Advertisement

q Inhibits Cardiac L-type Ca2+ Channels through Phosphatidylinositol 3-Kinase*

  • Zhongju Lu
    Affiliations
    Department of Physiology and Biophysics and the Institute of Molecular Cardiology, Stony Brook, New York 11794
    Search for articles by this author
  • Ya-Ping Jiang
    Affiliations
    Department of Medicine, Stony Brook University, Stony Brook, New York 11794
    Search for articles by this author
  • Lisa M. Ballou
    Affiliations
    Department of Medicine, Stony Brook University, Stony Brook, New York 11794
    Search for articles by this author
  • Ira S. Cohen
    Affiliations
    Department of Physiology and Biophysics and the Institute of Molecular Cardiology, Stony Brook, New York 11794
    Search for articles by this author
  • Richard Z. Lin
    Correspondence
    To whom correspondence should be addressed: Dept. of Medicine, Division of Hematology, Stony Brook University, Stony Brook, NY 11794-8151. Tel.: 631-444-2059; Fax: 631-444-7530;
    Affiliations
    Department of Physiology and Biophysics and the Institute of Molecular Cardiology, Stony Brook, New York 11794

    Department of Medicine, Stony Brook University, Stony Brook, New York 11794

    Department of Veterans Affairs Medical Center, Northport, New York 11768
    Search for articles by this author
  • Author Footnotes
    * This work was supported by grants from the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to R. Z. L.) and National Institutes of Health Grants DK62722 (to R. Z. L.) and HL70161, HL28958, and HL67101 (to I. S. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:September 26, 2005DOI:https://doi.org/10.1074/jbc.M508441200
      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).
      The abbreviations used are: LTCC
      L-type Ca2+ channel
      PI
      phosphatidylinositol
      PI3K
      PI 3-kinase
      PI(3,4,5)P3
      PI 3,4,5-trisphosphate
      PI(4,5)P2
      PI 4,5-bisphosphate
      PI(3,5)P2
      PI 3,5-bisphosphate
      PLCβ
      phospholipase Cβ
      4-HT
      4-hydroxytamoxifen
      PH-Grp1
      glutathione S-transferase fused to the pleckstrin homology domain of Grp1
      WT
      wild-type
      F
      farad.
      2The abbreviations used are: LTCC
      L-type Ca2+ channel
      PI
      phosphatidylinositol
      PI3K
      PI 3-kinase
      PI(3,4,5)P3
      PI 3,4,5-trisphosphate
      PI(4,5)P2
      PI 4,5-bisphosphate
      PI(3,5)P2
      PI 3,5-bisphosphate
      PLCβ
      phospholipase Cβ
      4-HT
      4-hydroxytamoxifen
      PH-Grp1
      glutathione S-transferase fused to the pleckstrin homology domain of Grp1
      WT
      wild-type
      F
      farad.
      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 (
      • Blair L.A.
      • Marshall J.
      ,
      • Macrez N.
      • Mironneau C.
      • Carricaburu V.
      • Quignard J.F.
      • Babich A.
      • Czupalla C.
      • Nurnberg B.
      • Mironneau J.
      ). 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 (
      • Macrez N.
      • Mironneau C.
      • Carricaburu V.
      • Quignard J.F.
      • Babich A.
      • Czupalla C.
      • Nurnberg B.
      • Mironneau J.
      ,
      • Le Blanc C.
      • Mironneau C.
      • Barbot C.
      • Henaff M.
      • Bondeva T.
      • Wetzker R.
      • Macrez N.
      ). 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 (
      • Viard P.
      • Butcher A.J.
      • Halet G.
      • Davies A.
      • Nurnberg B.
      • Heblich F.
      • Dolphin A.C.
      ).
      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 (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Ahmadi K.
      • Timms J.
      • Katso R.
      • Driscoll P.C.
      • Woscholski R.
      • Parker P.J.
      • Waterfield M.D.
      ). Cardiac myocytes contain p85α and p85β regulatory subunits bound to either p110α or p110β catalytic subunits in unknown stoichiometric proportions (
      • Oudit G.Y.
      • Sun H.
      • Kerfant B.G.
      • Crackower M.A.
      • Penninger J.M.
      • Backx P.H.
      ). Cardiac myocytes also express p110γ (
      • Oudit G.Y.
      • Sun H.
      • Kerfant B.G.
      • Crackower M.A.
      • Penninger J.M.
      • Backx P.H.
      ). 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 (
      • Kurosu H.
      • Maehama T.
      • Okada T.
      • Yamamoto T.
      • Hoshino S.
      • Fukui Y.
      • Ui M.
      • Hazeki O.
      • Katada T.
      ).
      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 (
      • Kurosu H.
      • Maehama T.
      • Okada T.
      • Yamamoto T.
      • Hoshino S.
      • Fukui Y.
      • Ui M.
      • Hazeki O.
      • Katada T.
      ,
      • Kerchner K.R.
      • Clay R.L.
      • McCleery G.
      • Watson N.
      • McIntire W.E.
      • Myung C.S.
      • Garrison J.C.
      ), 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 (
      • Ballou L.M.
      • Lin H.Y.
      • Fan G.
      • Jiang Y.P.
      • Lin R.Z.
      ). Using purified recombinant proteins, we demonstrated that Gαq binds directly to the enzyme to inhibit its activity.
      L. 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 (
      • Ballou L.M.
      • Lin H.Y.
      • Fan G.
      • Jiang Y.P.
      • Lin R.Z.
      ). 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 (
      • Ballou L.M.
      • Lin H.Y.
      • Fan G.
      • Jiang Y.P.
      • Lin R.Z.
      ,
      • Venkatakrishnan G.
      • Exton J.H.
      ,
      • Fan G.
      • Ballou L.M.
      • Lin R.Z.
      ).
      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.
      Fan, 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.
      Fan, 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.

