Modulation of GABAA Receptor Phosphorylation and Membrane Trafficking by Phospholipase C-related Inactive Protein/Protein Phosphatase 1 and 2A Signaling Complex Underlying Brain-derived Neurotrophic Factor-dependent Regulation of GABAergic Inhibition
2006; Elsevier BV; Volume: 281; Issue: 31 Linguagem: Inglês
10.1074/jbc.m603118200
ISSN1083-351X
AutoresTakashi Kanematsu, Atsushi Yasunaga, Yoshito Mizoguchi, Akiko Kuratani, Josef T. Kittler, Jasmina N. Jovanovic, Kei Takenaka, Keiichi I. Nakayama, Kiyoko Fukami, Tadaomi Takenawa, Stephen J. Moss, Junichi Nabekura, Masato Hirata,
Tópico(s)Ion channel regulation and function
ResumoBrain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABAA receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABAA receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABAA receptor β3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABAA receptors. We demonstrate that PRIP associates with phosphatases as well as with β subunits. PRIP was found to colocalize with GABAA receptor clusters in cultured neurons and with recombinant GABAA receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to β subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABAA receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABAA receptors by mediating the specific association between β subunits of these receptors with protein phosphatases. Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABAA receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABAA receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABAA receptor β3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABAA receptors. We demonstrate that PRIP associates with phosphatases as well as with β subunits. PRIP was found to colocalize with GABAA receptor clusters in cultured neurons and with recombinant GABAA receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to β subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABAA receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABAA receptors by mediating the specific association between β subunits of these receptors with protein phosphatases. The neurotrophin brain-derived neurotrophic factor (BDNF) 4The abbreviations used are: BDNF, brain-derived neurotrophic factor; GABA, γ-aminobutyric acid; PP, protein phosphatase; PRIP, phospholipase C-related but catalytically inactive protein; DKO, double knock-out; WT, wild type; Pn, postnatal day n; DIV, days in vitro; GST, glutathione S-transferase; PKC, protein kinase C; PIPES, 1,4-piperazinediethanesulfonic acid. has been shown to modulate directly both excitatory and inhibitory synaptic transmission. Acting via the TrkB tyrosine kinase receptor, BDNF exerts rapid effects both presynaptically, by modulating transmitter release, and postsynaptically, by changing the properties of ionotropic receptors (1Rose C.R. Blum R. Kafitz K.W. Kovalchuk Y. Konnerth A. Bioessays. 2004; 26: 1185-1194Crossref PubMed Scopus (104) Google Scholar). At inhibitory synapses, acute application of BDNF depresses inhibitory synaptic transmission mediated through γ-aminobutyric acid type A (GABAA) receptors in hippocampal slices (2Tanaka T. Saito H. Matsuki N. J. Neurosci. 1997; 17: 2959-2966Crossref PubMed Google Scholar) and reduces miniature inhibitory postsynaptic currents acutely or following a transient increase, in cultured hippocampal (3Brunig I. Penschuck S. Berninger B. Benson J. Fritschy J.M. Eur. J. Neurosci. 2001; 13: 1320-1328Crossref PubMed Google Scholar, 4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar) and cerebellar granule neurons (5Cheng Q. Yeh H.H. J. Physiol. 2003; 548: 711-721Crossref PubMed Scopus (62) Google Scholar). Modulation of the strength of synaptic inhibition by BDNF may be important for the maturation of inhibitory synapses (6Vicario-Abejon C. Owens D. McKay R. Segal M. Nat. Rev. Neurosci. 