Calcium Influx through L-type Channels Generates Protein Kinase M to Induce Burst Firing of Dopamine Cells in the Rat Ventral Tegmental Area
2007; Elsevier BV; Volume: 282; Issue: 12 Linguagem: Inglês
10.1074/jbc.m610230200
ISSN1083-351X
AutoresYudan Liu, Jules J.E. Doré, Xihua Chen,
Tópico(s)Neurotransmitter Receptor Influence on Behavior
ResumoEnhanced activity of the dopaminergic system originating in the ventral tegmental area is implicated in addictive and psychiatric disorders. Burst firing increases dopamine levels at the synapse to signal novelty and salience. We have previously reported a calcium-dependent burst firing of dopamine cells mediated by L-type channels following cholinergic stimulation; this paper describes a cellular mechanism resulting in burst firing following L-type channel activation. Calcium influx through L-type channels following FPL 64176 or (S)-(–)-Bay K8644 induced burst firing independent of dopamine, glutamate, or calcium from the internal stores. Burst firing induced as such was completely blocked by the substrate site protein kinase C (PKC) inhibitor chelerythrine but not by the diacylglycerol site inhibitor calphostin C. Western blotting analysis showed that FPL 64176 and (S)-(–)-Bay K8644 increased the cleavage of PKC to generate protein kinase M (PKM) and the specific calpain inhibitor MDL28170 blocked this increase. Prevention of PKM production by inhibiting calpain or depleting PKC blocked burst firing induction whereas direct loading of purified PKM into cells induced burst firing. Activation of the N-methyl-d-aspartic acid type glutamate or cholinergic receptors known to induce burst firing increased PKM expression. These results indicate that calcium influx through L-type channels activates a calcium-dependent protease that cleaves PKC to generate constitutively active and labile PKM resulting in burst firing of dopamine cells, a pathway that is involved in glutamatergic or cholinergic modulation of the central dopamine system. Enhanced activity of the dopaminergic system originating in the ventral tegmental area is implicated in addictive and psychiatric disorders. Burst firing increases dopamine levels at the synapse to signal novelty and salience. We have previously reported a calcium-dependent burst firing of dopamine cells mediated by L-type channels following cholinergic stimulation; this paper describes a cellular mechanism resulting in burst firing following L-type channel activation. Calcium influx through L-type channels following FPL 64176 or (S)-(–)-Bay K8644 induced burst firing independent of dopamine, glutamate, or calcium from the internal stores. Burst firing induced as such was completely blocked by the substrate site protein kinase C (PKC) inhibitor chelerythrine but not by the diacylglycerol site inhibitor calphostin C. Western blotting analysis showed that FPL 64176 and (S)-(–)-Bay K8644 increased the cleavage of PKC to generate protein kinase M (PKM) and the specific calpain inhibitor MDL28170 blocked this increase. Prevention of PKM production by inhibiting calpain or depleting PKC blocked burst firing induction whereas direct loading of purified PKM into cells induced burst firing. Activation of the N-methyl-d-aspartic acid type glutamate or cholinergic receptors known to induce burst firing increased PKM expression. These results indicate that calcium influx through L-type channels activates a calcium-dependent protease that cleaves PKC to generate constitutively active and labile PKM resulting in burst firing of dopamine cells, a pathway that is involved in glutamatergic or cholinergic modulation of the central dopamine system. Dopaminergic (DA) 2The abbreviations used are: DA, dopamine; PKC, protein kinase C; PKM, protein kinase M; VTA, ventral tegmental area; CaMKII, Ca2+-calmodulin-dependent kinase II; PKA, protein kinase A; ACSF, artificial cerebrospinal fluid; PMA, phorbol 12-myristate 13-acetate; ISI, interspike interval; CV, coefficient of variance; CPA, cyclopiazonic acid; SERCA, sarco-endoreticulum