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Control of Calcium Signal Propagation to the Mitochondria by Inositol 1,4,5-Trisphosphate-binding Proteins

2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês

10.1074/jbc.m411591200

ISSN

1083-351X

Autores

Xuena Lin, Péter Várnai, György Csordás, András Balla, Takeharu Nagai, Atsushi Miyawaki, Tamás Balla, György Hajnóczky,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Cytosolic Ca2+ ([Ca2+]c) signals triggered by many agonists are established through the inositol 1,4,5-trisphosphate (IP3) messenger pathway. This pathway is believed to use Ca2+-dependent local interactions among IP3 receptors (IP3R) and other Ca2+ channels leading to coordinated Ca2+ release from the endoplasmic reticulum throughout the cell and coupling Ca2+ entry and mitochondrial Ca2+ uptake to Ca2+ release. To evaluate the role of IP3 in the local control mechanisms that support the propagation of [Ca2+]c waves, store-operated Ca2+ entry, and mitochondrial Ca2+ uptake, we used two IP3-binding proteins (IP3BP): 1) the PH domain of the phospholipase C-like protein, p130 (p130PH); and 2) the ligand-binding domain of the human type-I IP3R (IP3R224–605). As expected, p130PH-GFP and GFP-IP3R224–605 behave as effective mobile cytosolic IP3 buffers. In COS-7 cells, the expression of IP3BPs had no effect on store-operated Ca2+ entry. However, the IP3-linked [Ca2+]c signal appeared as a regenerative wave and IP3BPs slowed down the wave propagation. Most importantly, IP3BPs largely inhibited the mitochondrial [Ca2+] signal and decreased the relationship between the [Ca2+]c and mitochondrial [Ca2+] signals, indicating disconnection of the mitochondria from the [Ca2+]c signal. These data suggest that IP3 elevations are important to regulate the local interactions among IP3Rs during propagation of [Ca2+]c waves and that the IP3-dependent synchronization of Ca2+ release events is crucial for the coupling between Ca2+ release and mitochondrial Ca2+ uptake. Cytosolic Ca2+ ([Ca2+]c) signals triggered by many agonists are established through the inositol 1,4,5-trisphosphate (IP3) messenger pathway. This pathway is believed to use Ca2+-dependent local interactions among IP3 receptors (IP3R) and other Ca2+ channels leading to coordinated Ca2+ release from the endoplasmic reticulum throughout the cell and coupling Ca2+ entry and mitochondrial Ca2+ uptake to Ca2+ release. To evaluate the role of IP3 in the local control mechanisms that support the propagation of [Ca2+]c waves, store-operated Ca2+ entry, and mitochondrial Ca2+ uptake, we used two IP3-binding proteins (IP3BP): 1) the PH domain of the phospholipase C-like protein, p130 (p130PH); and 2) the ligand-binding domain of the human type-I IP3R (IP3R224–605). As expected, p130PH-GFP and GFP-IP3R224–605 behave as effective mobile cytosolic IP3 buffers. In COS-7 cells, the expression of IP3BPs had no effect on store-operated Ca2+ entry. However, the IP3-linked [Ca2+]c signal appeared as a regenerative wave and IP3BPs slowed down the wave propagation. Most importantly, IP3BPs largely inhibited the mitochondrial [Ca2+] signal and decreased the relationship between the [Ca2+]c and mitochondrial [Ca2+] signals, indicating disconnection of the mitochondria from the [Ca2+]c signal. These data suggest that IP3 elevations are important to regulate the local interactions among IP3Rs during propagation of [Ca2+]c waves and that the IP3-dependent synchronization of Ca2+ release events is crucial for the coupling between Ca2+ release and mitochondrial Ca2+ uptake. Inositol 1,4,5-trisphosphate (IP3) 1The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; IP3BP, IP3-binding protein; PH, pleckstrin homology; FRAP, fluorescence recovery after photobleaching; tlag, the lag time needed to reach half of the maximum cytosolic Ca2+ response; FGFP, GFP fluorescence; RFP, red fluorescence protein; YFP, yellow fluorescence protein; R0/RP, prestimulation