2002; 3: 965-974Crossref PubMed Scopus (205) Google Scholar) and, in addition, may have important implications for synaptic plasticity and information processing in the brain. Two possible mechanisms for the BDNF-dependent regulation of GABAA receptor function have been proposed: alterations in GABAA receptor cell surface numbers (3Brunig I. Penschuck S. Berninger B. Benson J. Fritschy J.M. Eur. J. Neurosci. 2001; 13: 1320-1328Crossref PubMed Google Scholar, 5Cheng Q. Yeh H.H. J. Physiol. 2003; 548: 711-721Crossref PubMed Scopus (62) Google Scholar) and/or modulation of GABAA receptor phosphorylation (4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar), which may be mutually related (7Kittler J.T. Chen G. Honing S. Bogdanov Y.B. McAinsh K. Arancibia-Carcamo I.L. Jovanovic J.N. Pangalos M.N. Haucke V. Yan Z. Moss S.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14871-14876Crossref PubMed Scopus (142) Google Scholar). However, the precise mechanism regarding the molecular machinery underlying these events requires further investigation. Phospholipase C-related but catalytically inactive protein type 1 (PRIP-1), a novel d-myo-inositol 1,4,5-trisphosphate-binding protein, is a molecule similar to phospholipase C-δ1 but is catalytically inactive and is expressed predominantly in the brain (8Kanematsu 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, 9Kanematsu T. Misumi Y. Watanabe Y. Ozaki S. Koga T. Iwanaga S. Ikehara Y. Hirata M. Biochem. J. 1996; 313: 319-325Crossref PubMed Scopus (90) Google Scholar, 10Kanematsu T. Yoshimura K. Hidaka K. Takeuchi H. Katan M. Hirata M. Eur. J. Biochem. 2000; 267: 2731-2737Crossref PubMed Scopus (56) Google Scholar, 11Yoshida M. Kanematsu T. Watanabe Y. Koga T. Ozaki S. Iwanaga S. Hirata M. J. Biochem. (Tokyo). 1994; 115: 973-980Crossref PubMed Scopus (61) Google Scholar, 12Takeuchi H. Kanematsu T. Misumi Y. Yaakob H.B. Yagisawa H. Ikehara Y. Watanabe Y. Tan Z. Shears S.B. Hirata M. Biochem. J. 1996; 318: 561-568Crossref PubMed Scopus (61) Google Scholar, 13Takeuchi H. Kanematsu T. Misumi Y. Sakane F. Konishi H. Kikkawa U. Watanabe Y. Katan M. Hirata M. Biochim. Biophys. Acta. 1997; 1359: 275-285Crossref PubMed Scopus (90) Google Scholar, 14Takeuchi 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 (54) Google Scholar, 15Matsuda M. Kanematsu T. Takeuchi H. Kukita T. Hirata M. Neurosci. Lett. 1998; 257: 97-100Crossref PubMed Scopus (32) Google Scholar). PRIP-1 has a number of binding partners, including the catalytic subunit of protein phosphatase 1α (PP1c) (16Yoshimura K. Takeuchi H. Sato O. Hidaka K. Doira N. Terunuma M. Harad A.K. Ogawa Y. Ito Y. Kanematsu T. Hirata M. J. Biol. Chem. 2001; 276: 17908-17913Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 17Terunuma M. Jang I.S. Ha S.H. Kittler J.T. Kanematsu T. Jovanovic J.N. Nakayama K.I. Akaike N. Ryu S.H. Moss S.J. Hirata M. J. Neurosci. 2004; 24: 7074-7084Crossref PubMed Scopus (91) Google Scholar), GABAA receptor-associated protein (18Wang H. Bedford F.K. Brandon N.J. Moss S.J. Olsen R.W. Nature. 1999; 397: 69-72Crossref PubMed Scopus (491) Google Scholar, 19Kanematsu 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. Nakayama K. EMBO J. 2002; 21: 1004-1011Crossref PubMed Scopus (117) Google Scholar), and GABAA receptor β subunits (17Terunuma M. Jang I.S. Ha S.H. Kittler J.T. Kanematsu T. Jovanovic J.N. Nakayama K.I. Akaike N. Ryu S.H. Moss S.J. Hirata M. J. Neurosci. 