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• W2054792009 abstract "Kv4 potassium channels regulate action potentials in neurons and cardiac myocytes. Co-expression of EF hand-containing Ca2+-binding proteins termed KChIPs with pore-forming Kv4 α subunits causes changes in the gating and amplitude of Kv4 currents (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553–556). Here we show that KChIPs profoundly affect the intracellular trafficking and molecular properties of Kv4.2 α subunits. Co-expression of KChIPs1–3 causes a dramatic redistribution of Kv4.2, releasing intrinsic endoplasmic reticulum retention and allowing for trafficking to the cell surface. KChIP co-expression also causes fundamental changes in Kv4.2 steady-state expression levels, phosphorylation, detergent solubility, and stability that reconstitute the molecular properties of Kv4.2 in native cells. Interestingly, the KChIP4a isoform, which exhibits unique effects on Kv4 channel gating, does not exert these effects on Kv4.2 and negatively influences the impact of other KChIPs. We provide evidence that these KChIP effects occur through the masking of an N-terminal Kv4.2 hydrophobic domain. These studies point to an essential role for KChIPs in determining both the biophysical and molecular characteristics of Kv4 channels and provide a molecular basis for the dramatic phenotype of KChIP knockout mice. Kv4 potassium channels regulate action potentials in neurons and cardiac myocytes. Co-expression of EF hand-containing Ca2+-binding proteins termed KChIPs with pore-forming Kv4 α subunits causes changes in the gating and amplitude of Kv4 currents (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553–556). Here we show that KChIPs profoundly affect the intracellular trafficking and molecular properties of Kv4.2 α subunits. Co-expression of KChIPs1–3 causes a dramatic redistribution of Kv4.2, releasing intrinsic endoplasmic reticulum retention and allowing for trafficking to the cell surface. KChIP co-expression also causes fundamental changes in Kv4.2 steady-state expression levels, phosphorylation, detergent solubility, and stability that reconstitute the molecular properties of Kv4.2 in native cells. Interestingly, the KChIP4a isoform, which exhibits unique effects on Kv4 channel gating, does not exert these effects on Kv4.2 and negatively influences the impact of other KChIPs. We provide evidence that these KChIP effects occur through the masking of an N-terminal Kv4.2 hydrophobic domain. These studies point to an essential role for KChIPs in determining both the biophysical and molecular characteristics of Kv4 channels and provide a molecular basis for the dramatic phenotype of KChIP knockout mice. Voltage-gated potassium or Kv channels, specifically those mediating low threshold, rapidly inactivating Ito and IA currents, are known to regulate cardiac and neuronal membrane excitability, respectively (1Hille B. Ionic Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Inc., Sunderland, MA2001: 136-139Google Scholar). In cardiac cells, Ito is a major determinant of the falling phase of the cardiac action potential (2Oudit G.Y. Kassiri Z. Sah R. Ramirez R.J. Zobel C. Backx P.H. J. Mol. Cell. Cardiol. 2001; 33: 851-872Abstract Full Text PDF PubMed Scopus (170) Google Scholar). In hippocampal pyramidal neurons, dendritic IA can limit the peak of back-propagating action potentials as well as modulate incoming synaptic information, exerting profound effects on information processing (3Johnston D. Hoffman D.A. Magee J.C. Poolos N.P. Watanabe S. Colbert C.M. Migliore M. J. Physiol. (Lond.). 