Ca2+-dependent ATPase; DAT, dopamine transporter; NMDA, N-methyl-d-aspartic acid; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; GABAA, γ-aminobutyric acid, type A.2The abbreviations used are: DA, dopamine; PKC, protein kinase C; PKM, protein kinase M; VTA, ventral tegmental area; CaMKII, Ca2+-calmodulin-dependent kinase II; PKA, protein kinase A; ACSF, artificial cerebrospinal fluid; PMA, phorbol 12-myristate 13-acetate; ISI, interspike interval; CV, coefficient of variance; CPA, cyclopiazonic acid; SERCA, sarco-endoreticulum Ca2+-dependent ATPase; DAT, dopamine transporter; NMDA, N-methyl-d-aspartic acid; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; GABAA, γ-aminobutyric acid, type A. projections from the ventral tegmental area (VTA) constitute the mesolimbocortical system that underlies drug abuse and schizophrenia, primarily as the result of increased DA transmission (1Kelley A.E. 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DA cells in slices predominantly display pacemaker-like firing; burst firing can be induced by surrogate synaptic stimulation with bath application of glutamatergic or cholinergic agonists (11Johnson S.W. Seutin V. North R.A. Science. 1992; 258: 665-667Crossref PubMed Scopus (367) Google Scholar, 12Zhang L. Liu Y. Chen X. J. Physiol. (Lond.). 2005; 568: 469-481Crossref Scopus (41) Google Scholar) that mobilize Ca2+. Ca2+ entry has been shown to be pivotal in regulating firing patterns of DA neurons. Intracellular administration of Ca2+ evokes burst firing, while intracellular Ca2+ chelators block it (13Grace A.A. Bunney B.S. J. Neurosci. 1984; 4: 2877-2890Crossref PubMed Google Scholar). Our previous study shows that carbachol, a general cholinergic agonist, induces burst firing primarily by promoting Ca2+ influx through L-type channels (12Zhang L. Liu Y. Chen X. J. Physiol. (Lond.). 2005; 568: 469-481Crossref Scopus (41) Google Scholar). These channels are responsible for approximately one third of total Ca2+ currents of DA neurons (14Cardozo D.L. Bean B.P. J. Neurophysiol. 1995; 74: 1137-1148Crossref PubMed Scopus (117) Google Scholar, 15Takada M. Kang Y. Imanishi M. Eur. J. Neurosci. 2001; 13: 757-762Crossref PubMed Scopus (43) Google Scholar, 16Durante P. Cardenas C.G. Whittaker J.A. Kitai S.T. Scroggs R.S. J. Neurophysiol. 2004; 91: 1450-1454Crossref PubMed Scopus (66) Google Scholar) and contribute preferentially to whole-cell Ca2+ currents evoked by small depolarizations (16Durante P. Cardenas C.G. Whittaker J.A. Kitai S.T. Scroggs R.S. J. Neurophysiol. 2004; 91: 1450-1454Crossref PubMed Scopus (66) Google Scholar, 17Xu W. Lipscombe D. J. Neurosci. 2001; 21: 5944-5951Crossref PubMed Google Scholar). In line with this, L-type channels have been shown to be involved in spontaneous and burst firing (12Zhang L. Liu Y. Chen X. J. Physiol. (Lond.). 2005; 568: 469-481Crossref Scopus (41) Google Scholar, 18Nedergaard S. Flatman J.A. Engberg I. J. Physiol. (Lond.). 1993; 466: 727-747Google Scholar, 19Mercuri N.B. Bonci A. Calabresi P. Stratta F. Stefani A. Bernardi G. Br. J. Pharmacol. 1994; 113: 831-838Crossref PubMed Scopus (126) Google Scholar, 20Johnson S.W. Wu Y.N. Brain Res. 2004; 1019: 293-296Crossref PubMed Scopus (51) Google Scholar). Activation of L-type channels has been shown to modulate synaptic strength in DA cells (21Bonci A. Grillner P. Mercuri N.B. Bernardi G. J. Neurosci. 1998; 18: 6693-6703Crossref PubMed Google Scholar). Additionally, L-type channels gate Ca2+-release from internal Ca2+ stores, activate plasma membrane Ca2+-dependent K+ channels, as well as several Ca2+-dependent kinases such as protein kinase C (PKC), Ca2+-calmodulin-dependent kinase II (CaMKII), and protein kinase A (PKA) (22Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4141) Google Scholar). These kinases are capable of phosphorylating a variety of ion channels to regulate the excitability of neurons (23Levitan I.B. Nat. Neurosci. 