2004; 24: 7074-7084Crossref PubMed Scopus (91) Google Scholar). We have recently reported that PRIP-1 plays an important role in regulating GABAA receptor activity based on pharmacological and behavioral phenotype of mice lacking the PRIP-1 gene (PRIP-1 KO) (19Kanematsu 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. Nakayama K. EMBO J. 2002; 21: 1004-1011Crossref PubMed Scopus (117) Google Scholar). In addition, PRIP-1 activity is important for phospho-dependent modulation of GABAA receptors in response to cAMP-dependent protein kinase A signaling pathways by modulating the binding and phosphatase activity of PP1 (17Terunuma M. Jang I.S. Ha S.H. Kittler J.T. Kanematsu T. Jovanovic J.N. Nakayama K.I. Akaike N. Ryu S.H. Moss S.J. Hirata M. J. Neurosci. 2004; 24: 7074-7084Crossref PubMed Scopus (91) Google Scholar). In addition to PRIP-1, the identification of the second PRIP isoform, PRIP-2, with a broad tissue distribution, including the brain, has been reported recently (20Kikuno R. Nagase T. Ishikawa K. Hirosawa M. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1999; 6: 197-205Crossref PubMed Scopus (175) Google Scholar, 21Otsuki M. Fukami K. Kohno T. Yokota J. Takenawa T. Biochem. Biophys. Res. Commun. 1999; 266: 97-103Crossref PubMed Scopus (51) Google Scholar, 22Uji A. Matsuda M. Kukita T. Maeda K. Kanematsu T. Hirata M. Life Sci. 2002; 72: 443-453Crossref PubMed Scopus (59) Google Scholar). PRIP-2 also interacts with both PP1c and GABAA receptor-associated protein (22Uji A. Matsuda M. Kukita T. Maeda K. Kanematsu T. Hirata M. Life Sci. 2002; 72: 443-453Crossref PubMed Scopus (59) Google Scholar, 23Yanagihori S. Terunuma M. Koyanao K. Ryu S.H. Kanematsu T. Hirata M. Adv. Enz. Regul. 2006; (in press)PubMed Google Scholar), but the contribution of PRIP-2 to signaling pathways regulating GABAA receptors is currently unknown (24Takenaka K. Fukami K. Otsuki M. Nakamura Y. Kataoka Y. Wada M. Tsuji K. Nishikawa S. Yoshida N. Takenawa T. Mol. Cell Biol. 2003; 23: 7329-7338Crossref PubMed Scopus (40) Google Scholar). Given the functional similarity between PRIP-1 and -2 proteins at least in vitro, it is of interest to investigate the regulation of GABAA receptors in the absence of both isoforms. PRIP-1 and -2 double knock-out (PRIP-DKO) mice were therefore generated to help establish the functional significance of these proteins in GABAA receptor phosphorylation and trafficking. In the present study, we examined the specific role(s) of PRIP proteins in BDNF-mediated modulation of GABAA receptors. We observed a complete lack of BDNF-dependent inhibition of GABA-evoked currents in PRIP-DKO mice because of altered properties of the BDNF signaling pathway regulating phosphorylation and cell surface expression of GABAA receptors. Collectively, our results suggest that PRIP-1 and -2 play important roles in the modulation of GABAA receptor function and may be involved in long term changes in the efficacy of inhibitory synaptic transmission. Generation of PRIP-DKO Mice—The PRIP-1 KO mice (19Kanematsu 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. Nakayama K. EMBO J. 2002; 21: 1004-1011Crossref PubMed Scopus (117) Google Scholar) and PRIP-2 KO mice (24Takenaka K. Fukami K. Otsuki M. Nakamura Y. Kataoka Y. Wada M. Tsuji K. Nishikawa S. Yoshida N. Takenawa T. Mol. Cell Biol. 2003; 23: 7329-7338Crossref PubMed Scopus (40) Google Scholar) that were both back-crossed against the C57BL/6J background (n = 7 and n = 2, respectively), were crossed to generate a PRIP-DKO mouse strain and corresponding wild type (WT). Homozygous PRIP-DKO and WT mice were mated inter se to obtain the required number of mice, and only F1 and F2 generations of both genotypes were used for experiments. The handling of mice and all procedures performed on them were approved by the Animal Care Committee of Kyushu University, following the guidelines of the Japanese Council on Animal Care. Neuron Culture Preparation—High density cultures of dissociated hippocampal or cortical neurons were prepared from both WT and PRIP-DKO mice (postnatal day (P)0 or P1) as described previously (19Kanematsu 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. Nakayama K. EMBO J. 2002; 21: 1004-1011Crossref PubMed Scopus (117) Google Scholar). Neurons were cultured for 14-21 days (14-21 days in vitro (DIV)) before experiments. Electrophysiological Recordings—Cultured hippocampal neurons (14-18 DIV) were used for electrophysiological recording. Electrical measurements were performed by the nystatin-perforated patch recording method described in Mizoguchi et al. (25Mizoguchi Y. Ishibashi H. Nabekura J. J. Physiol. 