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• W2066063313 abstract "In fast-spiking neurons such as those in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, Kv3.1 potassium channels are required for high frequency firing. The Kv3.1b splice variant of this channel predominates in the mature nervous system and is a substrate for phosphorylation by protein kinase C (PKC) at Ser-503. In resting neurons, basal phosphorylation at this site decreases Kv3.1 current, reducing neuronal ability to follow high frequency stimulation. We used a phospho-specific antibody to determine which PKC isozymes control serine 503 phosphorylation in Kv3.1b-tranfected cells and in auditory neurons in brainstem slices. By using isozyme-specific inhibitors, we found that the novel PKC-δ isozyme, together with the novel PKC-ε and conventional PKCs, contributed to the basal phosphorylation of Kv3.1b in MNTB neurons. In contrast, only PKC-ε and conventional PKCs mediate increases in phosphorylation produced by pharmacological activation of PKC in MNTB neurons or by metabotropic glutamate receptor activation in Kv3.1/mGluR1-cotransfected cells. We also measured the time course of dephosphorylation and recovery of basal phosphorylation of Kv3.1b following brief high frequency electrical stimulation of the trapezoid body, and we determined that the recovery process is mediated by both novel PKC-δ and PKC-ε isozymes and by conventional PKCs. The association between Kv3.1b and PKC isozymes was confirmed by reciprocal coimmunoprecipitation of Kv3.1b with multiple PKC isozymes. Our results suggest that the Kv3.1b channel is regulated by both conventional and novel PKC isozymes and that novel PKC-δ contributes specifically to the maintenance of basal phosphorylation in auditory neurons. In fast-spiking neurons such as those in the medial nucleus of the trapezoid body (MNTB) in the auditory brainstem, Kv3.1 potassium channels are required for high frequency firing. The Kv3.1b splice variant of this channel predominates in the mature nervous system and is a substrate for phosphorylation by protein kinase C (PKC) at Ser-503. In resting neurons, basal phosphorylation at this site decreases Kv3.1 current, reducing neuronal ability to follow high frequency stimulation. We used a phospho-specific antibody to determine which PKC isozymes control serine 503 phosphorylation in Kv3.1b-tranfected cells and in auditory neurons in brainstem slices. By using isozyme-specific inhibitors, we found that the novel PKC-δ isozyme, together with the novel PKC-ε and conventional PKCs, contributed to the basal phosphorylation of Kv3.1b in MNTB neurons. In contrast, only PKC-ε and conventional PKCs mediate increases in phosphorylation produced by pharmacological activation of PKC in MNTB neurons or by metabotropic glutamate receptor activation in Kv3.1/mGluR1-cotransfected cells. We also measured the time course of dephosphorylation and recovery of basal phosphorylation of Kv3.1b following brief high frequency electrical stimulation of the trapezoid body, and we determined that the recovery process is mediated by both novel PKC-δ and PKC-ε isozymes and by conventional PKCs. The association between Kv3.1b and PKC isozymes was confirmed by reciprocal coimmunoprecipitation of Kv3.1b with multiple PKC isozymes. Our results suggest that the Kv3.1b channel is regulated by both conventional and novel PKC isozymes and that novel PKC-δ contributes specifically to the maintenance of basal phosphorylation in auditory neurons. There is a good correlation between the ability of neurons to discharge at high rates and the expression of Kv3 family potassium channels, particularly the Kv3.1 channel (1Lenz S. Perney T.M. Qin Y. Robbins E. Chesselet M.F. Synapse. 