      EXPERIMENTAL PROCEDURES

      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 (
      • Ballou L.M.
      • Lin H.Y.
      • Fan G.
      • Jiang Y.P.
      • Lin R.Z.
      ).
      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.
      Fan, 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.
      Fan, 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 (
      • Ballou L.M.
      • Cross M.E.
      • Huang S.
      • McReynolds E.M.
      • Zhang B.X.
      • Lin R.Z.
      ). 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.

      RESULTS

      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.
      Fan, 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 (
      • Macrez N.
      • Mironneau C.
      • Carricaburu V.
      • Quignard J.F.
      • Babich A.
      • Czupalla C.
      • Nurnberg B.
      • Mironneau J.
      ,
      • Le Blanc C.
      • Mironneau C.
      • Barbot C.
      • Henaff M.
      • Bondeva T.
      • Wetzker R.
      • Macrez N.
      ).
      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.
      Figure thumbnail gr1
      FIGURE 1Effect of PI(3,4,5)P3 on the activation of ICa,L. Myocytes were isolated from tamoxifen-treated WT, GαqQ209L-hbER (QL), and GαqQ209L-AA-hbER (QL-AA) mice, and average peak ICa,L densities were measured by whole-cell patch clamping. The membrane was held at -50 mV and depolarized for 300 ms to +10 mV. Internal solution with or without phosphoinositides (1 μm) was infused through the patch pipette. ** signifies a statistically significant increase in peak ICa,L density induced by PI(3,4,5)P3 as compared with the matched control. The number of cells examined in each group is indicated in parentheses.
      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.
      Figure thumbnail gr2
      FIGURE 2I-V relationships of myocytes infused with PI(3,4,5)P3. Myocytes were isolated from tamoxifen-treated mice. Data were generated using 300-ms depolarizing voltage steps from -50 to +50 mV in 10-mV increments from a holding potential of -50 mV. Top and middle panels show representative traces of ICa,L activation in cells infused with internal solution without (control (Con)) or with 1 μm PI(3,4,5)P3. Bottom panels plot the mean peak inward current density for each data set at each voltage potential. A, WT, n = 12 for control and n = 7 for PI(3,4,5)P3; B,GαqQ209L-hbER (QL), n = 5 for control and n = 7 for PI(3,4,5)P3; C,GαqQ209L-AA-hbER (QL-AA), n = 5 for both groups.
      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.
      Figure thumbnail gr3
      FIGURE 3Time-dependent effect of PI(3,4,5)P3 on ICa,L activation. Myocytes were isolated from tamoxifen-treated GαqQ209L-hbER (QL) and GαqQ209L-AA-hbER (QL-AA) mice. Cells were patched and infused with internal solution without (Con) or with 1 μm PI(3,4,5)P3. Peak ICa,L densities were measured following repeated (every 10 s) voltage steps from -50 mV to +10 mV (300 ms duration). Values shown are average ICa,L densities normalized to the value of the first ICa,L recording, which is taken immediately after breaking into the whole-cell mode. Left panel, n = 7; middle and right panels, n = 5 for both groups.
      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 (
      • Oudit G.Y.
      • Sun H.
      • Kerfant B.G.
      • Crackower M.A.
      • Penninger J.M.
      • Backx P.H.
      ), 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.
      Figure thumbnail gr4