2003; 548: 703-709Crossref PubMed Scopus (78) Google Scholar). The synthetic peptide incorporating residues 553-566 of rat PRIP-1 (EGEVTDEDEEAEMS in an abbreviation by a single character of amino acids) or scrambled control peptide (EETEMDAGDEVSEE) were added to the pipette solution at a concentration of 3 μg/ml for the measurement of GABA-evoked Cl− currents (IGABA). All of the experiments were performed at the room temperature (25 ± 1 °C). All of the data are normalized to the amplitude just before BDNF application and expressed as the means ± S.E. Generation of Plasmids—A series of C-terminal truncated rat (r)PRIP-1 constructs (see Fig. 5A) was constructed in pSG5 vector. A series of N-terminally truncated rPRIP-1 and N-terminally truncated human (h)PRIP-2 constructs (see Fig. 5A) were generated in pETHis6-30 vector (19Kanematsu 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. Nakayama K. EMBO J. 2002; 21: 1004-1011Crossref PubMed Scopus (117) Google Scholar). To make rPRIP-1/pDsRedN1, a pcMT31 clone (9Kanematsu T. Misumi Y. Watanabe Y. Ozaki S. Koga T. Iwanaga S. Ikehara Y. Hirata M. Biochem. J. 1996; 313: 319-325Crossref PubMed Scopus (90) Google Scholar) was introduced into a pDsRedN1 vector (Clontech Laboratories, Palo Alto, CA). For construction of a PRIP-1-binding peptide plasmid that disrupts the association between PRIP and β subunits, the PCR-amplified fragment (amino acid residues 553-565 of rPRIP-1) was introduced into pIRES2-EGFP vector (Clontech Laboratories). Constructing strategies of Myc-tagged PP2A in pRK5 vector, GABAA receptor α1Myc and β2Myc subunit in pGW1 vector, and glutathione S-transferase (GST)-fused intracellular loop regions of α1, β1, β2, and β3 subunits were previously described (4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar, 26Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 27Brandon N.J. Uren J.M. Kittler J.T. Wang H. Olsen R. Parker P.J. Moss S.J. J. Neurosci. 1999; 19: 9228-9234Crossref PubMed Google Scholar). Immunoblot Analysis of GABAA Receptor β3 Subunits Phosphorylation—To assess changes in phosphorylation of GABAA receptors, anti-phospho(P)-β3 antibody, which recognizes the phosphorylated Ser408/Ser409 residues in the β3 subunit (4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar), was used for immunoblot analysis. The procedures for this analysis used were similar to those described previously (4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar). Primary antibodies used were as follows: rabbit anti-PRIP-1 antibody (19Kanematsu 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. Nakayama K. EMBO J. 2002; 21: 1004-1011Crossref PubMed Scopus (117) Google Scholar), rabbit anti-PRIP-2 antibody (24Takenaka K. Fukami K. Otsuki M. Nakamura Y. Kataoka Y. Wada M. Tsuji K. Nishikawa S. Yoshida N. Takenawa T. Mol. Cell Biol. 2003; 23: 7329-7338Crossref PubMed Scopus (40) Google Scholar), rabbit anti-PP1 antibody, mouse anti-β2/3 antibody (clone 62-3G1), rabbit anti-pan PKC antibody that recognizes PKC α, β, and γ (number 06-870; Upstate Biotechnology, Lake Placid, NY), mouse anti-TrkB antibody (clone 47), mouse anti-PP1 antibody (clone 24), mouse anti-PP2A antibody (clone 46) (BD Transduction Laboratories, Lexington, KY), mouse anti-β tubulin antibody (Roche Applied Science), and mouse anti-Myc antibody (9E10). Anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibody (Amersham Biosciences) were used as secondary antibodies. Prestained protein markers (broad range; New England BioLabs, Beverly, MA; precision plus, Bio-Rad) were used. The chemiluminescent signals were detected and quantified using a LAS-1000 plus gel documentation system (Fujifilm, Tokyo, Japan). Immunoprecipitation and GST Protein Pulldown Assay—Co-immunoprecipitation assays and GST pulldown assays were performed as described previously (17Terunuma M. Jang I.S. Ha S.H. Kittler J.T. Kanematsu T. Jovanovic J.N. Nakayama K.I. Akaike N. Ryu S.H. Moss S.J. Hirata M. J. Neurosci. 2004; 24: 7074-7084Crossref PubMed Scopus (91) Google Scholar, 19Kanematsu 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. Nakayama K. EMBO J. 2002; 21: 1004-1011Crossref PubMed Scopus (117) Google Scholar). Briefly, the lysate obtained from COS7 cells transfected with the full-length rPRIP-1/pSG5 and PP2Ac/pRK5-Myc plasmid, cultured rat cortical neurons (21 DIV), or mouse brains was subjected to immunoprecipitation with rabbit control IgG, rabbit polyclonal anti-Myc antibody (c-Myc (A-14); Santa Cruz Biotechnology, Santa Cruz, CA), or rabbit polyclonal anti-PRIP-1 antibody, followed by the addition of 20 μl of 50% slurry of protein G-Sepharose beads (Amersham Biosciences). For GST protein pulldown analysis, GST-α1, -β1, -β2, and -β3 and GST immobilized on glutathione-Sepharose beads were incubated with recombinant proteins of interest or with the rat brain lysate. A series of recombinant PRIP-1 and PRIP-2 mutants (see Fig. 5A) were expressed and labeled by in vitro transcription/translation using the Transcend™ chemiluminescent translation detection system and analyzed by Western blotting with streptavidin-horseradish peroxidase (Promega, Madison, WI). Immunocytochemistry—For detection of GABAA receptors expressed at the cell surface, COS7 or HEK293 cells were transfected with rPRIP-1/pDsRedN1, GABAA receptor α1Myc and β2Myc subunit constructs in pGW1 and PRIP-1-binding peptide in pIRES2-EGFP, using the procedure described previously (28Kittler J.T. Delmas P. Jovanovic J.N. Brown D.A. Smart T.G. Moss S.J. J. Neurosci. 2000; 20: 7972-7977Crossref PubMed Google Scholar). Co-localization between PRIP-1 and GABAA receptors in cultured neurons was investigated as follows: mouse anti-GABAA receptor β2/3 antibody (clone 62-3G1) or mouse anti-β subunits antibody (MAB341; Chemicon Inc., Pittsburgh, PA) was added to the culture medium to bind to cell surface-expressed β subunits prior to fixation with 4% paraformaldehyde and subsequent permeabilization using 0.1% saponin in 80 mm PIPES, pH 7.2, 1 mm MgCl2, 1 mm EGTA. The cells were then stained with rabbit anti-PRIP-1 antibody followed by incubation with Cy3-conjugated anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa-488-conjugated anti-rabbit antibody (Molecular Probes, Eugene, OR). The signals were visualized by confocal microscopy (Bio-Rad). Cell Surface Receptor Assay ([3H]Muscimol Binding Assay)—Cultured cortical neurons (14-18 DIV) plated on a 96-well plate were washed with Neurobasal-A medium (Invitrogen) three times and were incubated with BDNF alone (100 ng/ml) or BDNF (100 ng/ml) plus K252a (200 nm) (Sigma-Aldrich) for appropriate time at 37 °C, followed by washing with ice-cold Neurobasal-A medium. The assay buffer comprising Neurobasal-A medium containing 120 nm [3H]muscimol (specific radioactivity, 1110.0 GBq/mmol) (PerkinElmer Life Sciences) with or without 150 μm muscimol (Sigma-Aldrich) was added to each well. The plate was incubated for 30 min on ice, followed by three quick washes with ice-cold neurobasal-A medium. Hundred microliters of liquid scintillator (MicroScint Mixture-20; PerkinElmer Life Sciences) was added to each well, and the radioactivity was counted on a TopCount NXT (PerkinElmer Life Sciences). Nonspecific binding