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CELLULAR AND MOLECULAR

Regulation of Choline Transporter Surface Expression and Phosphorylation by Protein Kinase C and Protein Phosphatase 1/2A

Department of Anatomy and Neurobiology, University of Kentucky Medical Center, Lexington, Kentucky (J.G., S.A.); and Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee (S.M.F., R.D.B.)

Received February 10, 2004; accepted April 2, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The Na⁺/Cl^–-dependent, hemicholinium-3-sensitive choline transporter (CHT) provides choline for acetylcholine biosynthesis. Recent studies show that CHT contains canonical protein kinase C (PKC) serine and threonine residues. We examined the ability of PKC and serine/threonine protein phosphatase 1/2A (PP1/PP2A) to regulate CHT function, surface expression, and phosphorylation. In mouse crude striatal and hippocampal synaptosomes, PKC activators -phorbol 12-myristate 13-acetate (-PMA) and -phorbol 12,13-dibutyrate produced time- and concentration-dependent reductions in CHT function. PP1/PP2A inhibitors okadaic acid (OKA) and calyculin A (CL-A) produced a time- and concentration-dependent decrease in CHT function. However, tautomycin (PP1 inhibitor) and cyclosporin A (PP2B inhibitor) failed to alter CHT-mediated choline uptake. Choline transport kinetic studies following -PMA, OKA, and CL-A treatment revealed a reduction in the maximal choline transport velocity (V_max) with no change in K_m for choline. These modulators also produced no change in the total levels of CHT protein in the crude hippocampal and striatal synaptosomes; however, surface biotinylation studies using the membrane-impermeant N-hydroxysuccinimide-biotin in crude synaptosomes following treatment with -PMA, OKA, and CL-A indicate significant reductions of CHT levels in biotinylated fractions. Pretreatment with OKA alone, but not -PMA, significantly augmented the phosphorylation level of CHT proteins. Our findings suggest that neuronal PKC and PP1/PP2A activity may establish the level of function and surface expression of CHT. These studies also provide the first evidence that CHT is a phosphoprotein and that the basal PP1/PP2A activity may have a dominant role in controlling the levels of CHT phosphorylation.

In cholinergic neurons, a distinct hemicholinium-3 (HC-3)-sensitive, Na⁺/Cl^–-dependent choline transporter (CHT) provides choline for ACh synthesis (Apparsundaram et al., 2000, 2001; Okuda and Haga, 2000, 2003; Okuda et al., 2000; Ferguson and Blakely, 2004). CHT mediates high-affinity choline uptake, a process considered to be rate-limiting in ACh synthesis and a potential target for modulating cholinergic function (Knipper et al., 1992; Apparsundaram et al., 2000, 2001; Okuda and Haga, 2000, 2003; Okuda et al., 2000; Ferguson and Blakely, 2004). Disruption of CHT in mice is lethal to neonates (R. Blakely, unpublished data), this further validates the importance of CHT in biological function.

Neuronal depolarization (Barker, 1976; Murrin and Kuhar, 1976; Roskoski, 1978; Sherman et al., 1978; O'Regan and Collier, 1981), second messengers (Cancela et al., 1995; Vogelsberg et al., 1997; Ford et al., 1999), and acute drug treatments (Guyenet et al., 1973; Lowenstein and Coyle, 1986; Cooke and Rylett, 1997) have been shown to rapidly regulate CHT function. However, the mechanisms underlying these effects have not been studied thoroughly. Recent studies of Na⁺- and Cl^–-dependent neurotransmitter transporters, including dopamine, norepinephrine, and serotonin, reveal that protein kinase C (PKC) and protein phosphatase 1/2A (PP1/PP2A) regulate these transporter proteins via rapid changes in transporter surface expression and phosphorylation (Torres et al., 2003).

The presence of consensus PKC, serine/threonine phosphorylation sites in CHT protein provides a compelling possibility that changes in the phosphorylation state may be involved in CHT regulation (Okuda and Haga, 2000; Apparsundaram et al., 2001). Recent findings also revealed the localization of CHT protein in multiple compartments, including the plasma membrane, a subpopulation of synaptic vesicles and endosomal vesicles (Ferguson et al., 2003; Ribeiro et al., 2003; Ferguson and Blakely, 2004). These observations suggest that CHT may also be potentially regulated via changes in CHT distribution. Indeed, previous studies in locust brain synaptosomes (Knipper et al., 1992) and Limulus brain slices (Ford et al., 1999) have reported PKC-mediated alterations in CHT function. In parallel studies using HC-3 binding, these investigators also showed that PKC activation alters the surface redistribution of CHT proteins (Knipper et al., 1992; Ford et al., 1999). Besides PKC, PP1/PP2A inhibitors calyculin A and okadaic acid also displayed specific and profound inhibitory effects on high-affinity choline uptake (Issa et al., 1996). The focus of the present study was to determine the influence of PKC and protein phosphatases on CHT function, surface distribution, and phosphorylation in the mouse brain.

