Dopamine D4 Receptors Regulate GABAA Receptor Trafficking via an Actin/Cofilin/Myosin-dependent Mechanism
2009; Elsevier BV; Volume: 284; Issue: 13 Linguagem: Inglês
10.1074/jbc.m807387200
ISSN1083-351X
AutoresNicholas Graziane, Eunice Y. Yuen, Zhen Yan,
Tópico(s)Receptor Mechanisms and Signaling
ResumoThe GABAA receptor-mediated inhibitory transmission in prefrontal cortex (PFC) is implicated in cognitive processes such as working memory. Our previous study has found that GABAAR current is subject to the regulation of dopamine D4 receptors, a PFC-enriched neuromodulator critically involved in various mental disorders associated with PFC dysfunction. In this study, we have investigated the cellular mechanism underlying D4 modulation of GABAARs. We found that the density of surface clusters of GABAAR β2/3 subunits was reduced by D4, suggesting that the D4 reduction of GABAAR current is associated with a decrease in functional GABAARs at the plasma membrane. Moreover, the D4 reduction of GABAAR current was blocked by the actin stabilizer phalloidin and was occluded by the actin destabilizer latrunculin, suggesting that D4 regulates GABAAR trafficking via an actin-dependent mechanism. Cofilin, a major actin depolymerizing factor whose activity is strongly increased by dephosphorylation at Ser3, provides the possible link between D4 signaling and the actin dynamics. Because myosin motor proteins are important for the transport of vesicles along actin filaments, we also tested the potential involvement of myosin in D4 regulation of GABAAR trafficking. We found that dialysis with a myosin peptide, which competes with endogenous myosin proteins for actin-binding sites, prevented the D4 reduction of GABAAR current. These results suggest that D4 receptor activation increases cofilin activity presumably via its dephosphorylation, resulting in actin depolymerization, thus causing a decrease in the myosin-based transport of GABAAR clusters to the surface. The GABAA receptor-mediated inhibitory transmission in prefrontal cortex (PFC) is implicated in cognitive processes such as working memory. Our previous study has found that GABAAR current is subject to the regulation of dopamine D4 receptors, a PFC-enriched neuromodulator critically involved in various mental disorders associated with PFC dysfunction. In this study, we have investigated the cellular mechanism underlying D4 modulation of GABAARs. We found that the density of surface clusters of GABAAR β2/3 subunits was reduced by D4, suggesting that the D4 reduction of GABAAR current is associated with a decrease in functional GABAARs at the plasma membrane. Moreover, the D4 reduction of GABAAR current was blocked by the actin stabilizer phalloidin and was occluded by the actin destabilizer latrunculin, suggesting that D4 regulates GABAAR trafficking via an actin-dependent mechanism. Cofilin, a major actin depolymerizing factor whose activity is strongly increased by dephosphorylation at Ser3, provides the possible link between D4 signaling and the actin dynamics. Because myosin motor proteins are important for the transport of vesicles along actin filaments, we also tested the potential involvement of myosin in D4 regulation of GABAAR trafficking. We found that dialysis with a myosin peptide, which competes with endogenous myosin proteins for actin-binding sites, prevented the D4 reduction of GABAAR current. These results suggest that D4 receptor activation increases cofilin activity presumably via its dephosphorylation, resulting in actin depolymerization, thus causing a decrease in the myosin-based transport of GABAAR clusters to the surface. Prefrontal cortex (PFC), 2The abbreviations used are: PFC, prefrontal cortex; GABA, γ-aminobutyric acid; GABAA, GABA, type A; GABAAR, GABAA receptor; ANOVA, analysis of variance; IPSC, inhibitory postsynaptic currents; mIPSC, miniature IPSC; p-cof, p-cofilin; PP1, protein phosphatase 1. a brain region strongly associated with cognitive and emotional processes (1Miller E.K. Neuron. 1999; 22: 15-17Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), is particularly critical for working memory, a mechanism for encoding and maintaining newly acquired, task-relevant information (2Goldman-Rakic P.S. Neuron. 