1994; 18: 55-66Crossref PubMed Scopus (87) Google Scholar, 2Weiser M. Bueno E. Sekirnjak C. Martone M.E. Baker H. Hillman D. Chen S. Thornhill W. Ellisman M. Rudy B. J. Neurosci. 1995; 15: 4298-4314Crossref PubMed Google Scholar, 3Du J. Zhang L. Weiser M. Rudy B. McBain C.J. J. Neurosci. 1996; 16: 506-518Crossref PubMed Google Scholar, 4Perney T.M. Kaczmarek L.K. J. Comp. Neurol. 1997; 386: 178-202Crossref PubMed Scopus (112) Google Scholar, 5Rudy B. Chow A. Lau D. Amarillo Y. Ozaita A. Saganich M. Moreno H. Nadal M.S. Hernandez-Pineda R. Hernandez-Cruz A. Erisir A. Leonard C. De Miera E.V.-S. Ann. N. Y. Acad. Sci. 1999; 868: 304-343Crossref PubMed Scopus (260) Google Scholar). Neurons that express Kv3.1 at high levels include those in the medial nucleus of the trapezoid body (MNTB) 2The abbreviations used are: MNTB, the medial nucleus of the trapezoid body; PKC, protein kinase C; cPKC, conventional protein kinase C; nPKC, novel protein kinase C; aPKC, atypical protein kinase C; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c] carbazole; GF109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; PMA, phorbol 12-myristate 13-acetate; Ro 31-7549, bisindolylmaleimide VIII; Me2SO, dimethyl sulfoxide; CHO, Chinese hamster ovary; DHPG, dihydroxyphenylglycine; MCPG, (RS)-α-methyl-4-carboxylphenylglycine. within the auditory brainstem (6Perney T.M. Kaczmarek L.K. Curr. Opin. Cell Biol. 1991; 3: 663-666Crossref PubMed Scopus (62) Google Scholar, 7Trussell L.O. Annu. Rev. Physiol. 1999; 61: 477-496Crossref PubMed Scopus (341) Google Scholar, 8Grigg J.J. Brew H.M. Tempel B.L. Hear. Res. 2000; 140: 77-90Crossref PubMed Scopus (114) Google Scholar). The MNTB is key element of neural pathways that detect differences in the level or timing of interaural stimuli to compute sound localization. MNTB neurons are able to fire at frequencies of hundreds of Hz (9Leao R.M. Von Gersdorff H. J. Neurophysiol. 2002; 87: 2297-2306Crossref PubMed Scopus (52) Google Scholar, 10Kopp-Scheinpflug C. Lippe W.R. Dorrscheidt G.J. Rubsamen R. J. Assoc. Res. Otolaryngol. 2003; 4: 1-23Crossref PubMed Scopus (103) Google Scholar) and to lock their action potentials precisely to the phase of auditory stimuli at frequencies of up to 2–4 kHz or to rapid fluctuations in the amplitude of high frequency sounds (11Joris P.X. Yin T.C. J. Neurophysiol. 1995; 73: 1043-1062Crossref PubMed Scopus (230) Google Scholar). One important physiological specialization enabling MNTB neurons to fire at such high frequencies is the high level of expression of Kv3.1 potassium channels. Genetic knock-out of the Kv3.1 gene, as well as pharmacological and computer modeling studies, confirms that a high threshold component of potassium current in MNTB neurons is carried by Kv3.1 channels and that its elimination impairs the neuronal response to high frequency stimulation (12Brew H.M. Forsythe I.D. J. Neurosci. 1995; 15: 8011-8022Crossref PubMed Google Scholar, 13Wang L.Y. Gan L. Forsythe I.D. Kaczmarek L.K. J. Physiol. (Lond.). 1998; 509: 183-194Crossref Scopus (291) Google Scholar, 14Macica C.M. von Hehn C.A. Wang L.Y. Ho C.S. Yokoyama S. Joho R.H. Kaczmarek L.K. J. Neurosci. 2003; 23: 1133-1141Crossref PubMed Google Scholar). Nevertheless, high levels of Kv3.1b current degrade the accuracy of action potential timing at lower frequencies of firing (14Macica C.M. von Hehn C.A. Wang L.Y. Ho C.S. Yokoyama S. Joho R.H. Kaczmarek L.K. J. Neurosci. 