The availability of CHT-specific antibodies that recognize CHT proteins in mammalian tissues (Ferguson et al., 2003; Kus et al., 2003) present new opportunities for investigating whether or not such regulatory mechanisms control CHT function. Our studies reveal that: 1) CHT is a phosphoprotein, and 2) PKC and PP1/PP2A may influence CHT function via changes in CHT surface distribution. These changes in CHT phosphorylation and surface distribution could have profound effects on the intensity and duration of cholinergic synaptic transmission.

	Materials and Methods

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Reagents. Okadaic acid (OKA), phorbol 12-myristate 13-acetate (-PMA), and phorbol 12,13-dibutyrate were obtained from Alexis Corporation (Läufelfingen, Switzerland). Calyculin A (CL-A), tautomycin, and cyclosporin A (CsA) were all purchased from LC Laboratories (Woburn, MA). Sulfo-NHS-biotin and monomeric avidin beads were purchased from Pierce Chemical (Rockford, IL), and staurosporine was obtained from Q-Bio (Carlsbad, CA). D-Biotin and HC-3 were purchased from Sigma-Aldrich (St. Louis, MO); [methyl-³H]choline chloride (80 Ci/mmol) and [³²P]orthophosphate (11 Ci/mmol) were obtained from Amersham Biosciences Inc. (Piscataway, NJ). Rabbit polyclonal antibody raised against the carboxy terminal 15 amino acids (VDSSPEGSGTEDNLQ, residues 566–580) coupled to keyhole limpet hemocyanin (Research Genetics, Huntsville, AL) and the mouse monoclonal antibody raised against the COOH terminus of the human CHT conjugated to glutathione S-transferase (Allen Levey, Emory University, Atlanta, GA) were used in this study. These antibodies have been well characterized in recent studies (Ferguson et al., 2003; Kus et al., 2003) for their specificity in detecting CHT in native tissues. Rabbit polyclonal calnexin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and mouse monoclonal syntaxin 1A (Sigma-Aldrich) antibodies also were used.

Preparation of Mouse Crude Striatal and Hippocampal Synaptosomes. All the experimental procedures were in accordance with the guidelines of the Policies on the Use of Animals and Humans in Neuroscience Research, and all protocols were approved by the local university Animal Care Committee. In all experiments, male C57BL/6 mice (25–30 g b.wt., 8–10 weeks old) (The Jackson Laboratory, Bar Harbor, ME) were decapitated under chloral hydrate anesthesia (0.4 g/kg dose/intraperitoneal administration). The brains were removed and partially dissected into 1-mm sections using a rodent brain matrix. The hippocampus and striatum were isolated on an ice-cold Petri dish under a dissecting microscope. Isolated tissues were homogenized in ice-cold 0.32 M sucrose using a glass homogenizer with Teflon pestle (clearance 0.1 mm), eight strokes at 2500 rpm (Wheaton overhead stirrer). The homogenate was spun at 1000g for 15 min. The resulting supernatant was spun at 12,500g for 15 min, and the pellet-containing synaptosomes were suspended in Krebs bicarbonate buffer (KBB) containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, Na₂EDTA, 1.7 mM CaCl₂, 10 mM glucose, supplemented with 100 µM ascorbic acid and 10 µM physostigmine, and bubbled with 95:5 mixture of O₂/CO₂. Aliquots (10 µl) of crude synaptosomal suspension were used for protein determination using the Bradford method (Bio-Rad, Hercules, CA).

Choline Transport Assays. For choline transport assays, aliquots (50 µl) of the synaptosomal suspension, typically 30 to 50 µgof protein, were incubated for 5 min at 37°C in the presence and absence of 10 µM HC-3. Then, synaptosomes were further incubated with the indicated modulating agent(s) at 37°C for the indicated times. Choline transport was initiated by the addition of 0.1 ml of 100 nM [³H]choline, and the incubation was continued for 5 min at 37°C. The incubation was stopped by transferring the tubes to an ice bath followed by rapid filtration using the Brandel filtration apparatus (Brandel Inc., Gaithersburg, MD). Kinetic studies were carried out using 1 nM to 5 µM choline with 5% to 20% labeled choline. Specific CHT-mediated choline uptake is defined as total choline uptake minus choline uptake in the presence of HC-3.

