and rat cerebellar granule neurons (1Blair L.A. Marshall J. Neuron. 1997; 19: 421-429Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 2Macrez N. Mironneau C. Carricaburu V. Quignard J.F. Babich A. Czupalla C. Nurnberg B. Mironneau J. Circ. Res. 2001; 89: 692-699Crossref PubMed Scopus (87) Google Scholar). In contrast, we found that exogenous PI(3,4,5)P3 had no effect on ICa,L activation in WT mouse cardiac myocytes (Figs. 1 and 2). We have also found that infusion of canine cardiac myocytes with PI(3,4,5)P3 does not potentiate ICa,L activation (data not shown). One possible explanation for these differences between cell types could be that cardiac forms of the LTCC are highly sensitive to the level PI(3,4,5)P3 and are maximally active at the level found in these cells. Alternatively, PI3K signaling might be maximally activated in cardiac myocytes, so addition of exogenous lipid second messenger would not have an effect. This possibility seems remote because basal Akt activity in the mouse heart is very low but can be strongly increased by insulin treatment (Fig. 8). We have also measured PI3K activity in p110α, p110β, or p110γ immunoprecipitates from freshly prepared heart lysates and found the activities to be very low (data not shown). We believe that LTCC function or localization at the plasma membrane is near maximal in cardiac myocytes, despite the low PI3K activity. Therefore, increasing PI3K signaling does not further stimulate ICa,L, but inhibition of this signaling pathway by activated Gαq, PH-Grp1, or PTEN can lead to a reduction in ICa,L. Activation of Gq-coupled receptors might have complex effects on the modulation of the LTCC. G protein-coupled receptors can activate more than one type of Gα subunit, and these could modulate LTCC function in diverse ways. Furthermore, some of the released βγ subunits could activate p110γ or p110β. In addition, p110α can complex with different p85 isoforms, and these heterodimers may be differentially affected by Gαq. Interestingly, Macrez and co-workers (18Quignard J.F. Mironneau J. Carricaburu V. Fournier B. Babich A. Nurnberg B. Mironneau C. Macrez N. J. Biol. Chem. 2001; 276: 32545-32551Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) found that angiotensin II stimulation of the portal vein myocyte LTCC is mediated by Gβγ activation of p110γ. The Gβγ dimer in question appears to be released from Gα13 rather than Gαq (19Macrez N. Morel J.L. Kalkbrenner F. Viard P. Schultz G. Mironneau J. J. Biol. Chem. 1997; 272: 23180-23185Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Not all βγ dimers are equivalent in activating PI3K. Gβγ dimers containing β5 are least able to activate p110γ and p110β, while those containing γ11 are least able to activate p110γ (8Kerchner K.R. Clay R.L. McCleery G. Watson N. McIntire W.E. Myung C.S. Garrison J.C. J. Biol. Chem. 2004; 279: 44554-44562Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 20Maier U. Babich A. Macrez N. Leopoldt D. Gierschik P. Illenberger D. Nurnberg B. J. Biol. Chem. 2000; 275: 13746-13754Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We predict that stimulation of a Gq-coupled receptor will negatively modulate the LTCC if (a) the activated Gαq inhibits p110α and (b) its released Gβγ dimer is a weak activator of p110β and p110γ. More studies are needed to determine how cells integrate these competing signals to mount an appropriate Ca2+ channel response. LTCCs are composed of α1, β, and δ/α2 subunits and, in some forms, an additional γ subunit. There are also multiple isoforms of each subunit. The α1 subunit forms the pore of the channel and the intracellular β subunit regulates cellular localization. Cardiac LTCCs are mostly composed of α1C and β2a isoforms, although other β variants are also present in the heart (21Striessnig J. Cell Physiol. Biochem. 1999; 9: 242-269Crossref PubMed Scopus (162) Google Scholar). In a heterologous expression system, PI3K signaling stimulated ICa,L through Akt-mediated phosphorylation of β2a subunits, leading to increased trafficking of the LTCC to the plasma membrane (4Viard P. Butcher A.J. Halet G. Davies A. Nurnberg B. Heblich F. Dolphin A.C. Nat. Neurosci. 2004; 7: 939-946Crossref PubMed Scopus (207) Google Scholar). Akt specifically phosphorylates the β2a subunit on a consensus sequence that is not present in β1b, β3, or β4 (4Viard P. Butcher A.J. Halet G. Davies A. Nurnberg B. Heblich F. Dolphin A.C. Nat. Neurosci. 2004; 7: 939-946Crossref PubMed Scopus (207) Google Scholar). Inhibition of Akt by Gαq may reduce trafficking of the cardiac LTCC to the plasma membrane. Additional studies are planned to determine whether infusion of purified activated Akt proteins reverses the inhibition of ICa,L in our transgenic myocytes and if these cells show a decreased amount of LTCC proteins in the plasma membrane. Finally, it would be of interest to explore whether G protein-coupled receptors, Gαq, and p110α PI3K form a macromolecular signaling complex with the LTCC at the myocyte plasma membrane so that the current through these channels can be specifically regulated. In conclusion, results from this study indicate that negative modulation of the LTCC by activated Gαq in cardiac myocytes is mediated by inhibition of PI3K, perhaps specifically by the p110α isoform. Further studies are needed to determine whether L-type and other types of Ca2+ channels present in other excitable cell types are also inhibited by Gαq through a similar mechanism.
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