      FIGURE 4Modulation of ICa,L activation by PI3K isozymes. Myocytes were isolated from tamoxifen-treated GαqQ209L-AA-hbER mice, and average peak ICa,L densities were measured using the protocol described in the legend of . Cells were infused with internal solution without (Con) or with 20 nm PI3Ks plus or minus 1 μm PI(4,5)P2. ** signifies a statistically significant increase in ICa,L density induced by PI3K in the presence of PI(4,5)P2 as compared with the matched control with enzyme alone. The number of cells analyzed in each group is indicated in parentheses.
      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).
      Figure thumbnail gr5
      FIGURE 5I-V relationships for myocytes infused with PI3K isozymes. Myocytes were isolated from tamoxifen-treated GαqQ209L-AA-hbER mice, and ICa,L was elicited using the protocol described in the legend of , representative recordings of ICa,L activation in cells infused with 20 nm of PI3K in the absence (A) or presence (B) of 1 μm PI(4,5)P2. Mean I-V relationships in the absence (C) or presence (D) of 1 μm PI(4,5)P2. Con, n = 5; p110α/p85α, n = 5; p110β/p85α, n = 4; and p110γ, n = 4.
      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).
      Figure thumbnail gr6
      FIGURE 6Time-dependent effect of PI3K isozymes plus PI(4,5)P2 on ICa,L activation. Myocytes were isolated from tamoxifen-treated GαqQ209L-AA-hbER mice, and ICa,L densities were measured following repeated depolarization steps and normalized as described in the legend of . Cells were infused with 20 nm PI3K isozymes plus 1 μm PI(4,5)P2 (n = 4 for all three conditions).
      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 (
      • Gray A.
      • Van Der Kaay J.
      • Downes C.P.
      ) and has been used to block PI3K signaling (
      • Viard P.
      • Butcher A.J.
      • Halet G.
      • Davies A.
      • Nurnberg B.
      • Heblich F.
      • Dolphin A.C.
      ). 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).
      Figure thumbnail gr7
      FIGURE 7Reduction of ICa,L density in WT myocytes by decreasing endogenous PI(3,4,5)P3. Cardiac myocytes were isolated from WT mice not injected with tamoxifen, and ICa,L was measured in the absence of 4-HT using the protocol described in the legend of sample traces of ICa,L activation in the presence of control internal solution (top panel) or 20 nm PH-Grp1 (middle panel) and the corresponding I-V relationships (bottom panel; n = 7 for both groups). B, representative recordings of ICa,L activation in myocytes infused with PTEN buffer diluted into internal solution (top panel) or 20 nm PTEN (middle panel) and the corresponding I-V relationships (bottom panel; n = 7 for PTEN buffer and n = 20 for PTEN).
      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.
      Fan, 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.
      Figure thumbnail gr8
      FIGURE 8Akt activity in hearts from WT and GαqQ209L-AA-hbER mice. Mice were injected with 1 mg of tamoxifen daily for 28 days, fasted overnight, and then injected intraperitoneally with 10 units of insulin/kg of body weight or saline as control. Animals were sacrificed 10 min later and the hearts removed. A, Akt activity was measured in heart lysates after immunoprecipitation with an Akt antibody. The number of mice used per group is shown in parentheses. ** signifies a significant difference between GαqQ209L-AA-hbER (QL-AA) + insulin and WT + insulin (t test, p < 0.01). B, Western blot analysis of heart lysate proteins prepared from mice that were injected with insulin. The blot was probed with an Akt antibody (top panel). Signals were quantitated using the Odyssey Infrared Imaging System. WT, 3.86 ± 0.23 arbitrary units; QL-AA, 4.07 ± 0.35 arbitrary units. The difference is not statistically significant (t test). The blot was stripped and reprobed with a phospho-specific Erk1/2 antibody (bottom panel).