2000; 525: 75-81Crossref Scopus (212) Google Scholar). Kv channels are composed of homo- or heterotetramers of transmembrane pore-forming and voltage-sensing α subunits (4Chandy K.G. Gutman G.A. North R.A. Ligand- and Voltage-gated Ion Channels. CRC Press, Inc., Boca Raton, FL1995: 1-71Google Scholar, 5Coetzee W.A. Amarillo Y. Chiu J. Chow A. Lau D. McCormack T. Moreno H. Nadal M.S. Ozaita A. Pountney D. Saganich M. Vega-Saenz de Miera E. Rudy B. Ann. N. Y. Acad. Sci. 1999; 868: 233-285Crossref PubMed Scopus (977) Google Scholar). Available evidence suggests that Shal or Kv4 family α subunits underlie Ito in cardiac myocytes, and IA in dendrites of many central nervous system neurons. The expression of specific Kv4 family mRNAs correlates precisely with the level of Ito in cardiac myocytes (6Dixon J.E. Shi W. Wang H.S. McDonald C. Yu H. Wymore R.S. Cohen I.S. McKinnon D. Circ. Res. 1996; 79: 659-668Crossref PubMed Scopus (393) Google Scholar, 7Guo W. Xu H. London B. Nerbonne J.M. J. Physiol. (Lond.). 1999; 521: 587-599Crossref Scopus (191) Google Scholar) and dendritic IA in neurons (8Serodio P. Rudy B. J. Neurophysiol. 1998; 79: 1081-1091Crossref PubMed Scopus (300) Google Scholar, 9Song W.J. Tkatch T. Baranauskas G. Ichinohe N. Kitai S.T. Surmeier D.J. J. Neurosci. 1998; 18: 3124-3137Crossref PubMed Google Scholar, 10Shibata R. Wakazono Y. Nakahira K. Trimmer J.S. Ikenaka K. Dev. Neurosci. 1999; 21: 87-93Crossref PubMed Scopus (34) Google Scholar, 11Tkatch T. Baranauskas G. Surmeier D.J. J. Neurosci. 2000; 20: 579-588Crossref PubMed Google Scholar). Robust staining with antibodies specific for Kv4 family members is observed in cardiac myocytes (12Barry D.M. Trimmer J.S. Merlie J.P. Nerbonne J.M. Circ. Res. 1995; 77: 361-369Crossref PubMed Google Scholar) and in neurons (13Sheng M. Tsaur M.L. Jan Y.N. Jan L.Y. Neuron. 1992; 9: 271-284Abstract Full Text PDF PubMed Scopus (400) Google Scholar, 14Maletic-Savatic M. Lenn N.J. Trimmer J.S. J. Neurosci. 1995; 15: 3840-3851Crossref PubMed Google Scholar, 15Tsaur M.L. Chou C.C. Shih Y.H. Wang H.L. FEBS Lett. 1997; 400: 215-220Crossref PubMed Scopus (52) Google Scholar, 16An W.F. Bowlby M.R. Betty M. Cao J. Ling H.P. Mendoza G. Hinson J.W. Mattsson K.I. Strassle B.W. Trimmer J.S. Rhodes K.J. Nature. 2000; 403: 553-556Crossref PubMed Scopus (834) Google Scholar). Moreover, experimental knockdown of Kv4 expression in cardiac myocytes (17Johns D.C. Nuss H.B. Marban E. J. Biol. Chem. 1997; 272: 31598-31603Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 18Barry D.M. Xu H. Schuessler R.B. Nerbonne J.M. Circ. Res. 1998; 83: 560-567Crossref PubMed Scopus (253) Google Scholar, 19Hoppe U.C. Marban E. Johns D.C. J. Clin. Invest. 2000; 105: 1077-1084Crossref PubMed Scopus (64) Google Scholar) and in neurons (20Malin S.A. Nerbonne J.M. J. Neurosci. 2000; 20: 5191-5199Crossref PubMed Google Scholar, 21Malin S.A. Nerbonne J.M. J. Neurosci. 2001; 21: 8004-8014Crossref PubMed Google Scholar) results in suppression of Ito and IA, respectively.Many Kv channels contain auxiliary subunits that can regulate the biophysical, biochemical, and cell biological characteristics of the resultant channel complexes (22Trimmer J.S. Curr. Opin. Neurobiol. 1998; 8: 370-374Crossref PubMed Scopus (95) Google Scholar). We have recently described a highly related family of four Kv4 Channel Interacting Proteins (KChIPs1–4) that binds to the cytoplasmic N-terminal domain of Kv4 α subunits (16An W.F. Bowlby M.R. Betty M. Cao J. Ling H.P. Mendoza G. Hinson J.W. Mattsson K.I. Strassle B.W. Trimmer J.S. Rhodes K.J. Nature. 2000; 403: 553-556Crossref PubMed Scopus (834) Google Scholar, 23Holmqvist M.H. Cao J. Hernandez-Pineda R. Jacobson M.D. Carroll K.I. Sung M.A. Betty M. Ge P. Gilbride K.J. Brown M.E. Jurman M.E. Lawson D. Silos-Santiago I. Xie Y. Covarrubias M. Rhodes K.J. Distefano P.S. An W.