2003; 23: 1133-1141Crossref PubMed Google Scholar, 15Song P. Yang Y. Barnes-Davies M. Hamann M. Bhattacharjee A. Forsythe I.D. Oliver D.L. Kaczmarek L.K. Nat. Neurosci. 2005; 8: 1335-1342Crossref PubMed Scopus (109) Google Scholar, 16Kaczmarek L.K. Bhattacharjee A. Desai R. Gan L. Song P. von Hehn C.A. Whim M.D. Yang B. Hear. Res. 2005; 206: 133-145Crossref PubMed Scopus (57) Google Scholar). Two isoforms of the Kv3.1 channel exist, Kv3.1a and Kv3.1b, that are generated by alternative splicing of the Kv3.1 gene. The Kv3.1b channel predominates in the mature nervous system and has a longer carboxyl terminus than that of Kv3.1a (2Weiser M. Bueno E. Sekirnjak C. Martone M.E. Baker H. Hillman D. Chen S. Thornhill W. Ellisman M. Rudy B. J. Neurosci. 1995; 15: 4298-4314Crossref PubMed Google Scholar, 17Perney T.M. Marshall J. Martin K.A. Hockfield S. Kaczmarek L.K. J. Neurophysiol. 1992; 68: 756-766Crossref PubMed Scopus (196) Google Scholar). Activators of PKC significantly reduce the amplitude of Kv3.1b current (14Macica C.M. von Hehn C.A. Wang L.Y. Ho C.S. Yokoyama S. Joho R.H. Kaczmarek L.K. J. Neurosci. 2003; 23: 1133-1141Crossref PubMed Google Scholar, 18Critz S.D. Wible B.A. Lopez H.S. Brown A.M. J. Neurochem. 1993; 60: 1175-1178Crossref PubMed Scopus (59) Google Scholar, 19Kanemasa T. Gan L. Perney T.M. Wang L.Y. Kaczmarek L.K. J. Neurophysiol. 1995; 74: 207-217Crossref PubMed Scopus (101) Google Scholar). Although activation of PKC stimulates phosphate incorporation into several serine residues in Kv3.1b, the specific actions of PKC on Kv3.1b currents have been shown to depend selectively on the phosphorylation of Ser-503 in the carboxyl-terminal region (14Macica C.M. von Hehn C.A. Wang L.Y. Ho C.S. Yokoyama S. Joho R.H. Kaczmarek L.K. J. Neurosci. 2003; 23: 1133-1141Crossref PubMed Google Scholar). By using a phospho-specific antibody to serine 503 of Kv3.1b, it has been found that Kv3.1b in MNTB neurons is basally phosphorylated by PKC in a quiet auditory environment, providing maximal timing accuracy at low firing frequencies. In vivo acoustic stimulation of animals, or high frequency stimulation of the afferent input of MNTB neurons in brainstem slices, results in a rapid and reversible decrease in the level of phosphorylation. This dephosphorylation permits neurons to fire at higher rates, albeit with lower temporal accuracy. Thus phosphorylation of Kv3.1b by PKC appears to be a mechanism that rapidly adjusts the intrinsic electrical properties of neurons to the pattern of incoming auditory stimuli (16Kaczmarek L.K. Bhattacharjee A. Desai R. Gan L. Song P. von Hehn C.A. Whim M.D. Yang B. Hear. Res. 2005; 206: 133-145Crossref PubMed Scopus (57) Google Scholar). PKC includes a family of Ser/Thr protein kinases that control many different aspects of neuronal function (20Malenka R.C. Madison D.V. Nicoll R.A. Nature. 1986; 321: 175-177Crossref PubMed Scopus (503) Google Scholar, 21Tanaka C. Nishizuka Y. Annu. Rev. Neurosci. 1994; 17: 551-567Crossref PubMed Scopus (509) Google Scholar, 22Malmberg A.B. Prog. Brain Res. 2000; 129: 51-59Crossref PubMed Scopus (38) Google Scholar, 23Metzger F. Kapfhammer J.P. Cerebellum. 