1995; 1: 477-485Abstract Full Text PDF Scopus (1885) Google Scholar). Working memory relies on the coordinated sustained firing of PFC pyramidal neurons between the temporary presentation of a stimulus cue and the later initiation of a behavioral response (2Goldman-Rakic P.S. Neuron. 1995; 1: 477-485Abstract Full Text PDF Scopus (1885) Google Scholar). The synchronization of pyramidal neuron activity during working memory processes is controlled by GABAergic interneurons (3Rao S.G. Williams G.V. Goldman-Rakic P.S. J. Neurosci. 2000; 20: 485-494Crossref PubMed Google Scholar, 4Constantinidis C. Williams G.V. Goldman-Rakic P.S. Nat. Neurosci. 2002; 5: 175-180Crossref PubMed Scopus (271) Google Scholar). 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A. 1996; 93: 1325-1329Crossref PubMed Scopus (521) Google Scholar) or long term treatment with the psychotomimetic drug phencyclidine (27Jentsch J.D. Redmond Jr., D.E. Elsworth J. Taylor J.R. Youngren K.D. Roth R.H. Science. 1997; 277: 953-955Crossref PubMed Scopus (352) Google Scholar, 28Jentsch J.D. Taylor J.R. Redmond Jr., D.E. Elsworth J.D. Youngren K.D. Roth R.H. Psychopharmacology. 1999; 142: 78-84Crossref PubMed Scopus (85) Google Scholar). D4 receptor-deficient mice show reduced novelty seeking and cortical hyperexcitability (29Dulawa S. Grandy D.K. Low M.J. Paulus M. Geyer M. J. Neurosci. 1999; 19: 9550-9556Crossref PubMed Google Scholar, 30Rubinstein M. Cepeda C. Hurst R.S. Flores-Hernandez J. Ariano M.A. Falzone T.L. Kozell L.B. Meshul C.K. Bunzow J.R. Low M.J. Levine M.S. Grandy D.K. J. Neurosci. 2001; 21: 3756-3763Crossref PubMed Google Scholar). To understand the mechanism of D4 actions in PFC, it is important to identify its cellular targets key to PFC functions such as working memory. One of our previous studies has demonstrated that GABAA receptors are subject to D4 regulation in PFC pyramidal neurons (31Wang X. Zhong P. Yan Z. J. Neurosci. 2002; 22: 9185-9193Crossref PubMed Google Scholar). In this study, we have revealed the mechanism underlying this regulation. Acute Dissociation Procedure and Primary Culture Preparation-PFC neurons from young adult (3–4 weeks postnatal) rats were acutely dissociated using procedures described previously (32Feng J. Cai X. Zhao J.H. Yan Z. J. Neurosci. 2001; 21: 6502-6511Crossref PubMed Google Scholar, 33Chen G. Greengard P. Yan Z. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2596-2600Crossref PubMed Scopus (181) Google Scholar). All of the experiments were carried out with the approval of State University of New York at Buffalo Animal Care Committee. After incubation of brain slices in NaHCO3-buffered saline, PFC was dissected and placed in an oxygenated chamber containing papain (0.8 mg/ml; Sigma) in HEPES-buffered Hanks' balanced salt solution (Sigma) at room temperature. After 35 min of enzyme digestion, the tissue was rinsed three times with a low Ca2+ saline and mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35-mm Lux Petri dish, which was then placed on the stage of a Zeiss Axiovert S100 inverted microscope. Rat PFC cultures were prepared as previously described (34Cai X. Gu Z. Zhong P. Ren Y. Yan Z. J. Biol. Chem. 2002; 277: 36553-36562Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Briefly, PFC was dissected from 18-day rat embryos, and the cells were dissociated by incubating with 0.25% trypsin for 30 min and subsequent trituration through a Pasteur pipette cells. The cells were plated on coverslips (coated with poly-l-lysine) in Dulbecco's modified Eagle's medium with 10% fetal calf serum at a density of 0.75 × 105 cells/cm2. When neurons attached to the coverslip within 4 h, the medium was changed to Neurobasal with B27 supplement. The neurons were maintained for 2–3 weeks before being used. Whole Cell Recording of Ionic Currents-Pyramidal neurons located in the intermediate and deep layers (III–VI) of the rat PFC were recorded. Recordings of whole cell GABAAR-mediated currents used standard voltage clamp techniques (31Wang X. Zhong P. Yan Z. J. Neurosci. 