in the presence of 150 μm muscimol (40-80 dpm) was subtracted from that in its absence (500-780 dpm) to yield the specific binding. Each assay was done in triplicate. Regulation of GABAA Receptor Currents by BDNF in Hippocampal Neurons Isolated from WT and PRIP-DKO Mice—The precise molecular mechanisms that underlie BDNF-mediated alterations in GABAA receptor activity remain unclear. We have previously reported that PRIP-1 plays an important role in the modulation of GABAA receptor function by regulating the receptor trafficking (19Kanematsu 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. Nakayama K. EMBO J. 2002; 21: 1004-1011Crossref PubMed Scopus (117) Google Scholar) and phosphorylation (17Terunuma M. Jang I.S. Ha S.H. Kittler J.T. Kanematsu T. Jovanovic J.N. Nakayama K.I. Akaike N. Ryu S.H. Moss S.J. Hirata M. J. Neurosci. 2004; 24: 7074-7084Crossref PubMed Scopus (91) Google Scholar). To test whether PRIP proteins contribute to the observed BDNF-dependent alterations of GABAA receptor activity (2Tanaka T. Saito H. Matsuki N. J. Neurosci. 1997; 17: 2959-2966Crossref PubMed Google Scholar, 3Brunig I. Penschuck S. Berninger B. Benson J. Fritschy J.M. Eur. J. Neurosci. 2001; 13: 1320-1328Crossref PubMed Google Scholar, 4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar, 5Cheng Q. Yeh H.H. J. Physiol. 2003; 548: 711-721Crossref PubMed Scopus (62) Google Scholar), we have carried out experiments using PRIP-DKO mice, because PRIP-DKO mice became available recently as described above. We first examined whether BDNF alters GABA responses in PRIP-DKO hippocampal neurons by measuring the whole cell current responses. Because presynaptic effects of BDNF on inhibitory synaptic transmission have been reported (29Frerking M. Malenka R.C. Nicoll R.A. J. Neurophysiol. 1998; 80: 3383-3386Crossref PubMed Scopus (170) Google Scholar), we measured GABA-evoked Cl− currents (IGABA). Hippocampal neurons cultured from both WT and PRIP-DKO neonatal mice (P0 or P1) were analyzed by voltage clamp recordings. The application of 10 μm GABA to neurons from both genotypes induced a GABAA receptor-mediated inward current with a similar amplitude, which was completely blocked by 10 μm bicuculline (n = 3 each genotype; data not shown). In agreement with previous findings (2Tanaka T. Saito H. Matsuki N. J. Neurosci. 1997; 17: 2959-2966Crossref PubMed Google Scholar, 3Brunig I. Penschuck S. Berninger B. Benson J. Fritschy J.M. Eur. J. Neurosci. 2001; 13: 1320-1328Crossref PubMed Google Scholar, 4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar, 5Cheng Q. Yeh H.H. J. Physiol. 2003; 548: 711-721Crossref PubMed Scopus (62) Google Scholar), the initial transient increase in the GABA-evoked currents within 2.5 min of BDNF (5 ng/ml) application in WT neurons (n = 3) was followed by a prominent decrease within 10 min (Fig. 1). These effects were blocked in the presence of 200 nm of K252a, a broad tyrosine kinase inhibitor commonly used to demonstrate involvement of signaling via TrkB receptors (data not shown). The inhibition of GABA-evoked current amplitude continued for ∼30 min, reaching 55 ± 14% of the initial control levels. Strikingly, the amplitude of IGABA in neurons from PRIP-DKO mice did not exhibit a decrease upon BDNF application but instead exhibited a gradual increase reaching 145 ± 27% of control over the same time course (Fig. 1). Similar differences in BDNF-mediated alterations in GABA-evoked current were also obtained in recordings from acutely dissociated P14 hippocampal CA1 pyramidal neurons (25Mizoguchi Y. Ishibashi H. Nabekura J. J. Physiol. 2003; 548: 703-709Crossref PubMed Scopus (78) Google Scholar) from either WT or PRIP-DKO mice (results not shown). Studies published previously have provided evidence for two possible mechanisms underlying BDNF-dependent regulation of GABAA receptor currents that include alterations in cell surface expression and/or phosphorylation of GABAA receptor β3 subunit (3Brunig I. Penschuck S. Berninger B. Benson J. Fritschy J.M. Eur. J. Neurosci. 