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1035-1040Crossref PubMed Scopus (152) Google Scholar). When co-expressed in heterologous cells, KChIP1, KChIP2, and KChIP3 dramatically alter the inactivation kinetics and rate of recovery from inactivation of Kv4 channels and boost the amplitude of obtained current (16An W.F. Bowlby M.R. Betty M. Cao J. Ling H.P. Mendoza G. Hinson J.W. Mattsson K.I. Strassle B.W. Trimmer J.S. Rhodes K.J. Nature. 2000; 403: 553-556Crossref PubMed Scopus (834) Google Scholar). The effects of the KChIP4a splice variant are distinct from other KChIPs, due to a unique N terminus (23Holmqvist M.H. Cao J. Hernandez-Pineda R. Jacobson M.D. Carroll K.I. Sung M.A. Betty M. Ge P. Gilbride K.J. Brown M.E. Jurman M.E. Lawson D. Silos-Santiago I. Xie Y. Covarrubias M. Rhodes K.J. Distefano P.S. An W.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1035-1040Crossref PubMed Scopus (152) Google Scholar). KChIPs may contribute to both constitutive and dynamic regulation of Kv4 channels. KChIPs are Ca2+-binding proteins. The conserved KChIP core region contains four EF hand-containing Ca2+-binding motifs (16An W.F. Bowlby M.R. Betty M. Cao J. Ling H.P. Mendoza G. Hinson J.W. Mattsson K.I. Strassle B.W. Trimmer J.S. Rhodes K.J. Nature. 2000; 403: 553-556Crossref PubMed Scopus (834) Google Scholar), and studies with a minimal KChIP isoform suggest Ca2+-dependent effects on inactivation (24Patel S.P. Campbell D.L. Strauss H.C. J. Physiol. (Lond.). 2002; 545: 5-11Crossref Scopus (64) Google Scholar). Cyclic AMP-dependent protein kinase (PKA) 1The abbreviations used are: PKA, cyclic AMP-dependent protein kinase; AP, alkaline phosphatase; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein.1The abbreviations used are: PKA, cyclic AMP-dependent protein kinase; AP, alkaline phosphatase; PBS, phosphate-buffered saline; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein. regulation of Kv4.2-encoded currents in heterologous cells also requires KChIP co-expression (25Schrader L.A. Anderson A.E. Mayne A. Pfaffinger P.J. Sweatt J.D. J. Neurosci. 2002; 22: 10123-10133Crossref PubMed Google Scholar). The essential role of KChIPs was acutely demonstrated by genetic ablation of KChIP2 expression in mice, which completely eliminated the cardiac Ito current resulting in a predisposition to cardiac arrhythmias (26Kuo H.C. Cheng C.F. Clark R.B. Lin J.J. Lin J.L. Hoshijima M. Nguyen-Tran V.T. Gu Y. Ikeda Y. Chu P.H. Ross J. Giles W.R. Chien K.R. Cell. 2001; 107: 801-813Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). KChIP3 knockout mice exhibit reduced sensitivity to pain (27Cheng H.Y. Pitcher G.M. Laviolette S.R. Whishaw I.Q. Tong K.I. Kockeritz L.K. Wada T. Joza N.A. Crackower M. Goncalves J. Sarosi I. Woodgett J.R. Oliveira-dos-Santos A.J. Ikura M. van der Kooy D. Salter M.W. Penninger J.M. Cell. 2002; 108: 31-43Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), although as KChIP3 has also been identified as DREAM, a calcium-dependent transcription factor (28Carrion A.M. Link W.A. Ledo F. Mellstrom B. Naranjo J.R. Nature. 1999; 398: 80-84Crossref PubMed Scopus (488) Google Scholar), and calsenilin, which interacts with presenilins (29Buxbaum J.D. Choi E.K. Luo Y. Lilliehook C. Crowley A.C. Merriam D.E. Wasco W. Nat. Med. 1998; 4: 1177-1181Crossref PubMed Scopus (301) Google Scholar), the molecular pathophysiology of the phenotype of this knockout is not as clear.In order to better understand the molecular mechanisms underlying KChIP regulation of cardiac and neuronal excitability, we have investigated the effects of KChIP co-expression on the intracellular trafficking and molecular characteristics of Kv4.2 channels. We found that co-expression