2002; 22: 9185-9193Crossref PubMed Google Scholar). The internal solution consisted of 180 mm N-methyl-d-glucamine, 40 mm HEPES, 4 mm MgCl2, 0.5 mm 1,2-bis(2-aminohenoxy)ethane-N,N,N′,N′-tetraacetic acid, 12 mm phosphocreatine, 2 mm Na2ATP, 0.2 mm Na3GTP, 0.1 mm leupeptin, pH 7.3, 270 mosm/liter. The external solution consisted of 135 mm NaCl, 20 mm CsCl, 1 mm MgCl2, 10 mm HEPES, 5 mm BaCl2, 10 mm glucose, 0.001 mm tetrodotoxin, pH 7.3, 300 mosm/liter. Recordings were obtained using an Axopatch 200B amplifier that is controlled and monitored with a computer running pClamp 8 with a DigiData 1320 series interface. Electrode resistances were typically 2–4 MΩ in the bath. After seal rupture, series resistance (4–10 MΩ) was compensated (70–90%) and periodically monitored. The cell membrane potential was held at 0 mV. GABA (50 μm) was applied for 2 s every 30 s to minimize desensitization-induced decrease of current amplitude. Drugs were applied using a gravity-fed "sewer pipe" system. The array of application capillaries (∼150-μm inner diameter) was positioned a few hundred micrometers from the cell being recorded. Solution changes were affected by the SF-77B fast-step solution stimulus delivery device (Warner Instruments, Hamden, CT). Data analyses were performed with Clampfit (Axon Instruments, Sunnyvale, CA) and Kaleidagraph (Albeck Software, Reading, PA). For analysis of statistical significance, ANOVA tests were performed to compare the differential degrees of current modulation between groups subjected to different treatments. Electrophysiological Recording of Synaptic Currents-Recording of miniature inhibitory postsynaptic currents (mIPSC) in cultured PFC neurons (days in vitro 12–14) used the whole cell patch technique. Electrodes (3–5 MΩ) were filled with the following internal solution: 100 mm CsCl, 30 mm N-methyl-d-glucamine, 10 mm HEPES, 4 mm NaCl, 1 mm MgCl2 5 mm EGTA, 5 mm MgATP, 0.5 mm Na2GTP, 12 mm phosphocreatine, 0.2 mm leupeptin, 2 mm QX-314, pH 7.2–7.3, 265–270 mosm/liter. Oxygenated artificial cerebral spinal fluid (130 mm NaCl, 3 mm KCl, 5 mm MgCl2, 1 mm CaCl2, 26 mm NaHCO3, 1.25 mm NaH2PO4, 10 mm glucose, pH 7.4, 300 mosm/liter) was used as the external solution. Tetrodotoxin (0.5 μm), D-AP5 (20 μm), and 6,7-dinitroquinoxaline-2,3-dione (20 μm) were added to cultures to block action potentials N-methyl-d-aspartic acid and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors, respectively. The cell membrane potential was held at –70 mV. A mini analysis program (Synaptosoft, Leonia, NJ) was used to analyze the spontaneous synaptic events. For each different condition, mIPSC recordings of 8 min were used for analysis. Statistical comparisons of the amplitude and frequency of mIPSC were made using the Kolmogorov-Smirnov test. Recording of evoked IPSC in PFC slices used the same internal solution as what was used for mIPSC recording in cultures. The slice (300 μm) was placed in a perfusion chamber attached to the fixed-stage of an upright microscope (Olympus) and submerged in continuously flowing oxygenated artificial cerebral spinal fluid containing D-AP5 (20 μm) and DNQX (20 μm). The cells were visualized with a 40× water immersion lens and illuminated with near infrared light, and the image was detected with an infrared-sensitive CCD camera (Olympus, Center Valley, PA). A Multiclamp 700A amplifier was used for slice recordings (Axon Instruments). Tight seals (2–10 GΩ) from visualized pyramidal neurons were obtained by applying negative pressure. The membrane was disrupted with additional suction and the whole cell configuration was obtained. The access resistances ranged from 13 to 18 mΩ and were compensated 50–70%. The cells were held at –70 mV. Clampfit (Axon Instruments) was used to analyze evoked synaptic activity. The agents used such as N-(methyl)-4-(2-cyanophenyl)piperazinyl-3-methybenzamide maleate (PD168077; Tocris, Ballwin, MO), colchicine, phalloidin, latrunculin B (Calbiochem, San Diego, CA), dynamin inhibitory peptide (Tocris, Ballwin, MO), p-cofilin peptide, cofilin peptide, and a scrambled peptide were made up as concentrated stocks in water or Me2SO and stored at –20 °C. The final Me2SO concentration in all applied solutions was <0.1%. No change on GABAAR currents has been observed with this concentration of Me2SO. Stocks were thawed and diluted immediately