2003; 2: 206-214Crossref PubMed Scopus (41) Google Scholar). PKC enzymes have been divided into three groups as follows: (i) conventional, Ca2+-dependent PKC (cPKC) (α, βI, βII, and γ); (ii) novel, Ca2+-independent PKC (nPKC) (δ, ε, η, and θ); and (iii) atypical PKC isozymes (aPKC) (ξ, λ, ι, and μ). Both cPKCs and nPKCs are activated by phorbol esters, whereas aPKCs are insensitive to either Ca2+ or to phorbol esters. In this study we have identified the PKC isozymes that control the basal level of Kv3.1b phosphorylation in MNTB neurons, those that mediate the PKC activator and receptor-induced phosphorylation, and also those that contribute to the recovery from stimulation-induced dephosphorylation of Kv3.1b channels in MNTB neurons. Materials—12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c] carbazole (Gö6976) was purchased from Alexis (Carlsbad, CA). Rottlerin and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X) were from Calbiochem. Phorbol 12-myristate 13-acetate (PMA) and bisindolylmaleimide VIII (Ro 31-7549) were from Sigma. (S)-3,5-Dihydroxyphenylglycine (S-DHPG; Tocris Neuramin, Ltd., Bristol, UK) were prepared in H2O. (RS)-α-Methyl-4-carboxylphenylglycine (MCPG) was prepared in an NaOH solution. All the other drugs were dissolved in dimethyl sulfoxide (Me2SO). Polyclonal antibody against Ser-503-phosphorylated Kv3.1b was made by PhosphoSolutions (Aurora, CO). Antibodies against PKC-α, -δ, and -ε were purchased from Santa Cruz Biotechnologies. Immunocytochemistry—Chinese hamster ovary (CHO) cells stably expressing Kv3.1a, Kv3.1b, or Kv3.1 mutant S503A were maintained in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% fetal bovine serum, 0.1 mm hypoxanthine, and 0.5 mg/ml geneticin (Invitrogen). CHO cells were grown on coverslips for 24–48 h in a 5% CO2 incubator at 37 °C preceding drug administration. Cells were preincubated for 30 min with PKC inhibitors at the indicated concentrations and then stimulated with the PKC activator PMA for 12 min. For transient transfection of Kv3.1-CHO cells with pcDNA-mGluR1 (a kind gift from Dr. Jarda Wroblewski, Georgetown University), the cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mm l-glutamine, and 4.5% proline. Cells were transfected using Lipofectamine 2000 (Invitrogen) and were treated 10 min with mGluR1 agonist DHPG (300 μm) at 24 h after transfection. After drug treatment, CHO cells were quickly washed with ice-cold 0.1 m phosphate-buffered saline and then fixed with 4% paraformaldehyde at 4 °C for 10 min. After blocking and permeabilization, cells were incubated with rabbit anti-phosphoserine 503 Kv3.1b polyclonal antibody (1:400) at 4 °C for 24 h and subsequently with Alexa Fluor® 488 goat anti-rabbit IgG (1:700) at 4 °C overnight. The coverslips were then mounted on glass slides with Citifluor Mountant Media (Ted Pella, Redding, CA), and images were taken on an Olympus BX60 microscope by using a SPOT RT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Preparation of Brainstem Slices—Sprague-Dawley rats (17–19 days old, Charles River Breeding Laboratories, Wilmington, MA) were killed by decapitation, and transverse brainstem slices containing MNTB were cut at a thickness of 350 μm in ice-cold gassed artificial cerebrospinal fluid (in mm: 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 0.1 CaCl2, 3.0 MgCl2, 3 myoinositol, 2 sodium pyruvate, 0.4 ascorbic acid, and 25 glucose, pH 7.4). The slices were incubated at 37 °C for 50 min and thereafter kept at room temperature in recoding solution containing (in mm) 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2,1 MgCl2, 3 myoinositol, 2 sodium pyruvate, 0.4 ascorbic acid, and 25 glucose, pH 7.4. Slices were stimulated at the midline with a bipolar electrode (8 V, 0.2 ms) or treated with PKC inhibitor and activator as done with CHO cells. After that the slices were quickly transferred to 4% paraformaldehyde prior to immunohistochemistry. All experiments were conducted in accordance with the NIH and institutional animal care guidelines. Immunohistochemistry—Coronal sections were cut