Determination of Plasma Membrane-Associated Choline Transporter: Biotinylation and Immunoblotting. We examined the plasma membrane content of CHT in crude synaptosomes using the membrane-impermeant biotinylating reagent, sulfosuccinimidobiotin (NHS-biotin) (Chi and Reith, 2003; Salvatore et al., 2003). Crude synaptosomes (500 µg of protein/tube) were incubated with the specific modulating agent for 45 min at 37°C and washed three times with 1 ml of ice-cold KBB to remove excess agent. Each wash step consisted of centrifugation at 4°C for 2 min at 4000g with 1 ml of wash buffer. The tissue was then incubated in 500 µl of 1.5 mg/ml sulfosuccinimidobiotin for 1 h at 4°C, washed in ice-cold PBS/Ca/Mg buffer (138 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 9.6 mM Na₂HPO₄, 1 mM MgCl₂, and 0.1 mM CaCl₂, pH 7.3). After washing away the biotinylating reagents, the pellet was resuspended and incubated for 30 min with ice-cold 100 mM glycine (pH 3.0) in PBS/Ca/Mg buffer. Crude synaptosomes were again washed three times with PBS/Ca/Mg buffer and then lysed with 500 µl of radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton, and 1% sodium deoxycholate) and supplemented with protease inhibitors (1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, and 250 µM phenylmethylsulfonyl fluoride) and PP1/2A inhibitor (10 µM OKA). The extracts were centrifuged at 21,000g at 4°C. Aliquots (100 µl) of the extract were saved for immunoblotting of total CHT protein, whereas the remaining extracts were incubated with monomeric avidin beads (100 µl/tube) in the same buffer for 1 h at room temperature for the separation of biotinylated and nonbiotinylated proteins. At the end of incubation period, the beads were separated by centrifugation and washed in lysis buffer three times, and proteins bound to avidin beads were eluted by incubating in 50 µl of Laemmli buffer (62.5 mM Tris, pH 6.8, 20% glycerol, 2% SDS, and 5% bromphenol blue, supplemented with 5% -mercaptoethanol) for 20 min at room temperature. To achieve complete separation of biotinylated proteins and nonbiotinylated proteins, bead incubation, centrifugation, and elution with Laemmli buffer were repeated with a second aliquot of avidin beads. The bead eluents were combined, and aliquots (60 µl) of total extract and bead eluents were used in SDS-PAGE for protein separation. Separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA). The blots were incubated with the mouse monoclonal CHT antibody (1:1000) in PBS-Tween (0.5%) buffer containing 5% nonfat dry milk. The CHT-immunoreactive bands were detected using chemiluminescence (Amersham Biosciences Inc.) and autoradiography (Hyperfilm; Amersham Biosciences Inc.). After CHT detection, the blots were stripped (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM -mercaptoethanol) and reprobed with rabbit polyclonal calnexin antibody (1:1000) or mouse monoclonal syntaxin 1A (1:3000) antibody. Calnexin determinations were carried out to monitor the extent of biotinylation of intracellular proteins by membrane-impermeant NHS biotin, whereas syntaxin 1A determinations were carried out as controls for monitoring protein loading of fractions derived from either vehicle- or drug-treated synaptosomes. The band densities of specific bands were quantified using Scion image software (Scion Corporation, Frederick, MD), and CHT protein band densities in different fractions were normalized using syntaxin and calnexin band densities.

Metabolic Labeling and Immunoprecipitation. Metabolic labeling of CHTs with ³²PO₄ in the mouse striatum was carried out as described previously with slight modifications (Halpain et al., 1990; Vaughan et al., 1997). Mouse striatum was dissected out as described earlier, and then striatal sections were sliced (50- x 50- x 50-µm slices) using the McElwain tissue chopper (Mickle Laboratory Engineering Co. Inc., Surrey, UK). Slices equivalent to 300 µg of protein were placed in tubes containing 1 ml of oxygenated phosphate-free KBB (25 mM NaHCO₃, 125 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂, 5 mM MgSO₄, and 10 mM glucose, pH 7.3) and incubated for 30 min at 37°C with gentle shaking. Slices were then incubated with 1 ml of fresh phosphate-free KBB buffer containing 1 mCi/ml ³²P-labeled orthophosphate, and the mixture was incubated for 2 h at 37°C. Test compounds or vehicle were added over the final 45 min. At the end of the metabolic labeling, slices were washed three times with ice-cold phosphate-free KBB (1 ml/wash followed by centrifugation 8000g; 4°C, 2 min). After the final wash, slices were solubilized in 400 µl of 1% Triton X-100 buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.1% Triton), supplemented with protease inhibitors (1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, and 250 µM phenylmethylsulfonyl fluoride) and PP1/PP2A inhibitor (10 µM OKA) with the aid of a Sonic dismembrator (Fisher Scientific Co., Pittsburgh, PA). Extracts were centrifuged at 21,000g for 20 min, the supernatants were precleared with 3 mg of protein A-Sepharose beads (Amersham Biosciences Inc.), and the precleared extracts were incubated with 5 µl of preimmune or CHT immune serum or 10 µl of CHT monoclonal antibody at 4°C for 16 h. Antigen-antibody immune complexes were then separated using 3 mg of protein A-Sepharose beads (1 h; room temperature). Beads were washed three times with the lysis buffer, and the bound proteins were eluted with 50 µl of Laemmli buffer (20 min, room temperature). Samples were electrophoresed in SDS-PAGE gels, and dried gels were subjected to autoradiography (Kodak Biomax film; Eastman Kodak, Rochester, NY). CHT-specific bands were detected and the band density quantified using Scion image software.

Data Analysis. Results are expressed as mean ± S.E.M. values of three individual experiments. All data were analyzed by Student's t test or one-way analysis of variance (repeated measures) followed by Newman-Keuls post hoc test as appropriate (Prism 4.0; GraphPad Software Inc., San Diego, CA). Statistical significance was evaluated at = 0.05 level of significance.