ratio versus peak response ratio; RyR, ryanodine receptor; PLC, phospholipase C; ER, endoplasmic reticulum; p130, 130-kDa phospholipase C-like protein; GABAA, γ-aminobutyric acid, type A; pericam-mt, pericam targeted to the mitochondria; mito, mitochondrial; BSA, bovine serum albumin; Tg, thapsigargin; ECM, extracellular matrix; EGF, epidermal growth factor; ICM, intracellular medium; [Ca2+]m, mitochondrial matrix [Ca2+]; PIP2, phosphatidylinositol 4,5-bisphosphate.1The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; IP3BP, IP3-binding protein; PH, pleckstrin homology; FRAP, fluorescence recovery after photobleaching; tlag, the lag time needed to reach half of the maximum cytosolic Ca2+ response; FGFP, GFP fluorescence; RFP, red fluorescence protein; YFP, yellow fluorescence protein; R0/RP, prestimulation ratio versus peak response ratio; RyR, ryanodine receptor; PLC, phospholipase C; ER, endoplasmic reticulum; p130, 130-kDa phospholipase C-like protein; GABAA, γ-aminobutyric acid, type A; pericam-mt, pericam targeted to the mitochondria; mito, mitochondrial; BSA, bovine serum albumin; Tg, thapsigargin; ECM, extracellular matrix; EGF, epidermal growth factor; ICM, intracellular medium; [Ca2+]m, mitochondrial matrix [Ca2+]; PIP2, phosphatidylinositol 4,5-bisphosphate.-induced Ca2+ liberation from intracellular stores results in a [Ca2+]c signal that controls a wide spectrum of cell functions, including energy metabolism, gene transcription, and cell proliferation. Appropriate exposure of the effectors to Ca2+ throughout the cell is supported by several mechanisms that include the propagation of Ca2+ release throughout the cell without attenuation, the recruitment of Ca2+ entry, and efficient delivery of the [Ca2+]c signal into organelles such as the nucleus and mitochondria (1.Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 517-529Crossref PubMed Scopus (4064) Google Scholar). Spreading of the [Ca2+]c signal is facilitated by regenerative mechanisms of Ca2+ mobilization, which may derive from interactions between adjacent Ca2+ release sites. Non-metabolizable IP3 analogs have been found to evoke [Ca2+]c oscillations, providing support to the idea that at a constant [IP3] Ca2+ released through IP3Rs is sufficient to trigger regenerative Ca2+ release via binding to and activating IP3Rs and ryanodine receptors (RyR) (2.Wakui M. Potter B.V. Petersen O.H. Nature. 1989; 339: 317-320Crossref PubMed Scopus (243) Google Scholar). Based on IP3 microinjection and uncaging studies, the regenerative Ca2+ release during [Ca2+]c waves was also claimed to be largely independent of [IP3] (3.Lechleiter J.D. Clapham D.E. Cell. 1992; 69: 283-294Abstract Full Text PDF PubMed Scopus (300) Google Scholar, 4.Callamaras N. Marchant J.S. Sun X.P. Parker I. J. Physiol. (Lond.). 1998; 509: 81-91Crossref Scopus (137) Google Scholar). Along this line, IP3R-mediated [Ca2+]c waves were observed in the absence of any stimulated IP3 formation (5.Rooney T.A. Renard D.C. Sass E.J. Thomas A.P. J. Biol. Chem. 1991; 266: 12272-12282Abstract Full Text PDF PubMed Google Scholar). However, released Ca2+ may also promote phospholipase C (PLC) activation and, in turn, increase IP3, providing a potential amplification mechanism for IP3R-mediated Ca2+ release waves (6.Harootunian A.T. Kao J.P. Paranjape S. Tsien R.Y. Science. 1991; 251: 75-78Crossref PubMed Scopus (248) Google Scholar, 7.Young K.W. Nash M.S. Challiss R.A. Nahorski S.R. J. Biol. Chem. 2003; 278: 20753-20760Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 8.Meyer T. Stryer L. Annu. Rev. Biophys. Biophys. Chem. 1991; 20: 153-174Crossref PubMed Scopus (299) Google Scholar). Ca2+ may activate the PLC coupled to the agonist receptor to stimulate cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane. The localization of phospholipase C enzyme isoforms (9.Rice A. Parrington J. Jones K.T. Swann K. Dev. Biol. 2000; 228: 125-135Crossref PubMed Scopus (100) Google Scholar, 10.Lee S.B. Varnai P. Balla A. Jalink K. Rhee S.G. Balla T. J. Biol. Chem. 2004; 279: 24362-24371Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and PIP2 (11.Watt S.A. Kular G. Fleming I.N. Downes C.P. Lucocq J.M. Biochem. J. 2002; 363: 657-666Crossref PubMed Scopus (294) Google Scholar) in intracellular membranes has also been documented. IP3 formed at the plasma membrane may rapidly diffuse throughout the cytoplasm (12.Allbritton N.L. Meyer T. Stryer L. Science. 