on a cryostat at a thickness of 35 μm. Similarly with immunocytochemistry, sections were incubated with phospho-specific antibody against Kv3.1b at 4 °C for 60 h and subsequently with Alexa Fluor 488 goat anti-rabbit IgG at 4 °C for 24 h. Sections were scanned by a Bio-Rad model 1024 UV laser confocal microscope system. Coimmunoprecipitation and Western Blotting—CHO cells or rat brainstems were homogenized in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% dodecyl β-d-maltoside, and a protease/phosphatase inhibitor mixture). The lysate was precleared with 1:1 slurry of protein A-Sepharose and equilibrated in lysis buffer at 4 °C for 1 h with shaking. After a short spin, the supernatant was incubated at 4 °C overnight with rabbit IgG or antibodies as indicated. Immune complexes were pooled down by protein A-Sepharose at 4 °C for 2 h with shaking. Immunoprecipitates were washed and dissolved in SDS sample buffer. The protein was loaded and separated by SDS-PAGE (10%) and then transferred to polyvinylidene difluoride membranes and probed with the indicated antibodies. Goat anti-rabbit horseradish peroxidase-coupled secondary antibodies were used for detection with West Femto chemiluminescence (Pierce). Image Analysis—Optical density of the immunostaining was measured using ImagePro Plus software (Media Cybernetics, Silver Spring, MD) and was referred to as “level of immunoreactivity” in the figures. OD values were subjected to statistical evaluation using Student's t test or one-way analysis of variance followed by post hoc comparison to confirm significant differences between the groups. Criteria for significance in all analyses were defined as p < 0.05. Data were presented as mean ± S.E. Detection of Phosphorylated Kv3.1b Potassium Channels Using a Ser-503 Phospho-specific Antibody—Immunohistochemistry and immunoblotting were performed to evaluate the specificity of the anti-phospho-Ser-503 Kv3.1b antibody that was used throughout this study. CHO cells were stably transfected with the wild-type Kv3.1b gene or with S503A mutant Kv3.1b, in which this PKC phosphorylation site is mutated. As described previously (15Song P. Yang Y. Barnes-Davies M. Hamann M. Bhattacharjee A. Forsythe I.D. Oliver D.L. Kaczmarek L.K. Nat. Neurosci. 2005; 8: 1335-1342Crossref PubMed Scopus (109) Google Scholar), no detectable basal immunostaining was observed in Kv3.1b-CHO cells until treatment with PMA (500 nm), an activator of PKC (Fig. 1A). Moreover, no immunoreactivity was detected after treatment with PMA in cells transfected with the S503A mutant Kv3.1b or with the Kv3.1a splice variant, both of which lack the Ser-503 phosphorylation site. Negative control experiments in which the primary antibody was either omitted or pre-adsorbed with the antigen peptide also revealed no immunostaining in PMA-treated Kv3.1b-CHO cells. In contrast, incubation of the primary antibody with a heterologous peptide did not affect the immunostaining in PMA-treated Kv3.1b-CHO cells (Fig. 1A). In contrast to the Kv3.1b-transfected CHO cells, profuse immunostaining was seen on the membrane of MNTB principal neurons in adult rats using the phospho-specific Kv3.1b antibody, even in the absence of pharmacological activation of PKC. This immunolabeling represented specific staining of Kv3.1b phosphorylated at Ser-503 because it was blocked by preincubating the primary antibody with the antigenic peptide but not with the heterologous peptide (Fig. 1B). Immunoblots of denatured protein extracts were carried out in transfected CHO cells. Consistent with previous studies using antibodies against other regions of the Kv3.1b protein (2Weiser M. Bueno E. Sekirnjak C. Martone M.E. Baker H. Hillman D. Chen S. Thornhill W. Ellisman M. Rudy B. J. Neurosci. 