2006; 9: 305-310Crossref PubMed Scopus (102) Google Scholar). Finding the mechanism that controls the firing mode of DA cells would provide a vital means of modulating the system in both normal and disease conditions. Here, we present results that Ca2+ influx through L-type channels activates a Ca2+-dependent protease, which in turn cleaves PKC to generate a labile fragment that is constitutively active (termed protein kinase M, PKM) to induce burst firing in DA neurons. Slice Preparation—All procedures involving animal handling and tissue harvesting were in accordance with guidelines set by the Institutional Animal Care Committee at the Memorial University of Newfoundland. Sprague-Dawley rat pups (9–21 days old) of either sex were deeply anesthetized with halothane and killed by chest compression. The skull was quickly opened to expose the brain, which was cooled in situ with ice-cold, carbogenated artificial cerebrospinal fluid (ACSF, composition: 126 mm NaCl, 2.5 mm KCl, 1.2 mm NaH2PO4, 1.2 mm MgCl2, 2.4 mm CaCl2, 18 mm NaHCO3, and 11 mm glucose, pH 7.4, when bubbled with 95% O2 and 5% CO2). The brain was removed, and a block containing the midbrain was cut on a Leica vibratome (VT 1000, Heidelberger, Germany). Tissue slices were allowed to recover at room temperature (22 °C) in carbogenated ACSF for at least 1 h prior to recording. Slices were further trimmed to fit into a recording chamber and continuously perfused with carbogenated ACSF at a rate of 2–3 ml min–1 at room temperature. For PKC depletion experiments, one of the two VTA slices from the same animal was incubated in ACSF with or without 1–2 μm phorbol 12-myristate 13-acetate (PMA) for 20–24 h at room temperature in a partially sealed beaker continuously bubbled with carbogen. Patch Clamp Recording—All recordings were made from the VTA identified under a dissecting microscope (Leica MZ6). Patch electrodes were prepared from KG-33 glass micropipettes (OD 1.5 mm, Garner Glass CO., Claremont, CA) on a P-97 Brown-Flaming micropipette puller (Sutter Instruments, Novato, CA). For nystatin-perforated patch clamp recording, glass electrodes were filled to the tip with intracellular solution (120 mm potassium acetate, 40 mm HEPES, 5 mm MgCl2, and 10 mm EGTA with pH adjusted to 7.35 using 0.1 n KOH) and then back-filled with the same solution containing 450 μg ml–1 nystatin and Pluronic F127, yielding a tip resistance of 4–8 MΩ. For conventional whole-cell recording, electrodes were filled with a solution containing 126 mm potassium gluconate, 10 mm KCl, 0.2 mm EGTA, 10 mm HEPES, 4 mm MgATP, and 0.3 mm Na3GTP with pH adjusted to 7.35 and osmolarity to 295 mosm, yielding a resistance of 2–4 MΩ. Gigaohm seals were made using a Warner PC-505B (Warner Instruments Inc., Hamden, CT) or a MultiClamp 700B (Axon Instruments, Foster City, CA) amplifier. Signals were sampled at 5 kHz and digitized by DigiData 1320A using pCLAMP (versions 8 and 9) software (Axon Instruments). Selection of nystatin-perforated cell recordings in current clamp mode was determined by the size of the action potential, since many VTA cells were spontaneously active. After adequate partitioning of nystatin into the membrane, action potentials overshot 0 mV and measured at least 50 mV. Quality of conventional whole-cell recordings was assessed by a brief voltage step (–20 mV, 10 ms) from the holding potential (–55 mV). Only cells that had an access resistance of 200 MΩ were included. Cells whose access resistance increased significantly during the course of recording (>20%) were discarded. Episodic protocols were used to induce hyperpolarization activated current (Ih) and derive passive characteristics of the cell such as current-voltage relationship and input resistance. Current pulses for Ih induction were of 1 s duration, and the intervals between pulses were 8 s to allow complete recovery of Ih channels. Cells that displayed a prominent Ih and an apparent DA-induced hyperpolarization were identified as putative DA cells (11Johnson S.W. Seutin V. North R.A. Science. 1992; 258: 665-667Crossref PubMed Scopus (367) Google Scholar, 12Zhang L. Liu Y. Chen X. J. Physiol. (Lond.). 