	Results

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Activation of PKC Reduces CHT Function in the Mouse Hippocampus and Striatum. To examine the influence of PKC activation on CHT, we analyzed the effect of the PKC activator, -PMA, on CHT-mediated choline uptake in the mouse crude hippocampal and striatal synaptosomes. -PMA produces a time- and concentration-dependent decline in CHT-mediated choline uptake (Fig. 1). In the crude hippocampal synaptosomes, a significant reduction was seen with 1 µM -PMA following a 30-min treatment (Fig. 1A). In the crude striatal synaptosomes, a significant effect was seen beginning at 15 min of -PMA (1 µM) with about 20 ± 2% reduction at 45 min (Fig. 1B). Phorbol ester, phorbol 12,13-dibutyrate (1 µM, 45 min), also produced significant decreases in CHT-mediated choline uptake in the crude hippocampal (33 ± 6% reduction, p < 0.05) and striatal (38 ± 7% reduction, p < 0.05) synaptosomes. On the other hand, the inactive phorbol ester, -PMA (1 µM, 45 min), failed to alter CHT-mediated choline uptake in both crude hippocampal and striatal synaptosomes (Fig. 2). Treatment of crude synaptosomes with staurosporine (1 µM, 45 min), a PKC inhibitor, did not reveal a significant change but abolished -PMA-mediated reduction in CHT function in both hippocampus and striatum (Fig. 2). A structurally distinct PKC inhibitor, calphostin C, also provided effects that are comparable with staurosporine (data not shown). Together, these results suggest that activation of PKC reduces CHT function in the mouse hippocampus and striatum.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Activation of PKC produces a time- and concentration-dependent reduction in CHT function in the mouse crude hippocampal and striatal synaptosomes. Crude hippocampal (A) and striatal (B) synaptosomes were incubated (37°C, 45 min) in the absence (0 min) or presence of -PMA (1 µM) at various time periods. Crude hippocampal (C) and striatal (D) synaptosomes were incubated (37°C, 45 min) in the presence of -PMA at various concentrations (10–8 to 10–5M). Data are presented as mean ± S.E.M. values of three experiments carried out in triplicate. Asterisk(s) indicate significant difference as compared with control.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Staurosporine blockade of -PMA-evoked reductions in CHT function in the mouse hippocampal and striatal synaptosomes. Crude hippocampal (A) and striatal (B) synaptosomes were incubated (37°C, 45 min) in the presence of -PMA (1 µM), -PMA (1 µM), or staurosporine (1 µM). Data are presented as mean ± S.E.M. values of three experiments carried out in triplicate. Asterisk(s) indicate significant difference as compared with control.

Blockade of Protein Phosphatase PP1/PP2A Reduces CHT Function in the Mouse Hippocampus and Striatum. We next examined the influence of serine/threonine protein phosphatases on CHT function in the mouse crude hippocampal and striatal synaptosomes. Inhibitors of PP1/PP2A, OKA or CL-A, produced a concentration-dependent reduction in CHT function in the striatal synaptosomes (Fig. 3). Pretreatment of striatal synaptosomes with either a selective PP1 inhibitor, tautomycin, or a selective PP2B inhibitor, cyclosporine A, failed to alter CHT function (Fig. 3). These results indicate that PP2A, rather than PP1, may be responsible for regulating CHT function.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effects of OKA, CL-A, tautomycin, and CsA on CHT-mediated choline uptake in the mouse hippocampal and striatal synaptosomes. Crude hippocampal (A) and striatal (B) synaptosomes were incubated (37°C, 45 min) in the absence (controls) or presence of various concentrations of OKA, CL-A, tautomycin, or CsA (10–5 to 10–8 M). Data are presented as mean ± S.E.M. values of four experiments carried out in triplicate. Asterisk(s) indicate significant difference as compared with control.

PKC Activation and Protein Phosphatase PP1/PP2A Inhibition Reduce the Maximal Transport Capacity of CHT in the Mouse Striatum. Reduction in choline transport following PKC activation, and PP1/PP2A inhibition might involve changes in K_m and/or V_max (maximal transport capacity) of CHT function. To examine these possibilities, we performed choline transport kinetics in the presence and absence of -PMA (1 µM, 45 min), OKA (10 µM, 45 min), and CL-A (10 µM, 45 min). These studies revealed a reduction in the maximal transport capacity, with minimal change in K_m for choline following treatment with -PMA (28% reduction), OKA (37% reduction), and CL-A (33% reduction) (Table 1).