of KChIP1, KChIP2, and KChIP3, but not KChIP4a, leads to a conspicuous subcellular redistribution of Kv4.2 channels, from being ER-retained to being expressed on the cell surface, via a mechanism that may involve masking of a cytoplasmic trafficking and/or solubility determinant. KChIPs also induce a striking transformation in the overall molecular properties (expression level, phosphorylation, detergent solubility, and stability) of Kv4.2, leading to characteristics more typical of those observed for Kv4.2 in native cells. The dramatic differences between different KChIP isoforms in inducing these effects suggest that the precise KChIP composition of Kv4 channels impacts diverse aspects of function. Thus KChIPs induce dramatic effects on not only the biophysical properties but also the cell biological and molecular characteristics of Kv4 α subunits that dramatically impact the function, abundance, and distribution of Kv4 channels in excitable cells.EXPERIMENTAL PROCEDURESAntibodies—We have recently 2K. J. Rhodes, K. I. Carroll, M. A. Sung, L. C. Doliveira, M. M. Monaghan, S. L. Burke, B. W. Strassle, L. Buchwalder, J. Cao, W. F. An, and J. S. Trimmer, submitted for publication. generated anti-Kv4.2 ectodomain (S1-S2 linker, amino acids 209–225 CGSSPGHIKELPSGERY) rabbit polyclonal (Kv4.2e) and mouse monoclonal antibodies (K57/1 (IgG1) and K57/40 (IgG3)). Mouse monoclonal antibodies were generated as described previously (31Bekele-Arcuri Z. Matos M.F. Manganas L. Strassle B.W. Monaghan M.M. Rhodes K.J. Trimmer J.S. Neuropharmacology. 1996; 35: 851-865Crossref PubMed Scopus (101) Google Scholar). We previously generated an anti-Kv4.2 C-terminal cytoplasmic domain (amino acids 484–502 CLEKTTNHEFVDEQVFEES) rabbit polyclonal antibody Kv4.2C (32Nakahira K. Shi G. Rhodes K.J. Trimmer J.S. J. Biol. Chem. 1996; 271: 7084-7089Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). We also previously generated a phosphorylation site-specific rabbit polyclonal antibody against a synthetic peptide containing phosphoserine-552 (CT-PKA) as described (33Adams J.P. Anderson A.E. Varga A.W. Dineley K.T. Cook R.G. Pfaffinger P.J. Sweatt J.D. J. Neurochem. 2000; 75: 2277-2287Crossref PubMed Scopus (212) Google Scholar, 34Anderson A.E. Adams J.P. Qian Y. Cook R.G. Pfaffinger P.J. Sweatt J.D. J. Biol. Chem. 2000; 275: 5337-5346Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We recently 2K. J. Rhodes, K. I. Carroll, M. A. Sung, L. C. Doliveira, M. M. Monaghan, S. L. Burke, B. W. Strassle, L. Buchwalder, J. Cao, W. F. An, and J. S. Trimmer, submitted for publication. generated the following anti-KChIP mouse monoclonal antibodies: anti-KChIP1 (K55/7 (IgG1) and K55/29 (IgG2a)), anti-KChIP2 (K60/41 (IgG1) and K60/73 (IgG1)), anti-KChIP3 (K66/29 (IgG2a), K66/38 (IgG2a), and K90A/19 (IgG1)), and anti-KChIP4 (1G2 (IgG2a)). A pan-KChIP mouse monoclonal antibody, K55/82 (IgG2a), was also generated from the mice immunized with the KChIP1 fusion protein (16An W.F. Bowlby M.R. Betty M. Cao J. Ling H.P. Mendoza G. Hinson J.W. Mattsson K.I. Strassle B.W. Trimmer J.S. Rhodes K.J. Nature. 2000; 403: 553-556Crossref PubMed Scopus (834) Google Scholar). 2K. J. Rhodes, K. I. Carroll, M. A. Sung, L. C. Doliveira, M. M. Monaghan, S. L. Burke, B. W. Strassle, L. Buchwalder, J. Cao, W. F. An, and J. S. Trimmer, submitted for publication. Anti-PSD-95 mouse monoclonal antibodies K28/43 (IgG2a) and K28/86 (IgG1) were described previously (35Tiffany A.M. Manganas L.N. Kim E. Hsueh Y.P. Sheng M. Trimmer J.S. J. Cell Biol. 2000; 148: 147-158Crossref PubMed Scopus (149) Google Scholar, 36Rasband M.N. Park E.W. Zhen D. Arbuckle M.I. Poliak S. Peles E. Grant S.G. Trimmer J.S. J. Cell Biol. 2002; 159: 663-672Crossref PubMed