1992; 258: 1812-1815Crossref PubMed Scopus (899) Google Scholar), but the presence of PIP2 and PLC in the vicinity of the IP3R could also provide for a local IP3 feedback. Thus, the role of IP3 fluctuations in Ca2+ wave propagation requires further investigation. Recharging of the ER Ca2+ stores during the agonist-induced [Ca2+]c signal involves Ca2+ entry mediated by so-called store-operated Ca2+-entry mechanisms that might be mediated by the canonical transient receptor potential channel family (13.Putney Jr., J.W. Trends Cell Biol. 2004; 14: 282-286Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). One of the proposed mechanisms for activation of Ca2+ entry during Ca2+ release is a conformational coupling between IP3Rs and store-operated Ca2+ channels (14.Putney Jr., J.W. Broad L.M. Braun F.J. Lievremont J.P. Bird G.S. J. Cell Sci. 2001; 114: 2223-2229Crossref PubMed Google Scholar). Although store-operated Ca2+ entry is activated by agents that directly target the ER Ca2+ store (15.Parekh A.B. J. Physiol. (Lond.). 2003; 547: 333-348Crossref Scopus (165) Google Scholar, 16.Takemura H. Hughes A.R. Thastrup O. Putney Jr., J.W. J. Biol. Chem. 1989; 264: 12266-12271Abstract Full Text PDF PubMed Google Scholar), IP3 binding to the IP3R has been claimed to have a role in activation of Ca2+ entry (17.Kiselyov K. Mignery G.A. Zhu M.X. Muallem S. Mol. Cell. 1999; 4: 423-429Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 18.Ma H.T. Venkatachalam K. Rys-Sikora K.E. He L.P. Zheng F. Gill D.L. Biochem. J. 2003; 376: 667-676Crossref PubMed Scopus (24) Google Scholar). Nonetheless, the question of whether an increase in [IP3] is required for the channel activation remains elusive. IP3-induced [Ca2+] spikes are also delivered to the mitochondria to control the activity of several enzymes that participate in ATP production as well as that of other proteins compartmentalized to the matrix space (19.Jouaville L.S. Pinton P. Bastianutto C. Rutter G.A. Rizzuto R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13807-13812Crossref PubMed Scopus (630) Google Scholar, 20.Hajnoczky G. Robb-Gaspers L.D. Seitz M.B. Thomas A.P. Cell. 1995; 82: 415-424Abstract Full Text PDF PubMed Scopus (943) Google Scholar, 21.Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Crossref PubMed Scopus (985) Google Scholar). Mitochondrial Ca2+ uptake sites display low affinity toward Ca2+ and appear to respond mostly to the large local [Ca2+]c transients that occur in the vicinity of the activated IP3Rs and RyRs (21.Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Crossref PubMed Scopus (985) Google Scholar, 22.Csordas G. Thomas A.P. Hajnoczky G. EMBO J. 1999; 18: 96-108Crossref PubMed Scopus (439) Google Scholar, 23.Szalai G. Csordas G. Hantash B.M. Thomas A.P. Hajnoczky G. J. Biol. Chem. 2000; 275: 15305-15313Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 24.Rizzuto R. Pinton P. Carrington W. Fay F.S. Fogarty K.E. Lifshitz L.M. Tuft R.A. Pozzan T. Science. 