before use. The amino acid sequence for the myosin peptide is KLFNDPNIGKKGARGKKGKKGRAQKGAN. Immunocytochemical Staining-After treatment, the cultures were fixed in 4% paraformaldehyde for 20 min and incubated in 5% bovine serum for 1 h. For GABAAR surface expression, cultured neurons (nonpermeabilized) were incubated with an antibody against GABAAR β2/3 extracellular region (1:50; Chemicon, Billerica, MA) for 2 h at room temperature. After washing, the neurons were permeabilized with 0.1% Triton for 10 min and then incubated with MAP2 antibody (1:500; Santa Cruz, Santa Cruz, CA) for 2 h at room temperature. Following washing, the neurons were incubated with Alexa 488-conjugated secondary antibody (1:200; Invitrogen) and Alexa 594-conjugated secondary antibody (1:500; Invitrogen) for 1 h at room temperature. After washing, the coverslips were mounted on slides with VECTASHIELD mounting media (Vector Laboratories, Burlingame, CA). Fluorescent images were obtained using a 100× objective with a cooled charge-coupled device camera mounted on a Nikon microscope. All of the specimens were imaged under identical conditions and analyzed with identical parameters using ImageJ software. Control and PD168077-treated neurons with similar MAP2 staining were selected for analysis. To define dendritic clusters, a single threshold was chosen manually, so that clusters corresponded to puncta of at least 1.5-fold intensity of the diffuse fluorescence on the dendritic shaft. Three to four independent experiments for each of the treatments were performed. On each coverslip, the cluster density, cluster size, and cluster fluorescence intensity of several neurons (two or three dendritic segments of at least 20 μm in length/neuron) were measured. Quantitative analyses were conducted blindly (without knowledge of experimental treatment). Western Blots-After treatment, equal amounts of protein from culture homogenates were separated on 7.5% acrylamide gels and transferred to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk for 1 h at room temperature and then were incubated with the anti-p-cofilin (1:250; Cell Signaling, Danvers, MA), anti-cofilin (1:250; Cell Signaling), or anti-actin (1:500; Cell Signaling) for 3 h at room temperature. After washing, the blots were incubated with the horseradish peroxidase-conjugated anti-rabbit antibody (1:1000; Amersham Biosciences) for 2 h at room temperature. After washing, the blots were exposed to the enhanced chemiluminescence substrate. Quantification was obtained from densitometric measurements of immunoreactive bands on films using National Institutes of Health Image software. Activation of D4 Receptors Reduces GABAAR Channel Currents and Surface Expression in PFC Pyramidal Neurons-To examine the impact of dopamine D4 receptors on GABAergic signaling in PFC, we first tested the effect of PD168077, a highly selective D4 receptor agonist (35Glase S.A. Akunne H.C. Georgic L.M. Heffner T.G. MacKenzie R.G. Manley P.J. Pugsley T.A. Wise L.D. J. Med. Chem. 1997; 40: 1771-1772Crossref PubMed Scopus (104) Google Scholar), on whole cell ionic currents mediated by both synaptic and extrasynaptic GABAARs in acutely dissociated PFC pyramidal neurons. GABA (50 μm) application evoked a partially desensitizing outward current in neurons (held at 0 mV) that could be completely blocked by the GABAAR antagonist bicuculline (30 μm; data not shown). As shown in Fig. 1A, application of PD168077 (30 μm) caused a reversible reduction of GABAAR current amplitudes in dissociated PFC pyramidal neurons (16.8 ± 1.7%, n = 15). Consistent with our previous findings (31Wang X. Zhong P. Yan Z. J. Neurosci. 