Scopus (71) Google Scholar). Anti-GAD mouse monoclonal antibody GAD-6 (IgG2a) was obtained from the Developmental Studies Hybridoma Bank, Iowa City, IA. An anti-calnexin rabbit polyclonal antibody was obtained from Stressgen (Vancouver, British Columbia, Canada). Fluorescent species-specific and mouse isotype-specific secondary antibodies were obtained from Molecular Probes (Eugene, OR). Horseradish-peroxidase-conjugated secondary antibodies were from ICN (Aurora, OH).Immunofluorescence Analyses of Transfected COS-1 Cells—COS-1 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% newborn calf serum (Hyclone Laboratories, Logan, UT), 50 units/ml penicillin, 50 μg/ml streptomycin (both from Invitrogen) in a humidified incubator at 37 °C under 5% CO2. Cells were maintained in plastic tissue culture dishes or on poly-l-lysine-coated glass coverslips in plastic Petri dishes. Cells were transfected with mammalian expression vectors for Kv4.2 and KChIPs by the calcium phosphate precipitation method (37Shi G. Kleinklaus A.K. Marrion N.V. Trimmer J.S. J. Biol. Chem. 1994; 269: 23204-23211Abstract Full Text PDF PubMed Google Scholar) or with LipofectAMINE 2000 (Invitrogen) or Polyfect (Qiagen) transfection reagents using the manufacturers' protocols. Cells expressing Kv4.2 and/or KChIPs were stained 48 h post-transfection using a surface immunofluorescence protocol (38Shi G. Nakahira K. Hammond S. Rhodes K.J. Schechter L.E. Trimmer J.S. Neuron. 1996; 16: 843-852Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 39Manganas L.N. Trimmer J.S. J. Biol. Chem. 2000; 275: 29685-29693Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), applying the ectodomain-directed K57/1 mouse monoclonal antibody prior to detergent permeabilization to detect the cell surface Kv4.2 pool. The total cellular Kv4.2 pool was detected by EGFP fluorescence or with cytoplasmic directed rabbit polyclonal antibody Kv4.2N following detergent permeabilization. No difference in the intrinsic localization or KChIP-induced effects was observed between wild type and EGFP-tagged Kv4.2.Bound primary antibodies were detected using Alexa 594-conjugated goat anti-mouse IgG and, if needed, Alexa 488-conjugated goat anti-rabbit IgG. Cells were viewed under indirect immunofluorescence on a Zeiss Axioskop 2 microscope. Cells with detectable surface staining (red) and with total green fluorescence or staining, indicating successful transfection, were scored under narrow wavelength Texas Red and fluorescein filter sets, respectively. For some experiments standard double immunofluorescence staining of permeabilized COS-1 cells was performed as described (35Tiffany A.M. Manganas L.N. Kim E. Hsueh Y.P. Sheng M. Trimmer J.S. J. Cell Biol. 2000; 148: 147-158Crossref PubMed Scopus (149) Google Scholar). Images of cells were captured into a Zeiss Axiocam (Oberkochen, Germany) cooled CCD 24-bit color digital camera mounted on a Zeiss Axioskop 2 microscope with a 100×, 1.4 numerical aperture objective, using the software supplied with the camera.Immunoprecipitation, SDS-PAGE, and Immunoblotting—Analyses of COS-1 cell lysates prepared from transfected cells were performed as described (37Shi G. Kleinklaus A.K. Marrion N.V. Trimmer J.S. J. Biol. Chem. 1994; 269: 23204-23211Abstract Full Text PDF PubMed Google Scholar, 39Manganas L.N. Trimmer J.S. J. Biol. Chem. 2000; 275: 29685-29693Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In brief, to harvest COS-1 cells and prepare detergent lysates, cells were first washed twice in ice-cold PBS and then lysed for 5 min on ice in 1 ml of an ice-cold lysis buffer solution containing TBS (10 mm Tris, 150 mm NaCl, pH 8.0), 5 mm EDTA, 0.4% Triton X-100, 1 mm iodoacetamide, and a protease