2005; 568: 469-481Crossref Scopus (41) Google Scholar, 24Lacey M.G. Mercuri N.B. North R.A. J. Neurosci. 1989; 9: 1233-1241Crossref PubMed Google Scholar). Components of extracellular and intracellular solutions were purchased from bulk distributors Fisher Scientific (Nepean, Ontario, Canada) and VWR International (Missisauga, Ontario, Canada). All other chemicals were obtained from Sigma and Tocris (Ellisville, MO). Chemicals were dissolved in deionized water or Me2SO as required. Aliquots of stock solutions were kept at –30 °C. Prior to application, an aliquot was diluted to working concentration and applied to the ACSF bath. PKM (Sigma) was kept at –80 °C and was diluted to 1 unit ml–1 immediately before use in the internal solution for conventional whole-cell recording. DA solution was made fresh daily with an equimolar concentration of the antioxidant disodium metabisulfite. Western Blotting—Phosphorylated PKC and PKM were detected by a phospho-PKC (pan) antibody (Cell Signaling) that recognizes PKCα,-βI, -βII, -δ,-ϵ,-η, and -θ isoforms only when phosphorylated at a carboxyl-terminal residue homologous to Ser660 of PKCβII. Ser-660 is in the catalytic domain of PKC and allows simultaneous visualization of phosphorylated PKC and PKM. VTA slices from the same animal were hemisected to give equivalent halves for control and experimental groups. Slices were trimmed to contain the VTA and part of the substantia nigra and were homogenized in RIPA (1× phosphate-buffered saline, 1% Triton X-100, 50 mm Tris-HCl, pH 7.4, 0.5% deoxycholate, 50 mm β-glycerophosphate, 50 mm sodium fluoride, 5 mm EDTA, 0.1% sodium orthovanadate, 0.1% SDS, 75 ng/ml phenylmethylsulfanyl fluoride and 1× complete protease inhibitor (Roche Diagnostics, Quebec, Canada)). Insoluble materials were removed by centrifugation (15,000 × g for 10 min), and protein concentration for each sample was determined by BCA™ assay using bovine serum albumin standard (Pierce). Equivalent mass of total protein from slice homogenates were separated on an 8.5% acrylamide gel and transferred to a polyvinylidene fluoride membrane. The blots were blocked with Blotto (5% w/v nonfat milk in TBST (15 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% (v/v) Tween 20); 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% Tween 20) for 1 h at room temperature, then incubated with phospho-PKC (pan) antibody (1: 1,000) diluted in Blotto, rocking overnight at 4 °C. Blots were washed five times in TBST and followed by goat anti-rabbit horseradish peroxidase-conjugated IgG diluted in Blotto (Cell Signaling, 1:15,000) for 90 min at room temperature. After five washes in TBST, blots were incubated in enhanced SuperSignal West Pico chemiluminescent substrate (Pierce) to visualize specific immune complexes using hyperfilm x-ray film (Amersham Biosciences). Blots were then stripped in a solution of 2% SDS, 63 mm Tris-HCl, pH 6.8, and 0.1 m β-mercaptoethanol for 30 min at 50 °C. Stripping solution was removed by two washes with TBST at room temperature, followed by blocking with Blotto and incubation with anti-β-actin antibody (Sigma, 1:4,000) to act as loading controls. Quantification was obtained from densitometric measurements of immunoreactive bands using Gel Logic100 imaging system with Gel Logic200 Software (Eastman Kodak Co.). Data Analysis—Electrophysiological data were analyzed off-line with Mini Analysis (Synaptosoft Inc., Decatur, GA) and pCLAMP software. Basal firing frequencies were averaged values of at least 5 min stable baseline recording. Ih was measured as the difference in current or voltage between instantaneous and steady-state readings. Analysis of firing behavior was based on interspike intervals (ISIs) measured with the Mini Analysis program. Averaged as well as instantaneous firing frequencies were derived from those intervals. Coefficient of variance (CV) was calculated as the mean of ISIs over a 1-min period divided by their standard deviation. To compare CV values between cell groups, they were normalized against the mean