      DISCUSSION

      Our initial study showing that ICa,L in GαqQ209L-hbER and GαqQ209L-AA-hbER transgenic myocytes is similarly depressed as compared with WT myocytes
      Fan, 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 (
      • Shapiro M.S.
      • Loose M.D.
      • Hamilton S.E.
      • Nathanson N.M.
      • Gomeza J.
      • Wess J.
      • Hille B.
      ) and N- and P/Q-type (
      • Dolphin A.C.
      ) 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 (
      • Dolphin A.C.
      ). 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 (
      • Bannister R.A.
      • Melliti K.
      • Adams B.A.
      ).
      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 (
      • Ballou L.M.
      • Cross M.E.
      • Huang S.
      • McReynolds E.M.
      • Zhang B.X.
      • Lin R.Z.
      ,
      • Ballou L.M.
      • Tian P.Y.
      • Lin H.Y.
      • Jiang Y.P.
      • Lin R.Z.
      ). Subsequently, we demonstrated that activated Gαq directly binds to and inhibits the p110α/p85α PI3K
      Fan, 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.
      (
      • Ballou L.M.
      • Lin H.Y.
      • Fan G.
      • Jiang Y.P.
      • Lin R.Z.
      ). By contrast, transfected GαqQ209L did not inhibit p110β immunoprecipitated from cotransfected cells (
      • Ballou L.M.
      • Lin H.Y.
      • Fan G.
      • Jiang Y.P.
      • Lin R.Z.
      ). We have also reported that activated Gαq does not bind to p110γ,
      Fan, 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 (
      • Blair L.A.
      • Marshall J.
      ,
      • Macrez N.
      • Mironneau C.
      • Carricaburu V.
      • Quignard J.F.
      • Babich A.
      • Czupalla C.
      • Nurnberg B.
      • Mironneau J.
      ). 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 (
      • Quignard J.F.
      • Mironneau J.
      • Carricaburu V.
      • Fournier B.
      • Babich A.
      • Nurnberg B.
      • Mironneau C.
      • Macrez N.
      ) 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 (
      • Macrez N.
      • Morel J.L.
      • Kalkbrenner F.
      • Viard P.
      • Schultz G.
      • Mironneau J.
      ). 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 (
      • Kerchner K.R.
      • Clay R.L.
      • McCleery G.
      • Watson N.
      • McIntire W.E.
      • Myung C.S.
      • Garrison J.C.
      ,
      • Maier U.
      • Babich A.
      • Macrez N.
      • Leopoldt D.
      • Gierschik P.
      • Illenberger D.
      • Nurnberg B.
      ). 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 (
      • Striessnig J.
      ). 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 (
      • Viard P.
      • Butcher A.J.
      • Halet G.
      • Davies A.
      • Nurnberg B.
      • Heblich F.
      • Dolphin A.C.
      ). Akt specifically phosphorylates the β2a subunit on a consensus sequence that is not present in β1b, β3, or β4 (
      • Viard P.
      • Butcher A.J.
      • Halet G.
      • Davies A.
      • Nurnberg B.
      • Heblich F.
      • Dolphin A.C.
      ). 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.