inhibitor mixture (2 μg/ml aprotinin, 1 μg/ml leupeptin, 2 μg/ml antipain, 10 μg/ml benzamidine, and 0.2 mm phenylmethylsulfonyl fluoride). The detergent lysate was centrifuged in a microcentrifuge for 2 min at 13,000 × g to pellet nuclei and debris, and the resulting supernatant (cleared lysate) was saved for analysis. For immunoblots, the cleared lysate was added to an equal volume of 2× reducing SDS sample buffer and fractionated on 9 (for Kv4.2) or 15% (for KChIPs) SDS-polyacrylamide gels. Note that “lauryl sulfate” (Sigma L-5750; 69% lauryl sulfate (SDS), 26% myristyl sulfate, 5% cetyl sulfate) was used in SDS gel recipes to accentuate electrophoretic mobility differences between different phosphorylation states of Kv4.2, as has been described previously for other proteins (37Shi G. Kleinklaus A.K. Marrion N.V. Trimmer J.S. J. Biol. Chem. 1994; 269: 23204-23211Abstract Full Text PDF PubMed Google Scholar, 40Ward G.E. Garbers D.L. Vacquier V.D. Science. 1985; 227: 768-770Crossref PubMed Scopus (62) Google Scholar). Quantitation of immunoreactivity was performed by densitometry and analysis using Image J software, a public domain Java image processing program (rsb.info.nih.gov/ij/index.html).For immunoprecipitation reactions, 200 μl of detergent lysate was diluted to 1 ml in ice-cold lysis buffer. Affinity-purified polyclonal Kv4.2C antibody (5 μg) was added, and the mixture was incubated on a tube rotator at 4 °C for 16 h. The antibody-antigen complex was immobilized by adsorption onto 15 μl of protein A-agarose (Pierce) by incubation on a tube rotator for 1 h at 4 °C. Protein A beads were washed six times in lysis buffer and then incubated with calf intestinal alkaline phosphatase (AP, 0.1 unit/ml) for 16 h at 37 °C. The beads were then resuspended in reducing SDS sample buffer and analyzed on 9% SDS-polyacrylamide gels.Cycloheximide Treatment—To evaluate the stability of Kv4.2 by blocking total cellular protein synthesis, cultured COS-1 cells 24 h post-transfection were treated with cycloheximide (100 μg/ml) for the indicated times and then analyzed by immunoblotting.Primary Rat Hippocampal Neuronal Cultures—Low density primary embryonic rat hippocampal cultures were prepared according to established protocols (41Banker G.A. Cowan W.M. Brain Res. 1977; 126: 397-425Crossref PubMed Scopus (947) Google Scholar, 42Lim S.T. Antonucci D.E. Scannevin R.H. Trimmer J.S. Neuron. 2000; 25: 385-397Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Briefly, an astrocyte culture was prepared from cerebral hemispheres of neonatal rats and cultured for 5 days in 6-well tissue culture plates in minimum essential medium containing 10% horse serum and 0.6% glucose prior to plating of hippocampal neurons. The day before the hippocampal dissection, the astrocyte medium was changed to neuronal maintenance medium (minimum essential medium containing N2 supplements, 0.1% ovalbumin, and 0.1 mm sodium pyruvate) for conditioning. Hippocampi dissected from embryonic day 18 rat embryos were dissociated by treatment with 0.25% trypsin at 37 °C for 15 min. The hippocampal preparation were plated in minimum essential medium supplemented with 10% horse serum and 0.06% glucose onto 22-mm square coverslips (72 cells/mm2) or 60-cm plastic tissue culture dishes (177 cells/mm2) previously coated with 1 mg/ml poly-l-lysine. Neurons were incubated in a humidified incubator at 5% CO2 for 4 h to attach to the coverslips, and then the coverslips transferred, inverted on wax pedestals, to the 6-well tissue culture plates that contained the previously established astrocyte cultures as a feeder layer. For neurons on plastic dishes, the media were changed to the serum-free media conditioned with a separate astrocyte culture. After 3 days, cytosine arabinoside was added to the media to 1 μm final concentration. Cultures were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. One-half of the culture medium was changed weekly.Immunofluorescence Staining of Cultured Neurons—Staining of cultured neurons was performed as described previously (43Antonucci D.E. Lim S.T. Vassanelli S. Trimmer J.S. Neuroscience. 