PKC Activation and Protein Phosphatase PP1/PP2A Inhibition Reduce Surface Distribution of CHTs in the Mouse Striatum. A reduction in the maximal transport capacity following PKC activation and/or PP1/PP2A inhibition may indicate a decrease in the density of CHT on the plasma membrane and/or a decrease in the intrinsic activity of individual CHT molecules resident on the plasma membrane. To further investigate these possibilities, we examined the surface distribution of CHT in crude synaptosomes using a surface biotinylation technique. This technique has been well established to document changes in surface distribution of plasma membrane resident proteins in cell line model systems (Melikian et al., 1996; Qian et al., 1997); recently, the method has been applied to crude synaptosomes (Chi and Reith, 2003; Salvatore et al., 2003). This approach has been used to reveal a reduction in the plasma membrane expression of dopamine transporters in the striatal synaptosomes in rats (Chi and Reith, 2003; Salvatore et al., 2003). In the present study, we pretreated mouse crude striatal synaptosomes with vehicle or -PMA (1 µM, 45 min) and subjected to biotinylation protocol (see Materials and Methods). We isolated biotinylated proteins and determined CHT levels using transporter-specific antibody (Fig. 4). We also examined the levels of endoplasmic reticular protein, calnexin, and SNARE protein, syntaxin 1A (Fig. 4). Calnexin determinations were carried out to monitor the extent of biotinylation of intracellular proteins by membrane-impermeant NHS-biotin, whereas syntaxin 1A determinations were carried as a marker for equal loading of protein fractions derived from either vehicle- or drug-treated synaptosomes. We calculated the level of CHT in total, biotinylated, and nonbiotinylated fraction-based band densities, protein loaded in the gel, and protein recovery from avidin beads. In vehicle-treated striatal synaptosomes, 30% of total CHT protein was found in the biotinylated fraction. The biotinylated fraction may also include CHT proteins in broken plasma membranes that may be present in crude synaptosomal preparations; therefore, the actual levels of surface CHT may be less than 30% of total CHT levels. These studies also revealed the presence of calnexin (about 10%) in the bead eluent, reflecting the possibility that NHS-biotin may label a small fraction of intracellular proteins derived from broken cell bodies during homogenization or may access intracellular proteins via leak pathways.

View larger version (28K): [in this window] [in a new window]   Fig. 4. -PMA reduces surface distribution of CHTs in the mouse striatum. A, crude striatal synaptosomes were incubated (37°C, 45 min) in the absence (controls) or presence of -PMA (1 µM). Crude synaptosomes were then incubated with membrane-impermeant NHS-biotin. After quenching the biotinylation reaction, tissues were extracted with RIPA buffer (see Materials and Methods). Biotinylated proteins were separated from total lysate using avidin beads. CHT immunoreactivity in the samples was detected using CHT-specific antibody. Blots were then stripped and reprobed with rabbit polyclonal calnexin antibody or mouse monoclonal syntaxin 1A-specific antibody. Representative blots probed with CHT, calnexin, and syntaxin 1A antibodies (A) and band density of CHT-immunoreactive bands (B) are shown. CHT-IR bands were quantified using Scion image software and presented as mean ± S.E.M. values of three experiments (p < 0.05). Asterisk(s) indicates significant difference as compared with control (Student's t test; p < 0.05).

Pretreatment of synaptosomes with -PMA (10 µM, 45 min) produced over a 30% reduction of CHT in the biotinylated fraction with no alteration of CHT levels in total lysates. We observed no difference in the levels of syntaxin 1A and calnexin in the biotinylated fractions, suggesting selective reduction of CHT levels in bead eluents following -PMA treatment. PKC inhibitor staurosporine failed to alter the levels of CHT in the total and biotinylated fraction. However, staurosporine pretreatment abolished -PMA-mediated reduction of CHT levels in the biotinylated fraction (Fig. 5).

View larger version (35K): [in this window] [in a new window]   Fig. 5. Staurosporine (STAUR.) blockade of -PMA-mediated reduction in CHT surface expression in the mouse striatum. Crude striatal synaptosomes were incubated (37°C, 45 min) in the absence (controls) or presence of -PMA (1 µM). Staurosporine (1 µM) treatment was initiated 10 min prior to the addition of -PMA (1 µM). Crude synaptosomes were then incubated with membrane-impermeant NHS-biotin. After quenching the biotinylation reaction, tissues were extracted with RIPA buffer (see Materials and Methods). Biotinylated proteins were separated from total lysate using avidin beads. CHT immunoreactivity in the samples was detected using CHT-specific antibody. Blots were then stripped and reprobed with rabbit polyclonal calnexin antibody or mouse monoclonal syntaxin 1A-specific antibody. Representative blots probed with CHT, calnexin, and syntaxin 1A antibodies and band density of CHT-immunoreactive bands are shown. CHT-IR bands were quantified using Scion image software and presented as mean ± S.E.M. values of three experiments (p < 0.05). Asterisk indicates significant difference as compared controls (one-way repeated measures analysis of variance followed by Newman-Keuls post hoc test).

In the mouse crude striatal synaptosomal preparations, PP1/PP2A inhibitors, OKA (Fig. 6A) and calyculin A (Fig. 6B), also produced a significant reduction in CHT levels in the biotinylated fraction (OKA 30% reduction; CL-A 31% reduction) with no apparent change in total lysates (protein). Together, these results provide evidence that PKC activation and PP1/PP2A inhibition reduce surface expression of CHT proteins with no significant change in the plasma membrane permeability and accessibility to proteins in intracellular compartments.

View larger version (44K): [in this window] [in a new window]   Fig. 6. Inhibitors of PP1/PP2A reduce surface expression of CHTs in the mouse striatum. Crude striatal synaptosomes were incubated (37°C, 45 min) with either vehicle or 10 µM OKA (A) or 10 µM CL-A (B) for 45 min. After drug treatment, crude synaptosomes were then incubated with membrane-impermeant NHS-biotin. Tissues were extracted with lysis buffer (see Materials and Methods), and biotinylated proteins were separated from total lysates using avidin beads. CHT-immunoreactivity in total lysates and bead eluents was detected using mouse monoclonal CHT-specific antibody. Blots were then stripped and reprobed with rabbit polyclonal calnexin or mouse monoclonal syntaxin 1A-specific antibody. Representative blots probed with CHT, calnexin, and syntaxin 1A antibodies and band densities of CHT-immunoreactive bands are shown. CHT-IR bands were quantified using Scion image software and presented as mean ± S.E.M. values of three experiments (p < 0.05). Asterisk(s) indicates significant difference as compared with control (Student's t test; p < 0.05).