2001; 13: 1320-1328Crossref PubMed Google Scholar, 4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar, 5Cheng Q. Yeh H.H. J. Physiol. 2003; 548: 711-721Crossref PubMed Scopus (62) Google Scholar). Given that GABAA receptor β subunits directly associate with AP2 proteins of endocytic protein machinery in a phosphorylation-dependent manner (7Kittler J.T. Chen G. Honing S. Bogdanov Y.B. McAinsh K. Arancibia-Carcamo I.L. Jovanovic J.N. Pangalos M.N. Haucke V. Yan Z. Moss S.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14871-14876Crossref PubMed Scopus (142) Google Scholar), the two proposed mechanisms may in fact act in a coordinated fashion to mediate the BDNF-dependent regulation of GABAA receptors. We tested this hypothesis by examining both phosphorylation of the β3 subunit and its expression at the neuronal cell surface in PRIP-DKO mice in response to BDNF. Regulation of GABAA Receptor β3 Subunit Phosphorylation by BDNF in PRIP-DKO Neurons—Activation of BDNF/TrkB receptor-dependent signaling pathways leads to an increase in PKC activity and PKC-dependent phosphorylation of serines 408 and 409 (Ser408/Ser409) in the large intracellular loop of the GABAA receptor β3 subunit, followed by a rapid dephosphorylation by PP2A, thus causing a biphasic change in the phosphorylation state of these receptors (4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar). We have previously reported that PRIP-1 binds to and inactivates another phosphatase, PP1c (16Yoshimura K. Takeuchi H. Sato O. Hidaka K. Doira N. Terunuma M. Harad A.K. Ogawa Y. Ito Y. Kanematsu T. Hirata M. J. Biol. Chem. 2001; 276: 17908-17913Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), as well as GABAA receptor β3 subunit (17Terunuma M. Jang I.S. Ha S.H. Kittler J.T. Kanematsu T. Jovanovic J.N. Nakayama K.I. Akaike N. Ryu S.H. Moss S.J. Hirata M. J. Neurosci. 2004; 24: 7074-7084Crossref PubMed Scopus (91) Google Scholar) and regulates β3 subunit phosphorylation at these same residues mediated by protein kinase A (17Terunuma M. Jang I.S. Ha S.H. Kittler J.T. Kanematsu T. Jovanovic J.N. Nakayama K.I. Akaike N. Ryu S.H. Moss S.J. Hirata M. J. Neurosci. 2004; 24: 7074-7084Crossref PubMed Scopus (91) Google Scholar). Therefore, PRIP proteins may play an important role in the regulation of GABAA receptor phosphorylation in response to BDNF. To test this hypothesis, we examined whether BDNF-mediated changes in phosphorylation levels of the β3 subunit were altered in cortical neurons from PRIP-DKO mice (Fig. 2A). Cultured cortical neurons (14-18 DIV) of both genotypes were treated with BDNF (100 ng/ml) for 5, 15, and 30 min, and cell lysates were analyzed by immunoblotting using an antibody that specifically recognizes Ser408 and Ser409 in their phosphorylated form (anti-P-β3 antibody) (4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar). In WT neurons, BDNF-dependent biphasic modulation of the β3 subunit phosphorylation was observed with an initial 2-3-fold increase in the phosphorylation of β3 subunit within 5 min, followed by a decrease to 69 ± 5% of the initial control level within 30 min, similar to findings previously observed in rat neurons (4Jovanovic J.N. Thomas P. Kittler J.T. Smart T.G. Moss S.J. J. Neurosci. 2004; 24: 522-530Crossref PubMed Scopus (230) Google Scholar). In contrast, phosphorylation levels of the β3 subunits in PRIP-DKO neurons exhibited a gradual increase to 155 ± 9% level of the control over the same period of time. To control for possible alterations in the expression levels of signaling proteins participating
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