2001; 108: 69-81Crossref PubMed Scopus (97) Google Scholar). Briefly, cultured neurons were fixed in 3% (w/v) paraformaldehyde, 3% sucrose in phosphate-buffered saline (PBS, 0.15 m NaCl, 10 mm sodium phosphate, pH 7.4) for 15 min, washed twice with PBS, and permeabilized in 0.1% (v/v) Triton X-100. Nonspecific binding sites were blocked with Blotto-T (4% (w/v) non-fat dry milk in 20 mm Tris, pH 8.0, 150 mm NaCl, and 0.1% Triton X-100). Cells were then incubated in primary antibodies for 3 h or overnight. Cells were then washed three times in Blotto-T to remove excess primary antibody, followed by incubation in the appropriate secondary antibodies (diluted to 1:2000 in Blotto-T) for 1 h. After three washes in PBS-T, coverslips were mounted on microscope slides in a 90% (v/v) glycerol solution containing 0.1 mg/ml p-phenylenediamine in PBS, pH 9.0.Biochemical Analysis of Proteins in Cultured Neurons—Hippocampal neurons cultured on plastic dishes were washed twice with ice-cold Locke's solution (154 mm NaCl, 5.6 mm KCl, 2.3 mm CaCl2,1mm MgCl2, 5 mm glucose, 5 mm HEPES, pH 7.4), harvested, and centrifuged at 12,000 × g for 30 min at 4 °C. The pellets were extracted by adding reducing SDS sample buffer, size-fractionated on 7.5% SDS-acrylamide gels, and immunoblotted for Kv4.2 (K57/1).RESULTSKChIP Co-expression Leads to Changes in the Intracellular Trafficking of Kv4.2—To begin to address the effects of KChIP co-expression on Kv4 channels, we analyzed the immunofluorescence staining pattern of COS-1 cells expressing either Kv4.2 alone or Kv4.2 plus KChIP1, KChIP2, KChIP3, or KChIP4a. We found that virtually all of the Kv4.2-expressing COS-1 cells had robust intracellular Kv4.2 staining in a perinuclear pattern typical for ER-retained membrane proteins (Fig. 1A). Moreover, no detectable cell surface staining of intact cells with anti-ectodomain Kv4.2 antibodies was observed (Fig. 1A). To determine whether the intracellular pool of Kv4.2 was in fact associated with the ER, we double-labeled COS-1 cells expressing Kv4.2 alone for Kv4.2 and the resident ER protein calnexin, which yielded precisely co-localizing perinuclear immunofluorescence staining patterns (Fig. 1F). Consistent with the ER localization of Kv4.2, double staining for Kv4.2 and the Golgi apparatus marker Lens culinaris agglutinin revealed no overlap (Supplemental Fig. 1).Co-expression of Kv4.2 with KChIP1, KChIP2, or KChIP3 at a Kv4:KChIP cDNA ratio of 2:1 resulted in a dramatic change in the subcellular distribution of Kv4.2 (Figs. 1 and 2). Kv4.2 staining now extended to the periphery of the cell, and the transfected cells now displayed robust cell surface Kv4.2 staining (Figs. 1 and 2). The residual intracellular Kv4.2 pool was no longer associated with the ER but with the Golgi, as indicated by the co-localization of intracellular Kv4.2 with Lens culinaris agglutinin (Fig. 1G) and loss of co-localization with calnexin (Supplemental Fig. 1). However, cells co-transfected with Kv4.2 and KChIP4a did not respond in this manner and resembled cells expressing Kv4.2 alone in both a lack of Kv4.2 surface staining and the presence of an ER-localized pool of Kv4.2 (Figs. 1E and 2H).Fig. 2KChIPs exhibit distinct localizations in transfected COS-1 cells. COS-1 cells were transiently transfected with KChIP1 (A), KChIP2 (B), KChIP3 (C), or