PKC Activation and Protein Phosphatase PP1/PP2A Inhibition Reduce V_max of Choline Transport and Surface Distribution of CHT in the Mouse Hippocampus. In parallel with striatal synaptosomes, we also carried out experiments in hippocampal synaptosomes. Choline transport kinetic studies in hippocampal synaptosomes in the presence and absence of -PMA (1 µM, 45 min), OKA (10 µM, 45 min), and CL-A (10 µM, 45 min) also revealed a reduction in the maximal transport capacity, with minimal change in K_m for choline (Fig. 7, A and B). Biotinylation and immunoblot studies also revealed a significant reduction in CHT immunoreactivity in bead eluents following treatment with -PMA (1 µM, 45 min), OKA (10 µM, 45 min), and CL-A (10 µM, 45 min); however, these treatments failed to alter the levels of CHT-immunoreactivity in total lysates (Fig. 7, C and D).

View larger version (31K): [in this window] [in a new window]   Fig. 7. -PMA, okadaic acid, and calyculin A reduce Vmax of choline transport and surface expression of CHT in mouse hippocampus. Crude hippocampal synaptosomes were incubated (37°C, 45 min) in the absence (controls) or presence of 1 µM -PMA/10 µM okadaic acid/10 µM calyculin A. Choline transport Km (A) and Vmax (B) values were derived using one-site saturation curve fit (Kaleidagraph; Abelbeck/Synergy, Reading, PA). Data are presented as mean ± S.E.M. values of four experiments carried out in triplicate. Asterisk(s) indicate significant difference as compared with control. In C and D, after treatment with 1 µM -PMA/10 µM okadaic acid/10 µM calyculin A, crude synaptosomes were incubated with membrane-impermeant NHS-biotin. After quenching the biotinylation reaction, tissues were extracted with lysis buffer (see Materials and Methods). Biotinylated proteins were separated from total lysate using avidin beads. CHT-immunoreactivity present in the samples was detected using CHT-specific antibody. Band densities of CHT-immunoreactive bands in total lysate (C) and bead eluents (D) are shown. CHT-IR bands were quantified using Scion image software and presented as mean ± S.E.M. values of three experiments (p < 0.05). Asterisk(s) indicates significant difference as compared with control (Student's t test; p < 0.05).

Immunoprecipitation of Phosphorylated CHT Protein in the Mouse Hippocampus and Striatum. To investigate whether or not changes in CHT function following PKC activation and PP1/PP2A inhibition is accompanied by changes in the levels of CHT phosphorylation, we metabolically labeled mouse striatal slices with ³²PO₄ in the presence or absence of -PMA (1 µM, 45 min) and OKA (10 µM, 45 min). Then, we immunoprecipitated CHT proteins from the striatal slice extracts and subjected the immunoprecipitates to SDS-PAGE and autoradiography. Our analysis revealed the presence of an intense broad band at 55 kDa in the samples treated with combined application of -PMA and OKA and immunoprecipitated with CHT-specific serum (Fig. 8A). The 55-kDa CHT band was also seen in the vehicle-treated tissue, although at lower levels. The ³²PO₄-labeled 55-kDa band was absent or manifested in extremely lower levels in tissue extracts immunoprecipitated with either control preimmune serum or CHT-specific antiserum that was preabsorbed with antigenic peptide-linked affigel beads. The monoclonal CHT antibody also produced immunoprecipitates that contained intense levels of 55-kDa protein species in -PMA- and OKA-treated slices as compared with vehicle-treated controls. Immunoprecipitates obtained using hippocampal extracts yielded similar results. The ability of both monoclonal and polyclonal antibodies to immunoprecipitate protein species of identical size suggests that the immunoprecipitated species may be more likely the CHT molecules. Thus, these results indicate that CHT is a phosphoprotein controlled by PKC and PP1/PP2A activity.