2002; 22: 9185-9193Crossref PubMed Google Scholar), this effect of PD168077 was blocked by the specific D4 antagonist L-74172 (10 μm, data not shown), suggesting the mediation by D4 receptors. To examine the impact of D4 receptors on GABAergic synaptic transmission, we further measured IPSC evoked by electrical stimulation of synaptic GABAA receptors. As shown in Fig. 1B, bath application of PD168077 (40 μm) to PFC slices caused a reversible reduction of IPSC amplitudes (34.6 ± 2.6%, n = 7), whereas IPSC amplitudes remained stable in control neurons when no PD168077 was applied. Moreover, we measured miniature IPSC, a response from quantal release of single GABA vesicles. As shown in Fig. 1C, PD168077 (30 μm) caused a reversible reduction of mIPSC amplitudes in cultured PFC pyramidal neurons (17.8 ± 3.5%, n = 23). Taken together, these results suggest that D4 receptors down-regulate GABAAR function at the synapse. Next, we tested whether the D4-induced down-regulation of GABAAR function was due to a decrease in GABAAR surface expression. We labeled surface GABAA receptors using an antibody that targets the extracellular region of GABAAR β2/3 subunit in PFC cultures. Neurons were co-stained with MAP2, a dendritic marker. As illustrated in Fig. 1D, surface GABAARs were clustered around the soma and proximal dendrites. In cells treated with PD168077 (30 μm, 10 min), GABAAR surface clusters were substantially reduced. Quantification of immunocytochemical images (Fig. 1E) indicates that the density of GABAAR surface clusters (number of clusters/20 μm dendrite) was significantly reduced by PD168077 (control: 13.9 ± 1.4, n = 12; PD168077: 9.4 ± 1.4, n = 12, p < 0.05, ANOVA). PD168077 did not cause a significant change in the size of GABAAR surface clusters or the fluorescence intensity (normalized to MAP2 immunofluorescence) of GABAAR surface clusters. These results suggest that D4 receptor activation leads to a decrease of GABAAR surface cluster density, which is associated with the D4-induced reduction of whole cell GABAAR current, evoked IPSC, and miniature IPSC amplitude. The Actin Cytoskeleton Is Involved in D4 Regulation of GABAAR Currents-Next, we examined the underlying mechanism for D4 reduction of GABAARs at the cell surface. Previous studies have shown that GABAA receptors are removed from the plasma membrane mainly by clathrin/dynamin-mediated endocytosis (36Tehrani M. Barnes E.J. J. Neurochem. 1993; 60: 1755-1761Crossref PubMed Scopus (41) Google Scholar, 37Kittler J.T. Moss S.J. Curr. Opin. Neuorbiol. 2003; 13: 341-347Crossref PubMed Scopus (246) Google Scholar). To test whether D4 receptor activation induces GABAAR endocytosis, we dialyzed neurons with a dynamin inhibitory peptide, which competitively blocks dynamin from binding to amphiphysin, thus preventing endocytosis (38Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (485) Google Scholar). The effectiveness of this peptide to block GABAAR endocytosis has been demonstrated in our previous studies (39Chen G. Kittler J.T. Moss S.J. Yan Z. J. Neurosci. 2006; 26: 2513-2521Crossref PubMed Scopus (86) Google Scholar). As shown in Fig. 2 (A and B), PD168077 reduced GABAAR current in the presence of dynamin inhibitory peptide (50 μm, 15.6 ± 2.5%, n = 6), which was similar to the effect of PD168077 in the absence of this peptide (16.8 ± 1.7%, n = 6). These results suggest that D4 reduction of GABAAR current is not through increased endocytosis of GABAARs. Previous studies have suggested the involvement of cytoskeleton proteins in regulating GABAA receptor current and surface stability (40Meyer D.K. Olenik C. Hofmann F. Barth H. Leemhuis J. Brunig I. Aktories K. Norenberg W. J. Neurosci. 2000; 20: 6743-6751Crossref PubMed Google Scholar, 41Loebrich S. Bahring R. Katsuno T. Tsukita S. Kneussel M. EMBO J. 2006; 25: 987-999Crossref PubMed Scopus (118) Google Scholar); thus we investigated the potential role of microtubules and/or actin in D4 regulation of GABAAR current. As shown in Fig. 2C, dialysis with the actin stabilizing compound phalloidin (12.5 μm) largely blocked the capability of PD168077 to reduce GABAAR current. Phalloidin itself had little effect on basal GABAAR current (5.1 ± 1.0%, n = 5). As summarized in Fig. 2D, the effect of PD168077 was significantly (p < 0.005, ANOVA) smaller in phalloidin-loaded neurons (6.1 ± 0.5%, n = 4), compared with control neurons (16.1 ± 1.2%, n = 6). Conversely, application of latrunculin B, an actin depolymerizing compound, caused a decline of GABAAR current (31.1 ± 2.0%, n = 7) and largely occluded the effect of subsequently applied PD168077 (Fig. 2E). However, the microtubule destabilizing compound colchicine, which reduced basal GABAAR current (27.2 ± 3.0%, n = 8), failed to alter the reducing effect of