KChIP4a (D) and stained with KChIP-specific antibodies (KChIP1, K55/7; KChIP2, K60/41; KChIP3, K90A/16; and KChIP4a, 1G2) after detergent permeabilization. E–H, permeabilized COS-1 cells expressing EGFP-Kv4.2 and KChIP1 (E), KChIP2 (F), KChIP3 (G), and KChIP4 (H) were stained with the relevant KChIP-specific antibodies (right panels); EGFP-Kv4.2 localization is shown as EGFP fluorescence (left panels). Scale bars, 10 μm.View Large Image Figure ViewerDownload (PPT)To determine whether the lack of KChIP4 effects on Kv4.2 localization was simply due to differences in steady-state expression level relative to other KChIPs, the dose dependence of the effects of KChIP2 and KChIP4a on Kv4.2 surface expression was examined (Fig. 1H). Increased KChIP2 expression yielded a dose-dependent increase in the fraction of Kv4.2-expressing cells with detectable surface expression, with initial effects apparent at a KChIP2:Kv4.2 cDNA ratio of 1:128 and maximal effects at 1:8–1:4. Interestingly, the percentage of Kv4.2-expressing cells that exhibited detectable surface staining reached a plateau at ≈60%. The basis for this observed lack of quantitative response is not known but is not due to simple lack of co-expression as >95% of the cells expressed both Kv4.2 and KChIP2. It should be noted that a similar ceiling was observed in our previous studies of Kvβ subunit effects on Kv1.2 surface expression (38Shi G. Nakahira K. Hammond S. Rhodes K.J. Schechter L.E. Trimmer J.S. Neuron. 1996; 16: 843-852Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). KChIP4a co-expression did not yield any detectable increases in Kv4.2 surface expression at any dose tested (Fig. 1H).Interestingly, KChIP4a also had a subcellular distribution distinct from the other KChIPs when expressed either alone or with Kv4.2. KChIPs1–3 expressed alone or with Kv4.2 in COS-1 cells generally exhibited a diffuse distribution throughout the cell (Fig. 2, A–C), although KChIP3-expressing cells also exhibited additional nuclear staining (Fig. 2C) consistent with its previously described DNA binding function (44Spreafico F. Barski J.J. Farina C. Meyer M. Mol. Cell. Neurosci. 2001; 17: 1-16Crossref PubMed Scopus (57) Google Scholar). In contrast, KChIP4a staining was perinuclear (Fig. 2D), suggesting an association with the ER.KChIPs Induce Profound Changes in the Molecular Characteristics of Kv4.2—COS-1 cells expressing Kv4.2 alone exhibit a single pool of expressed protein with an apparent mass of 65 kDa on SDS gels (Fig. 3). However, two distinct populations of Kv4.2 protein, at 65 and 70 kDa, are observed when Kv4.2 is co-expressed with KChIP1, -2, or -3 (Fig. 3, A–C). The 70-kDa form is similar to the M r of native rat brain Kv4.2 (see below). The dose dependence of the induction of the surface expression and increased M r values of Kv4.2 upon co-expression of KChIPs1–3 were quite similar (Fig. 3E). Increased steady-state expression levels of Kv4.2 were also observed with increased co-expression of KChIPs, suggesting that KChIPs may also affect Kv4.2 turnover rates (Fig. 3 and see below). Moreover, similar to its lack of effect on Kv4.2 surface expression, KChIP4a co-expression did not affect the M r or steady-state expression level of Kv4.2 at any dose teste" @default.
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• W2054792009 date "2003-09-01" @default.
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• W2054792009 title "A Fundamental Role for KChIPs in Determining the Molecular Properties and Trafficking of Kv4.2 Potassium Channels" @default.
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• W2054792009 doi "https://doi.org/10.1074/jbc.m306142200" @default.
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