      References

        • Blair L.A.
        • Marshall J.
        Neuron. 1997; 19: 421-429
        • Macrez N.
        • Mironneau C.
        • Carricaburu V.
        • Quignard J.F.
        • Babich A.
        • Czupalla C.
        • Nurnberg B.
        • Mironneau J.
        Circ. Res. 2001; 89: 692-699
        • Le Blanc C.
        • Mironneau C.
        • Barbot C.
        • Henaff M.
        • Bondeva T.
        • Wetzker R.
        • Macrez N.
        Circ. Res. 2004; 95: 300-307
        • Viard P.
        • Butcher A.J.
        • Halet G.
        • Davies A.
        • Nurnberg B.
        • Heblich F.
        • Dolphin A.C.
        Nat. Neurosci. 2004; 7: 939-946
        • Vanhaesebroeck 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: 535-602
        • Oudit G.Y.
        • Sun H.
        • Kerfant B.G.
        • Crackower M.A.
        • Penninger J.M.
        • Backx P.H.
        J. Mol. Cell. Cardiol. 2004; 37: 449-471
        • Kurosu H.
        • Maehama T.
        • Okada T.
        • Yamamoto T.
        • Hoshino S.
        • Fukui Y.
        • Ui M.
        • Hazeki O.
        • Katada T.
        J. Biol. Chem. 1997; 272: 24252-24256
        • Kerchner K.R.
        • Clay R.L.
        • McCleery G.
        • Watson N.
        • McIntire W.E.
        • Myung C.S.
        • Garrison J.C.
        J. Biol. Chem. 2004; 279: 44554-44562
        • Ballou L.M.
        • Lin H.Y.
        • Fan G.
        • Jiang Y.P.
        • Lin R.Z.
        J. Biol. Chem. 2003; 278: 23472-23479
        • Venkatakrishnan G.
        • Exton J.H.
        J. Biol. Chem. 1996; 271: 5066-5072
        • Fan G.
        • Ballou L.M.
        • Lin R.Z.
        J. Biol. Chem. 2003; 278: 52432-52436
        • Ballou L.M.
        • Cross M.E.
        • Huang S.
        • McReynolds E.M.
        • Zhang B.X.
        • Lin R.Z.
        J. Biol. Chem. 2000; 275: 4803-4809
        • Gray A.
        • Van Der Kaay J.
        • Downes C.P.
        Biochem. J. 1999; 344: 929-936
        • Shapiro 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: 10899-10904
        • Dolphin A.C.
        Pharmacol. Rev. 2003; 55: 607-627
        • Bannister R.A.
        • Melliti K.
        • Adams B.A.
        Biophys. J. 2002; 83: 3256-3267
        • Ballou L.M.
        • Tian P.Y.
        • Lin H.Y.
        • Jiang Y.P.
        • Lin R.Z.
        J. Biol. Chem. 2001; 276: 40910-40916
        • Quignard J.F.
        • Mironneau J.
        • Carricaburu V.
        • Fournier B.
        • Babich A.
        • Nurnberg B.
        • Mironneau C.
        • Macrez N.
        J. Biol. Chem. 2001; 276: 32545-32551
        • Macrez N.
        • Morel J.L.
        • Kalkbrenner F.
        • Viard P.
        • Schultz G.
        • Mironneau J.
        J. Biol. Chem. 1997; 272: 23180-23185
        • Maier U.
        • Babich A.
        • Macrez N.
        • Leopoldt D.
        • Gierschik P.
        • Illenberger D.
        • Nurnberg B.
        J. Biol. Chem. 2000; 275: 13746-13754
        • Striessnig J.
        Cell Physiol. Biochem. 1999; 9: 242-269