CV value of the first 5 min. Relative density of ISIs in 2-s bins were plotted to reveal the distribution of a given ISI series, and the resulting histogram was fitted to a Lowess function. Burst firing was defined as two spikes or more in each bursting cycle at a frequency higher than non-bursting periods and separated by a post-burst hyperpolarization. For Western blotting analysis, density of phospho-PKM and PKC bands were normalized against the density of β-actin from the same sample. Data were expressed as means and S.E. Statistical comparisons of electrophysiological data were performed using two-tailed unpaired Student's t test. Western blotting data were compared using paired t test since each control and experimental pair contained hemisected slices from the same animal. Values were considered significant when p < 0.05. Except when indicated, experiments were done using the nystatinperforated whole-cell recording method at room temperature. Dialysis of PKM into recorded cells was done using conventional whole-cell recording. Only DA cells in the VTA identified according to criteria out-lined under "Experimental Procedures" were included. In nystatinperforated recording, the average hyperpolarization following a brief application of 50 μm DA (within 90 s) was –8.29 ± 0.57 mV (n = 71, excluding cells used for PKC depletion test). Most cells (47 of 71, 66%) were spontaneously active with single spike firing at a low basal firing frequency of 0.41 ± 0.04 Hz; the remainder (24 of 71, 34%) were quiescent during base-line recordings. Opening of L-type Calcium Channels Converts Firing Patterns—Bath application of 1–4 μm FPL 64176, a benzoylpyrole site L-type calcium channel opener, for 5–10 min converted firing patterns from quiescent state or single spiking to burst firing in 80.3% of treated cells (57 of 71), application of 1 μm FPL 64176 induced burst firing in 63.5% cells (33 of 52). The responses were spike-dependent. Percentage of converted burst firing in spontaneously firing cells (41 of 47, 87.2%) was much higher than that in quiescent cells (16 of 24, 66.7%), a response that appeared to be related to their different resting membrane potentials (–49.35 ± 0.54 mV for spiking cells versus –52.52 ± 0.92 mV for quiescent cells, p < 0.05). The responses were dose-dependent: 2 μm FPL 64176 induced burst firing in 9 of 17 cells that did not respond to 1 μm and similarly, 4 μm induced burst firing in 4 of 10 cells that did not respond following a dose of 2 μm. In cells that were spontaneously firing, FPL 64176 first induced a membrane depolarization (2.00 ± 0.38 mV, n = 41) accompanied by a 48.8 ± 9.3% increase in firing rate in the first 5 min of drug application; burst firing started after a varying period of latency. The lag between the start of drug application and burst firing ranged between 5 and 25 min with an average of 13 min (Fig. 1, A and F). Within a burst firing cycle, action potentials were fired with increasing frequencies followed by a pronounced post-burst hyperpolarization (Fig. 1B). The average intra-burst firing frequency (1.12 ± 0.13 Hz, n = 41) was much higher than basal tonic frequency (0.43 ± 0.05 Hz). The development of burst firing in quiescent cells (n = 16) was slightly different: they responded to FPL 64176 with a membrane depolarization (1.38 ± 0.38 mV) followed by a sudden appearance of burst firing (Fig. 1D) or irregular single spiking that evolved into burst firing (Fig. 1E). The L-type channel-mediated burst firing was long lasting even after prolonged washout up to 3 h; however, it was readily inhibited by L-type channel blocker nifedipine at a range of doses (1–10 μm, n = 29). Density plots of ISIs in 2-s bins showed an ISI distribution that fits to a single Gaussian distribution under control conditions and to a mixed Gaussian distribution following FPL 64176 application (Fig. 1C). The leftward shift of the main peak indicates higher firing frequencies and the second peak at much lower frequency represents the long pauses of firing between adjacent bursts. Bath application of 5 μm (S)-(–)-Bay K8644, a dihydropyridine site L-type channel activator, also induced burst firing in three of five cells tested (Fig. 1G). The time course of the response was similar to that following FPL 64176, a depolarization and an increase in firing rates followed by a conversion of firing patterns 10–25 min after the start of application. FPL 64176 Induces Burst Firing Independent of an Intermediate Transmitter—L-type channels have been shown to enhance a slow NMDA current in DA neurons (21Bonci A. Grillner P. Mercuri N.B. Bernardi G. J. Neurosci. 