1998; 280: 1763-1766Crossref PubMed Scopus (1747) Google Scholar), but a rapid mode of Ca2+ uptake at relatively low [Ca2+] has also been documented (25.Sparagna G.C. Gunter K.K. Sheu S.S. Gunter T.E. J. Biol. Chem. 1995; 270: 27510-27515Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). A local interaction between RyRs and mitochondrial Ca2+ uptake sites may secure that even fundamental Ca2+ release events (Ca2+ sparks) induce a [Ca2+]m increase in the neighboring mitochondrion (Ca2+ mark) (26.Pacher P. Thomas A.P. Hajnoczky G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2380-2385Crossref PubMed Scopus (129) Google Scholar). However, a substantially larger [Ca2+]m rise occurs during a global [Ca2+]c signal, suggesting that numerous Ca2+ release events cooperate with each other to establish a [Ca2+]m signal in a mitochondrion. At the ER-mitochondrial interface, great quantities of IP3Rs have been visualized and enrichment in ER Ca2+ pumps and mitochondrial Ca2+ uniporters is probable (reviewed in Refs. 27.Rizzuto, R., Duchen, M. R., and Pozzan, T. (2004) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/53/re1Google Scholar and 28.Hajnoczky G. Csordas G. Madesh M. Pacher P. J. Physiol. (Lond.). 2000; 529: 69-81Crossref Scopus (173) Google Scholar). Morphology of the ER-mitochondrial associations and concentration of the Ca2+ transporters at the interface may play a role in controlling coordinated activation of the individual Ca2+ release events that give rise to the IP3-linked [Ca2+]m spikes. Thus, the role of IP3 in coordination of Ca2+ release events is of great interest in a variety of Ca2+ signaling mechanisms. We reasoned that mobile IP3 buffers should be useful to test the role of IP3 in [Ca2+]c wave propagation, activation of Ca2+ entry, and in IP3R-dependent activation of mitochondrial Ca2+ uptake. An intensely investigated IP3-binding module is the IP3-binding domain of the IP3R. The N-terminal cytoplasmic region of the human type-I IP3R (residues 1–604) binds IP3 with comparable affinity to the full-length IP3R (29.Maeda N. Niinobe M. Mikoshiba K. EMBO J. 1990; 9: 61-67Crossref PubMed Scopus (230) Google Scholar). Removal of residues 1–223 (suppressor region) further increases the affinity for IP3 (30.Yoshikawa F. Iwasaki H. Michikawa T. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 316-327Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Expression of IP3R224–605 has been shown to attenuate the ATP-induced [Ca2+]c signal in human embryonic kidney 293 and in COS-7 cells (31.Varnai P. Lin X. Lee S.B. Tuymetova G. Bondeva T. Spat A. Rhee S.G. Hajnoczky G. Balla T. J. Biol. Chem. 2002; 277: 27412-27422Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 32.Uchiyama T. Yoshikawa F. Hishida A. Furuichi T. Mikoshiba K. J. Biol. Chem. 2002; 277: 8106-8113Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Another structurally unrelated IP3-binding module is the PH domain of certain proteins. Some PH domains are known to bind IP3 and/or PIP2, for example, the PH domains of PLCδ1 and the PLC-like 130-kDa protein (p130). p130 was isolated from rat brain as an IP3BP (33.Kanematsu T. Takeya H. Watanabe Y. Ozaki S. Yoshida M. Koga T. Iwanaga S. Hirata M. J. Biol. Chem. 1992; 267: 6518-6525Abstract Full Text PDF PubMed Google Scholar) and has been shown to be important in the signaling by GABAA receptors (34.Kanematsu T. Jang I.S. Yamaguchi T. Nagahama H. Yoshimura K. Hidaka K. Matsuda M. Takeuchi H. Misumi Y. Nakayama K. Yamamoto T. Akaike N. Hirata M. EMBO J. 2002; 21: 1004-1011Crossref PubMed Scopus (115) Google Scholar). p130 shares 38.2% sequence homology to PLC-δ1 but lacks catalytic activity. The PH domain of p130 (residues 95–233) has also been shown to inhibit the [Ca2+]c signal evoked by IP3-linked agonists (31.Varnai P. Lin X. Lee S.B. Tuymetova G. Bondeva T. Spat A. Rhee S.G. Hajnoczky G. Balla T. J. Biol. Chem. 2002; 277: 27412-27422Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 35.Takeuchi H. Oike M. Paterson H.F. Allen V. Kanematsu T. Ito Y. Erneux C. Katan M. Hirata M. Biochem. J. 2000; 349: 357-368Crossref PubMed Scopus (53) Google Scholar). Our main objective was to evaluate the possible role of fluctuations of IP3 in the spatio-temporal organization of the calcium signal, utilizing IP3R224–605 and p130PH. Our results indicate that these two proteins are freely distributed in the cytosol, inhibit