PD168077 (Fig. 2E). As summarized in Fig. 2F, neurons dialyzed with latrunculin B showed a significantly (p < 0.005, ANOVA) smaller effect of PD168077 (6.1 ± 1.1%, n = 6), compared with control neurons (16.1 ± 1.2%, n = 6) or neurons perfused with colchicine (14.1 ± 1.2%, n = 14). Consistently, bath application of latrunculin B also occluded the effect of PD168077 on evoked IPSC in PFC slice recordings (Fig. 2G, 6.2 ± 2.0%, n = 5). These results suggest that D4 reduces GABAAR current via an actin-dependent mechanism. D4 Reduction of GABAAR Current Is Dependent upon the Actin Depolymerizing Factor Cofilin-Next, we investigated the link between D4 receptor signaling and actin cytoskeleton. The dynamics of actin assembly is regulated by cofilin, a major actin depolymerizing factor (42Dos Remedios C.G. Chhabra D. Kekic M. Dedova I.V. Tsubakihara M. Berry D.A. Nosworthy N.J. Physiol. Rev. 2003; 83: 433-473Crossref PubMed Scopus (774) Google Scholar). The actin depolymerizing activity of cofilin is greatly increased by dephosphorylation at Ser3 (43Morgan T.E. Lockerbie R.O. Minamide L.S. Browning M.D. Bamburg J.R. J. Cell Biol. 1993; 122: 623-633Crossref PubMed Scopus (143) Google Scholar, 44Agnew B.J. Minamide L.S. Bamburg J.R. J. Biol. Chem. 1995; 270: 17582-17587Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). In vitro studies have shown that protein phosphatase 1 (PP1) can lead to the dephosphorylation and activation of cofilin (45Ambach A. Saunus J. Konstandin M. Wesselborg S. Meuer S.C. Samstag Y. Eur. J. Immunol. 2000; 30: 3422-3431Crossref PubMed Scopus (140) Google Scholar). Our previous study has found that D4 regulation of GABAAR current depends on activation of the anchored PP1 (31Wang X. Zhong P. Yan Z. J. Neurosci. 2002; 22: 9185-9193Crossref PubMed Google Scholar). Thus, we speculated that D4 activation might induce actin depolymerization by dephosphorylating cofilin via PP1, thus leading to the reduced GABAAR synaptic trafficking along actin cytoskeleton. To test this, we first examined the impact of D4 on cofilin activity using a Ser3 phospho-cofilin antibody in cultured PFC neurons. As shown in Fig. 3A, application of PD168077 (30 μm, 10 min) significantly reduced the level of Ser3-phosphorylated (inactive) cofilin (65.1 ± 3.1% of control, n = 5; p < 0.005, ANOVA), and this effect was blocked by pretreatment with the PP1 inhibitor okadaic acid (1 μm, 40 min, 95.3 ± 3.1% of control, n = 3; p > 0.05, ANOVA). The level of total cofilin or actin was not changed. These results suggest that D4 activation leads to the dephosphorylation and activation of cofilin through a PP1-dependent mechanism. To further test the involvement of cofilin, we dialyzed neurons with the cofilin peptides consisting of 1–16 residues of cofilin with or without Ser3 phosphorylation (46Aizawa H. Wakatsuki S. Ishii A. Moriyama K. Sasaki Y. Ohashi K. Sekine-Aizawa Y. Sehara-Fujisawa A. Mizuno K. Goshima Y. Yahara I. Nat. Neurosci. 2001; 4: 367-373Crossref PubMed Scopus (297) Google Scholar, 47Zhou Q. Homma K.J. Poo M.M. Neuron. 2004; 44: 749-757Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar). The Ser3-phosphorylated cofilin peptide, p-cof[1–16] (MApSGVAVSDGVIKVFN), serves as an inhibitor of endogenous cofilin, because it binds to cofilin phosphatases and thus prevents the dephosphorylation and activation of endogenous cofilin. The nonphosphorylated cofilin peptide, cof[1–16], serves as a negative control. As shown in Fig. 3B, in cells dialyzed with p-cof[1–16] (50 μm), the D4-induced decrease of IPSC was largely blocked (5.3 ± 2.1%, n = 5), whereas the control peptide cof[1–16] (50 μm) did not alter the D4 effect on IPSC (30.5 ± 2.2%, n = 6). Similarly, in acutely dissociated PFC neurons (Fig. 3, C and D), the D4 effect on GABAAR current was significantly (p < 0.05, ANOVA) blocked by dialysis with p-cof[1–16] peptide (8.2 ± 1.6%, n = 13), but not cof[1–16] peptide (13.8 ± 1.5%, n = 8), compared with control conditions (16.1 ± 1.2%, n = 6). These results suggest that D4 suppresses GABAAR current via a mechanism requiring cofilin activity. The Actin Motor Protein, Myosin, Is Involved in D4 Regulation of GABAAR
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