1998; 18: 6693-6703Crossref PubMed Google Scholar), suggesting that L-type channel opening could induce burst firing by promoting glutamate transmission. Because FPL 64176-induced burst firing was long lasting, we examined the involvement of synaptic mechanisms in two ways: whether induced burst firing persisted in the presence of a mixture containing 100 μm APV, 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione, and 100 μm picrotoxin (blocking NMDA, AMPA, and GABAA receptors, respectively) and whether pretreatment with this mixture altered the ability of FPL 64176 to induce burst firing. FPL 64176-induced burst firing was robust following the application of the mixture for 10–30 min (n = 7, Fig. 2A). Similarly, cells that were treated with the mixture for 5–10 min prior to FPL 64176 (1 μm) application in the presence of the mixture still responded with burst firing (n = 3). These results suggest that L-type channel opening does not induce burst firing either directly through increased glutamatergic transmission or indirectly by way of GABAergic interneurons in the VTA. Activation of L-type channels may release DA from the soma and dendrites, since this release has been shown to be Ca2+-dependent (25Chen B.T. Rice M.E. J. Neurosci. 2001; 21: 7841-7847Crossref PubMed Google Scholar, 26Beckstead M.J. Grandy D.K. Wickman K. Williams J.T. Neuron. 2004; 42: 939-946Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). DA acting at somatodendritic autoreceptors forms the short loop negative feedback to regulate the excitability of DA cells, we therefore examined whether D2 receptors were involved in burst firing. The D2 receptor antagonist sulpiride (1–10 μm) applied for 10–15 min to 5 burst firing cells induced by FPL 64176 had no effect on firing patterns (Fig. 2B). Two cells to which 10 μm sulpiride was applied for 8 min prior to FPL 64176 application in the presence of sulpiride displayed burst firing similar to that induced by FPL 64176 alone. These data suggest that L-type channel activation does not result in somatodendritic DA release to induce burst firing. Internal Ca2+ Stores Are Not Involved—Ca2+ entry through L-type channels gates ryanodine receptors on internal Ca2+ stores to mediate Ca2+-induced Ca2+ release (22Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4141) Google Scholar), we tested whether this was involved in burst firing. Cyclopiazonic acid (CPA) and thapsigargin were used to inhibit the sarco-endoreticulum Ca2+-dependent ATPase (SERCA) that refills internal Ca2+ stores. After burst firing was induced by FPL 64176, CPA (20–30 μm) applied for 20–60 min (n = 4) or thapsigargin (1–2 μm) applied for 50–70 min (n = 3) did not alter the induced burst firing (Fig. 2C). In three cells that were pretreated with CPA (20 μm) for 30 min, FPL 64176 (1 μm) was still able to induce strong burst firing. These results indicate that internal Ca2+ stores are not necessary for L-type channel-mediated burst firing. Ca2+-dependent Protein Kinase Mediates Burst Firing—Increased intracellular Ca2+ activates Ca2+-sensitive protein kinases such as PKC, PKA, and CaMKII that can phosphorylate ion channels including L-type channels themselves and regulate the excitability of neurons (27Dzhura I. Wu Y. Colbran R.J. Balser J.R. Anderson M.E. Nat. Cell Biol. 2000; 2: 173-177Crossref PubMed Scopus (300) Google Scholar, 28Young C.E. Yang C.R. J. Neurosci. 