1995; 15: 4298-4314Crossref PubMed Google Scholar, 24Parameshwaran-Iyer S. Carr C.E. Perney T.M. J. Neurobiol. 2003; 55: 165-178Crossref PubMed Scopus (28) Google Scholar), a protein band of ∼100 kDa was detected in extracts from Kv3.1b-CHO cells using the phospho-specific Kv3.1b antibody pre-absorbed with the heterologous peptide (Fig. 1C). This band was absent when the immunoblots were treated with antibody that was pre-adsorbed with the antigenic peptide or when the cells were treated with Me2SO, the vehicle for PMA treatment. Moreover, no band was detected in PMA-treated CHO cells that had been transfected with the S503A mutant Kv3.1b gene. Therefore, this antibody recognized Kv3.1b channels only when the channels were phosphorylated by PKC at Ser-503. PKC Isozymes Responsible for PMA-induced Phosphorylation of Kv3.1b Channels in CHO Cells—To identify the PKC isozymes involved in the modulation of Kv3.1b channels, a variety of PKC inhibitors was used specifically to inhibit different groups of PKC isozymes (Table 1). Group I inhibitors were those that are known only to inhibit the conventional family of PKCs (cPKC), whereas the group II inhibitors act on both cPKC and nPKC. Gö6976 is a specific inhibitor of cPKCs and has no effect on the activity of nPKC or aPKC isozymes even at micromolar levels (25Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar, 26Wenzel-Seifert K. Schachtele C. Seifert R. Biochem. Biophys. Res. Commun. 1994; 200: 1536-1543Crossref PubMed Scopus (49) Google Scholar). GF109203X is, at a concentration of 50 nm, a specific inhibitor of cPKCs. At the much higher concentration of 1 μm, GF109203X inhibits both cPKCs and nPKCs (27Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 28Vilarino N. de la Rosa L.A. Vieytes M.R. Botana L.M. Cell. Signal. 2001; 13: 177-190Crossref PubMed Scopus (7) Google Scholar). Similarly, Ro 31-7549 is known to antagonize only cPKCs at a concentration of 50 nm but inhibits both cPKCs and nPKCs at the higher concentration of 4 μm (29Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar, 30Wilkinson S.E. Parker P.J. Nixon J.S. Biochem. J. 1993; 294: 335-337Crossref PubMed Scopus (495) Google Scholar). Finally, rottlerin (10 μm) has long been used as a selective inhibitor of nPKC-δ (31Gschwendt M. Kielbassa K. Kittstein W. Marks F. FEBS Lett. 1994; 347: 85-89Crossref PubMed Scopus (98) Google Scholar, 32Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (762) Google Scholar). At much higher concentrations, however, rottlerin acts on all classes of PKC.TABLE 1Properties of the PKC inhibitorsInhibitorConcentrationPKC isozymes inhibitedRefs.Group I Gö6976100 nm to 1 μmcPKCs25Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar, 26Wenzel-Seifert K. Schachtele C. Seifert R. Biochem. Biophys. Res. Commun. 1994; 200: 1536-1543Crossref PubMed Scopus (49) Google Scholar GF109203X50 nmcPKCs27Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 28Vilarino N. de la Rosa L.A. Vieytes M.R. Botana L.M. Cell. Signal. 2001; 13: 177-190Crossref PubMed Scopus (7) Google Scholar Ro 31-754950 nmcPKCs29Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar, 30Wilkinson S.E. Parker P.J. Nixon J.S. Biochem. J. 1993; 294: 335-337Crossref PubMed Scopus (495) Google ScholarGroup II GF109203X1 μmcPKC, nPKC27Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 28Vilarino N. de la Rosa L.A. Vieytes M.R. Botana L.M. Cell. Signal. 