View larger version (43K): [in this window] [in a new window]   Fig. 8. Phosphorylation of CHT proteins in the mouse hippocampus and striatum. A, mouse hippocampal and striatal slices were metabolically labeled with [32 P] orthophosphate in phosphate-free KBB for 2 h, with vehicle or 10 µM -PMA plus 10 µM OKA for the final 45 min. Slices were then washed and extracted with 1% Triton buffer (see Materials and Methods). Aliquots of tissue extracts derived from hippocampal and striatal slices that are treated with vehicle or combined application of -PMA and okadaic acid were incubated with monoclonal CHT antibody (mAb) (1:100 dilution) or preimmune serum (preimm.) (1:100 dilution), or immune serum (21) (1:100 dilution) containing polyclonal antibodies recognizing the CHT or immune serum 21 preabsorbed with antigenic peptide (VDSSPEGSGTEDNLQ) affigel column. Proteins in immunoprecipitates were separated by SDS-PAGE electrophoresis, and the dried gel was subjected to autoradiography. B, time course effect of okadaic acid on CHT phosphorylation (top panel) and choline uptake (bottom panel) in the mouse striatum. Striatal slices were metabolically labeled for 2 h in the absence and presence of 10 µM OKA for the indicated time. C, effect of -PMA alone or in combination with okadaic acid on CHT phosphorylation (top panel) and choline uptake (bottom panel) in the mouse striatum. Striatal slices were metabolically labeled for 2 h with vehicle, 10 µM -PMA, 10 µM OKA, and 10 µM -PMA plus 10 µM OKA in the final 45 min. In B and C (top panel), after drug treatment, slices were washed and extracted with 1% Triton buffer. Aliquots of tissue extracts were incubated with polyclonal CHT antibody (21) (1:100 dilution). CHT-IR bands were quantified using Scion image software and presented as mean ± S.E.M. values of four experiments (p < 0.05). In B and C (bottom panel), slices were incubated with 10 µM -PMA, 10 µM OKA, and 10 µM -PMA plus 10 µM OKA for 45 min, and the choline uptake assays were performed.

We also examined the effect of either -PMA or OKA alone on CHT phosphorylation in the mouse striatum. OKA produced a time-dependent increase in CHT phosphorylation levels in the striatal slices (Fig. 8B, top panel). A marked increase in CHT phosphorylation was obtained following 30 and 45 min of pretreatment with OKA (10 µM, 45 min) (Fig. 8B, top panel). OKA-evoked increases in CHT phosphorylation levels were paralleled by a significant decrease in CHT-mediated choline uptake (Fig. 8B, bottom panel). On the other hand, -PMA (10 µM, 45 min) pretreatment alone failed to alter the basal levels of CHT phosphorylation (Fig. 8C, top panel). However, in concert with striatal synaptosomes, -PMA (10 µM, 45 min) produced a significant reduction of CHT-mediated choline uptake in striatal slices (Fig. 8C, bottom panel). Moreover, coapplication of -PMA and OKA augmented CHT phosphorylation to levels that were comparable with the application of OKA alone (Fig. 8C), suggesting no additive effects of -PMA and OKA. Thus, these results establish that CHT is a target for dephosphorylation by endogenous PP1/PP2A.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Neurotransmitters, second messengers, drug treatments, and neuronal activity have been shown to tightly regulate HC-3-sensitive choline transport (Guyenet et al., 1973; Barker, 1976; Murrin and Kuhar, 1976; Roskoski, 1978; Sherman et al., 1978; O'Regan and Collier, 1981; Lowenstein and Coyle, 1986; Cancela et al., 1995; Cooke and Rylett, 1997; Vogelsberg et al., 1997; Ford et al., 1999). However, little is known about regulatory mechanisms that control choline transport function. Recent studies on neurotransmitter transporters, including the dopamine (DAT), norepinephrine (NET), and serotonin (SERT) transporter, reveal that transporters may undergo rapid internalization and recycling in response to changes in the activity of cellular protein kinases and phosphatases (Torres et al., 2003). Such dynamic changes in transporter distribution are thought to be a key regulatory mechanism for spatial and temporal control of neurotransmitter concentrations at synapses. In conjunction, evidence has been steadily accumulating, documenting changes in transporter phosphorylation via cellular kinases and phosphatases (Vaughan et al., 1997; Ramamoorthy et al., 1998; Foster et al., 2003; Torres et al., 2003). Changes in transporter phosphorylation levels, in some cases, might be linked to changes in its surface distribution (Vaughan et al., 1997; Ramamoorthy et al., 1998; Foster et al., 2003; Torres et al., 2003). The recent availability of monoclonal and polyclonal CHT-specific antibodies enabled the investigation of changes in the surface distribution and phosphorylation levels of CHT in response to PKC and PP1/PP2A modulation. In the present study, we reveal that: 1) the function of CHT is governed by neuronal PKC and PP1/PP2A, 2) both PKC and PP1/PP2A may be involved in establishing the plasma membrane levels of CHT, and 3) PP1/PP2A activity may determine the levels of CHT phosphorylation.

Recent studies reveal that CHT is primarily localized in cholinergic neurons, with only low levels of CHT in noncholinergic and non-neuronal cells (Ferguson et al., 2003; Kus et al., 2003). Therefore, changes in HC-3-sensitive choline transport in the mouse striatum and hippocampus that are reported in our investigation mainly reflect changes in the function of CHT proteins expressed in cholinergic neurons. Our findings that PKC activation and protein phosphatase 1/2A inhibition reduces CHT function in the mouse hippocampal and striatal synaptosomes are similar to the previous reports in the rat hippocampal slices (Issa et al., 1996), mouse hippocampal synaptosomes (Cooke and Rylett, 1997), and Limulus brain hemislices (Ford et al., 1999). However, in contrast to the effects seen in Limulus brain hemislices (Ford et al., 1999), blockade of PKC failed to alter basal choline transport in the mouse hippocampal and striatal synaptosomes (Fig. 2). The differential effect of PKC inhibitors in the mouse and Limulus brain regions may be due to plausible differences in the levels/activity of PKC and protein phosphatase 1/2A activity on CHT in these tissues. Investigating the relationship between levels of PKC/PP1/PP2A activity and CHT regulation would shed some insights into these tissue differences. Furthermore, in contrast to in vitro reductions of high-affinity choline uptake (Issa et al., 1996; Cooke and Rylett, 1997) following exposure to PP1/PP2A inhibition, diverse effects are observed upon in vivo administration of PP1/PP2A inhibitor, OKA (Vogelsberg et al., 1997).