2004; 24: 8-23Crossref PubMed Scopus (65) Google Scholar, 29Lee T.S. Karl R. Moosmang S. Lenhardt P. Klugbauer N. Hofmann F. Kleppisch T. Welling A. J. Biol. Chem. 2006; 281: 25560-25567Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 30Yang L. Liu G. Zakharov S.I. Morrow J.P. Rybin V.O. Steinberg S.F. Marx S.O. J. Biol. Chem. 2005; 280: 207-214Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). After burst firing was induced by FPL 64176, bath application of the PKA inhibitor H-89 (5 or 10 μm) for 40–130 min (n = 4), the CaMKII inhibitor KN-93 (1–10 μm) for 25–100 min (n = 6), or both for 60–70 min (n = 3) did not block burst firing (Fig. 3A). The substrate site PKC inhibitor, chelerythrine (40 μm), applied for 15–44 min (n = 6) completely blocked FPL 64176-induced burst firing and the accompanying membrane potential oscillation. This blockade was reversible since burst firing reappeared after washing out chelerythrine for 18–33 min (Fig. 3B). To establish the role of PKC in burst firing, we further used an inhibitor that binds to the diacylglycerol site of the regulatory domain. Calphostin C (1–2 μm) applied for 40–180 min (n = 4) was largely ineffective except in one cell where burst firing slowed marginally but persisted (Fig. 3C). These results suggest that the catalytic subunit of PKC itself or a kinase with a similar substrate site is involved in burst firing. Proteolytic Cleavage of PKC Mediates Burst Firing—To solve the apparent inconsistency between the two PKC inhibitors, we considered other modes of PKC activation. Ca2+ entry has been shown to activate the protease calpain that proteolytically cleaves PKC to form PKM which has been shown to be constitutively active with a very short half-life within the cell (31Kishimoto A. Kajikawa N. Shiota M. Nishizuka Y. J. Biol. Chem. 1983; 258: 1156-1164Abstract Full Text PDF PubMed Google Scholar, 32Al Z. Cohen C.M. Biochem. J. 1993; 296: 675-683Crossref PubMed Scopus (44) Google Scholar, 33Cressman C.M. Mohan P.S. Nixon R.A. Shea T.B. FEBS Lett. 1995; 367: 223-227Crossref PubMed Scopus (64) Google Scholar, 34Shea T.B. Beermann M.L. Griffin W.R. Leli U. FEBS Lett. 1994; 350: 223-229Crossref PubMed Scopus (50) Google Scholar). We therefore tested whether proteolytic cleavage of PKC could explain the inconsistency in response to the two PKC inhibitors. Cells were induced to burst fire by FPL 64176; the calpain inhibitor MDL 28170 (200 μm) was then applied. In all cells tested as such, burst firing reverted to single spike firing in 25–40 min and resumed after washing 20–30 min (n = 5, Fig. 4A). Prior application of MDL 28170 (200 μm) for 30–40 min completely prevented FPL 64176-induced (2–4 μm) burst firing (n = 3). To further validate the role of PKM in L-type channel-induced burst firing, we compared the levels of phospho-PKC and PKM by Western blotting total protein lysates from slices that were treated with (S)-(–)-Bay K8644 or FPL 64176 alone and with MDL 28170 followed by FPL 64176. As shown in Fig. 4B, phospho-PKC antibody raised against the catalytic domain visualized three bands: two at ∼82 kDa corresponding to intact PKC isoforms and another at 45 kDa corresponding to the PKC catalytic unit or PKM. Application of L-type channel opener FPL 64176 (2 μm) or (S)-(–)-Bay K8644 (5 μm) for 30 min caused a considerable increase in phospho-PKM expression (Fig. 4C). Densitometry of the bands corresponding to phospho-PKM showed that (S)-(–)-Bay K 8644 (224 ± 61% relative to untreated values, n = 8, p < 0.05) or FPL 64176 (244 ± 51% relative to untreated values, n = 6, p < 0.05) significantly increased phospho-PKM levels (Fig. 4D). The increase by FPL 64176 could be completely blocked by prior treatment with the calpain inhibitor MDL 28170 (200 μm for 30 min; 8 ± 1% of FPL 64176 alone, n = 5, p < 0.0001; Fig. 4, C and D). There were no detectable changes in full-length phospho-PKC levels following (S)-(–)-Bay K8644 (102 ± 10% of control values, n = 8, p > 0.05), FPL 64176 (104 ± 6% of control values, n = 6, p > 0.05) o
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