2001; 13: 177-190Crossref PubMed Scopus (7) Google Scholar Ro 31-75494 μmcPKC, nPKC29Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar, 30Wilkinson S.E. Parker P.J. Nixon J.S. Biochem. J. 1993; 294: 335-337Crossref PubMed Scopus (495) Google ScholarPKC-δ inhibitor Rottlerin10 μmnPKC-δ31Gschwendt M. Kielbassa K. Kittstein W. Marks F. FEBS Lett. 1994; 347: 85-89Crossref PubMed Scopus (98) Google Scholar, 32Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (762) Google Scholar Open table in a new tab It has been established that CHO cells express the cPKC-α, -β, -γ isozymes, the nPKC-δ and -ε isozymes, and the atypical PKC-ζ,-λ,-μ, and -ι isozymes (33Tippmer S. Quitterer U. Kolm V. Faussner A. Roscher A. Mosthaf L. Muller-Esterl W. Haring H. Eur. J. Biochem. 1994; 225: 297-304Crossref PubMed Scopus (59) Google Scholar, 34Strassheim D. May L.G. Varker K.A. Puhl H.L. Phelps S.H. Porter R.A. Aronstam R.S. Noti J.D. Williams C.L. J. Biol. Chem. 1999; 274: 18675-18685Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 35Zhang X.A. Bontrager A.L. Stipp C.S. Kraeft S.K. Bazzoni G. Chen L.B. Hemler M.E. Mol. Biol. Cell. 2001; 12: 351-365Crossref PubMed Scopus (53) Google Scholar). We found that preincubation of Kv3.1b-transfected CHO cells with group I inhibitors, i.e. 100 nm Gö6976, 1 μm Gö6976, 50 nm Ro 31-7549, or 50 nm GF109203X, diminished but did not abolish PMA-induced phosphorylation of Kv3.1b channels (Fig. 2A). In contrast, 4 μm Ro 31-7549 and 1 μm GF109203X, which are group II inhibitors, completely abolished PMA-induced immunoreactivity of Kv3.1b channels, indicating that both conventional and novel PKCs are involved in PMA-induced phosphorylation of Kv3.1b in CHO cells. Rottlerin (10 μm), which inhibits only PKC-δ, had no effect on the actions of PMA. Quantification of the phospho-Kv3.1b immunoreactivity showed that PMA treatment of Kv3.1b-transfected CHO cells produced a dramatic increase in the phosphorylation level over control cells treated with Me2SO (p < 0.0001) (Fig. 2B). The effect of PMA was significantly attenuated by group I inhibitors to values between 59 and 32% of that in PMA-treated cells, i.e.100 nm Gö6976 (p < 0.01 versus PMA and p < 0.05 versus control), 1 μm Gö6976 (p < 0.0001 versus PMA and p < 0.05 versus control), 50 nm Ro 31-7549 (p < 0.0001 versus PMA and p < 0.05 versus control), and 50 nm GF109203X (p < 0.0001 versus PMA and p < 0.05 versus control). The group II inhibitors, i.e. 4 μm Ro 31-7549 and 1 μm GF109203X, completely abolished the effect of PMA, attenuating staining to less than 4.0% of PMA treatment (for both cases, p < 0.0001 versus PMA and p > 0.05 versus control). The effects of group II inhibitors were significantly more potent than those of group I inhibitors (p < 0.01). The response to PMA was not significantly affected by 10 μm rottlerin (p > 0.05 versus PMA and p < 0.001 versus control). PKC Isozymes Responsible for mGluR1-mediated Phosphorylation of Kv3.1b Channels in CHO Cells—It is known that activation of mGluR1 leads to PKC activation, through increases in membrane-bound diacylglycerol and intracellular inositol 1,4,5-trisphosphate level. In CHO cells cotransfected with mGluR1 and Kv3.1b channels, treatment with the mGluR1 agonist DHPG (300 μm) led to an increase in the phosphorylation level of Kv3.1b channels (Fig. 3). This effect was partially inhibited by the cPKC inhibitor Gö6976 (1 μm), was fully blocked by the n- and c-PKC inhibitor GF109203X (1 μm) and the group I/II mGluR antagonist MCPG (1 mm), but was not affected by the PKC-δ inhibitor rottlerin (10 μm). Quantification of the phospho-Kv3.1b immunoreactivity showed that DHPG treatment