Ca2+ release induced by suboptimal IP3 but do not suppress Ca2+ release evoked by sensitization of the IP3R to IP3, and fail to attenuate non-IP3R-mediated Ca2+ release. Thus, IP3R224–605 and p130PH act as mobile cytosolic IP3 buffers. IP3R224–605 and p130PH were used next to explore the effects of IP3 buffering on the propagation of IP3-linked [Ca2+]c waves, store depletion-induced Ca2+ entry, and on [Ca2+]c signal propagation to the mitochondria. Our studies reveal that buffering of IP3 leads to a decrease in the velocity of [Ca2+]c waves and can effectively uncouple [Ca2+]m from the [Ca2+]c signal in the cell. DNA Constructs and Recombinant Proteins—The constructs encoding the fusion proteins of the PH domain of PLCδ1 or of the p130 protein and the (1,4,5)IP3-binding domain (residues 224–605) of the human type-I (1,4,5)IP3 receptor with cyan, green, or yellow fluorescent protein have been described previously (31.Varnai P. Lin X. Lee S.B. Tuymetova G. Bondeva T. Spat A. Rhee S.G. Hajnoczky G. Balla T. J. Biol. Chem. 2002; 277: 27412-27422Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In addition, the PH domain of p130 and the R134L mutant were also inserted into a plasmid encoding a monomeric red fluorescent protein (RFP-p130PH and RFP-p130PH-R134L) (36.Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (1961) Google Scholar). Ratiometric pericam targeted to the mitochondria (pericam-mt) has also been published previously (37.Nagai T. Sawano A. Park E.S. Miyawaki A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3197-3202Crossref PubMed Scopus (789) Google Scholar). Bacterial expression of the GFP-fused protein domains (p130PH-GFP, GFP-IP3R224–605, and GFP-PLCδ1PH R40L) was carried out as described previously (31.Varnai P. Lin X. Lee S.B. Tuymetova G. Bondeva T. Spat A. Rhee S.G. Hajnoczky G. Balla T. J. Biol. Chem. 2002; 277: 27412-27422Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Cell Culture—COS-7 cells (obtained from ATCC) were cultured in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in humidified air (5% CO2) at 37 °C. RBL-2H3 cells were cultured as described previously (22.Csordas G. Thomas A.P. Hajnoczky G. EMBO J. 1999; 18: 96-108Crossref PubMed Scopus (439) Google Scholar). For imaging experiments, cells were plated onto poly-d-lysine-treated glass coverslips at a density of 20,000–25,000/cm2 and were grown for 3–4 days. For cell suspension studies, cells were cultured for 4–6 days in 75-cm2 flasks. Transfection of Cells—Cells plated onto poly-d-lysine-coated coverslips were transfected with plasmid DNA (1.5 μg/ml) for 6–12 h using Lipofectamine (Invitrogen) and Opti-MEM I medium (Invitrogen) according to the manufacturer's instructions. Cells were observed 24–36 h after transfection. Measurement of [3H]IP3—COS-7 cells (5 × 104 cells/ml) were cultured on 12-well culture dishes for 1 day and transfected with either the RFP-p130PH or RFP-p130PH-R134L construct. After 24 h, cells were labeled in 0.75-ml inositol-free Dulbecco's modified essential medium containing 0.1% BSA, 2.5% fetal bovine serum, and myo-[3H]inositol (20 μCi/ml, Amersham Biosciences) for 24 h. After two washes, cells were preincubated for 30 min at 37 °C before stimulation with 50 μm ATP for the indicated times. Incubations were terminated by the addition of ice-cold perchloric acid (5% final). Inositol phosphates were extracted and separated on high pressure liquid chromatography as described previously (38.Nakanishi S. Catt K.J. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (308) Google Scholar). The (1,4,5)IP3 and (1,3,4)IP3 values were combined. Fluorescence Imaging Measurements in Intact COS-7 Cells—Before use, the cells were preincubated in an extracellular medium (2% BSA/ECM) consisting of 121 mm NaCl, 5 mm NaHCO3, 10 mm Na-HEPES, 4.7 mm KCl, 1.2 mm KH2PO4, 1.2 mm MgSO4, 2 mm CaCl2, 10 mm glucose, and 2% BSA, pH 7.4. To monitor [Ca2+]c and to measure Mn2+ entry (Mn2+ quench), cells were loaded with 5 μm Fura-2/AM for 20–30 min in the presence of 200 μm sulfinpyrazone and 0.003% (w/v) pluronic acid at room temperature. Sulfinpyrazone was also present during the imaging measurements to minimize dye loss. To monitor [Ca2+]m, cells were loaded with 5 μm rhod2/AM for 50 min in the presence of 0.003% (w/v) pluronic acid at 35 °C. After loading, cells were washed and incubated in the same medium containing 0.25% BSA but no Ca2+ tracer was added. In some experiments, Ca2+ was not added to 0.25% BSA/ECM and the final [Ca2+]ECM was 95% cells were trypan blue-positive. Compartmentalized fura2FF has been shown to occur in the mitochondria of RBL-2H3 cells. Permeabilized cells were resuspended in ICM supplemented with succinate 2 mm and 0.25 μm rhod2/FA and maintained in a stirred thermostated cuvette at 35 °C. Rhod2/FA was added to monitor [Ca2+] in the intracellular medium that exchanges readily with the cytosolic space. Fluorescence was monitored in a multi-wavelength-excitation dual wavelength-emission fluorimeter using 340- and 380-nm excitation and 500-nm emission for fura2FF and 540-nm excitation and 580-nm emission for rhod2. In every experiment, five data triplets were obtained per second. Calibration of the Ca2+ signals was carried out at the end of each measurement as described previously (41.Csordas G. Hajnoczky G. Cell Calcium. 2001; 29: 249-262Crossref PubMed Scopus (85) Google Scholar). Cellular Localization of IP3- and Inositol Lipid-binding Domains Fused to GFP—We first visualized by confocal microscopy the subcellular localization of the IP3-binding domains, IP3R224–605 and p130PH, in COS-7 cells, which were expressed as GFP fusion proteins. GFP-N was used as a control for cytosolic distribution (Fig. 1A, i), whereas PLCδ1PH-GFP that binds to PIP2 through its PH domain was used as a reference for plasma membrane localization (Fig. 1A, iv). As indicated by the GFP fluorescence, GFP-IP3R224–605 and p130PH-GFP were found to be present in the cytoplasm (Fig. 1A, ii and iii). The cells were stimulated next with ATP that induces PLC-mediated cleavage of PIP2 to enhance IP3 formation. In ATP-stimulated COS-7 cells, no change was observed in the distribution of GFP-N, GFP-IP3R224–605, and p130PH-GFP (Fig. 1A, v, vi, and vii). The ATP-induced hydrolysis of PIP2 was confirmed by the partial translocation of PLCδ1PH-GFP from the membrane to the cytosol, which appeared as a fluorescence decrease at the plasma membrane and an increase in the cytosol (Fig. 1A, viii). FRAP studies were used to assess the mobility of the IP3BPs in the cytosol (Fig. 1B). When a large area of a p130PH-GFP-expressing cell was illuminated, an essentially homogeneous decrease in fluorescence appeared throughout the cell within 3s (Fig. 1B, i versus v), suggesting fast cytosolic distribution of the fluorescent protein. Furthermore, when small regions of the GFP-IP3R224–605-and p130PH-GFP-expressing cells were photobleached, the post-bleach fluorescence in these regions was not different from the pre-bleach signal, indicating that the fluorescence was recovered completely within 3s (Fig. 1B, ii versus vi and iii versus vii). As a positive control, GFP targeted to the mitochondrial matrix, mito-GFP, which shows slower redistribution than the cytosolic GFP (42.Verkman A.S. Trends Biochem. Sci. 2002; 27: 27-33Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar), was also photobleached. In this case, the loss of fluorescence in the region of bleaching was apparent in the post-bleach image (Fig. 1B, iv versus viii; 29 ± 3%, n = 12). Although the rate of image acquisition was too low to determine the half-recovery time for GFP-IP3R224–605 and p130PH-GFP, it is likely to be in the subsecond range, similarly to the half-recovery time measured for a freely diffusible cytosolic PH domain-GFP construct (PLCδ1PH(R40L)-GFP, 0.2s) (43.van der Wal J.

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