of mGluR1, Kv3.1b-cotransfected CHO cells produced a very marked increase in the phosphorylation level over control cells that were transfected with Kv3.1b and pcDNA vector alone (p < 0.001) (Fig. 3B). The effect of DHPG was significantly attenuated by the group I inhibitor Gö6976 (1 μm, p < 0.001 versus DHPG and p < 0.05 versus control). The group II inhibitor GF109203X (1 μm) completely abolished the effect of DHPG (p < 0.001 versus DHPG and p > 0.05 versus control). The effect of GF109203X was more potent than Gö6976 (p < 0.05). The response to DHPG was not significantly affected by the nPKC-δ inhibitor rottlerin (10 μm, p > 0.05 versus DHPG and p < 0.001 versus control). PKC Isozymes That Regulate the Basal Phosphorylation of Kv3.1b in Brainstem Slices—The PKC isozymes that have been identified in the brain and spinal cord are cPKC-α, -βI, -βII, -γ, and nPKC-δ, -ε, and aPKC-ζ (21Tanaka C. Nishizuka Y. Annu. Rev. Neurosci. 1994; 17: 551-567Crossref PubMed Scopus (509) Google Scholar, 36Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. FEBS Lett. 1987; 226: 125-128Crossref PubMed Scopus (134) Google Scholar). As shown in Fig. 4, dense basal phosphorylation of Kv3.1b potassium channels at Ser-503 was observed in MNTB neurons in brainstem slices of 17–19-day-old rats. The basal phosphorylation was diminished by pretreatment with the group I inhibitor, 1 μm Gö6976, and was completely abolished by the group II inhibitor, 1 μm GF109203X. In contrast to its lack of effect in PMA-treated CHO cells, rottlerin (10 μm), an inhibitor of nPKC-δ, also reduced the basal phosphorylation of Kv3.1b channels in MNTB neurons. Quantification showed that the basal Kv3.1b phosphorylation in MNTB neurons in control slices was reduced to 40% of control levels by 1 μm Gö6976 (p < 0.001 versus control) and to less than 6% of control by GF109203X (1 μm)(p < 0.0001 versus control and p < 0.001 versus Gö6976). Rottlerin (10 μm) reduced the basal staining to 71% of the control (p < 0.05). These data indicate that both cPKCs and nPKCs contribute to the basal phosphorylation of Kv3.1b channels in MNTB neurons and that nPKCs account for at least 34% of the basal staining. Because inhibition of PKC-δ alone by rottlerin produced a smaller inhibition of the basal staining, these results suggest that both nPKC-δ and nPKC-ε regulate the basal level of Kv3.1b phosphorylation. PKC Isozymes That Mediate PMA-induced Phosphorylation of Kv3.1b in MNTB Neurons—We next tested the actions of PKC inhibitors on PMA-induced phosphorylation of Kv3.1b channels in MNTB neurons. As shown in Fig. 5, treatment of brainstem slices with PMA (700 nm) significantly enhanced Kv3.1b phosphorylation in MNTB neurons. The group I inhibitors (100 nm Gö6976, 1 μm Gö6976, 50 nm Ro 31-7549, or 50 nm GF109203X) diminished Ser-503 phosphorylation but did not abolish it. In contrast, group II inhibitors (4 μm Ro 31-7549 or 1 μm GF109203X) completely abolished K3.1b phosphorylation in the presence of PMA, suggesting that both cPKCs and nPKCs contribute to PMA-induced phosphorylation of Kv3.1b channels. In contrast to the case of basal phosphorylation in MNTB neurons, the PKC-δ inhibitor rottlerin (10 μm) had no effect" @default.
• W2066063313 created "2016-06-24" @default.
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• W2066063313 date "2006-06-01" @default.
• W2066063313 modified "2023-10-14" @default.
• W2066063313 title "Modulation of Kv3.1b Potassium Channel Phosphorylation in Auditory Neurons by Conventional and Novel Protein Kinase C Isozymes" @default.
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