Previous studies have shown changes in surface distribution of neurotransmitter transporters, using surface biotinylation approaches, in response to PKC and PP1/PP2A modulation (Qian et al., 1997; Pristupa et al., 1998; Daniels and Amara, 1999). We attempted to examine the surface expression of CHTs using this surface biotinylation technique in crude synaptosomal preparations. The lack of significant change in the total CHT levels suggested the absence of CHT degradation following PKC activation and PP1/PP2A inhibition in the mouse striatal and hippocampal synaptosomes. We found reductions in the levels of CHT-immunoreactivity in avidin bead eluent fractions derived from tissues following exposure to PKC activators or PP1/PP2A inhibitors, suggesting reductions in plasma membrane resident CHTs rather than a change in total protein. However, based on the quantitation of calnexin in total extracts and bead eluents, we found 10% contamination of intracellular proteins in bead eluents. Since there was no significant difference in the band density of calnexin in vehicle- and drug-treated conditions, the impact of contamination from intracellular compartments would not negate differences in CHT levels observed in bead eluents between drug- and vehicle-treated samples. Moreover, the absence of change in the levels of syntaxin 1A in bead eluents indicates a selective change in the surface distribution of CHT. Such selective changes in CHT distribution may occur via dynamin-clathrin-mediated pathways as suggested for trafficking of G-protein-coupled receptors and neurotransmitter transporters (Ferguson et al., 1998; Daniels and Amara, 1999; Torres et al., 2003). In support of this speculation, recent studies provide evidence for the involvement of clathrin-mediated pathway in the trafficking of CHT (Ribeiro et al., 2003).

The existence of multiple population of CHT has been previously reported using choline uptake and HC-3 binding studies (Ivy et al., 2001). These studies reveal the presence of CHT in recycling endosomes (Ivy et al., 2001). Recent studies employing CHT-specific antibodies reveal the existence of CHT in the subpopulation of ACh-containing synaptic vesicles in cholinergic neurons and other vesicular compartments (Ferguson et al., 2003; Ribeiro et al., 2003; Ferguson and Blakely, 2004). -PMA, okadaic acid, and CL-A may redistribute plasma membrane resident CHT into other intracellular compartments. Such translocations may involve changes in the phosphorylation state of CHT proteins as was suggested for DAT, NET, and SERT proteins.

Previous investigators have speculated phosphorylation of CHT proteins as a potential mechanism for the regulation of CHT function (Knipper et al., 1992; Okuda and Haga, 2003). Our investigations provide the first evidence for the phosphorylation of CHT proteins and modulation of CHT phosphorylation in native tissues. The robust effects of PP1/PP2A inhibition coupled with no effect of PKC activation on the phosphorylation levels of CHT proteins suggest marked dephosphorylation of CHT under basal conditions. These results are qualitatively similar to DAT phosphorylation and regulation in striatal slices where DATs are regulated by rapid dephosphorylation under steady-state conditions (Vaughan et al., 1997; Foster et al., 2003). The lack of PKC activation on CHT phosphorylation may be due to the: 1) rapid dephosphorylation of CHT via PP1/PP2A, 2) no influence of PKC on CHT phosphorylation, and 3) different time course of PKC activation and CHT phosphorylation. Our initial studies using staurosporine revealed a significant reduction in OKA-evoked enhancement of CHT phosphorylation in the striatum (data not shown). These studies suggest the possibility that a staurosporine-sensitive pathway may be involved in phosphorylating CHT proteins under basal conditions, and the phosphorylated CHT proteins may be rapidly dephosphorylated via PP1/PP2A-regulated mechanisms. Moreover, whether the staurosporine-sensitive pathway revealed in the presence of OKA involves PKC remains to be explored. Additional studies are warranted to more thoroughly characterize kinases involved in mediating phosphorylation of CHT under basal condition and identify serine and threonine residues involved in CHT phosphorylation by PKC and PP1/PP2A.

The close correlation of changes in CHT function, phosphorylation, and surface distribution following OKA may suggest a direct relationship between changes in CHT phosphorylation and surface distribution. However, the lack of changes in basal levels of CHT phosphorylation by PKC activators would challenge this relationship. As seen in the case of dopamine transporters, it is also possible that phosphorylation of CHT may not be required for its internalization (Granas et al., 2003). More in-depth investigation of temporal relationships among PKC and PP1/PP2A modulators on CHT function, phosphorylation, and surface expression may shed more light on the role of phosphorylation and dephosphorylation in CHT function.

Although we provide evidence for CHT phosphorylation, it is not known whether the differences in the phosphorylation state of CHT dictate the so-called "active" and "inactive" population of CHTs. The localization of CHT in a subpopulation of synaptic vesicles and also in endosomal vesicles warrant the investigation of phosphorylation state of CHT in different compartments under basal and drug-exposed conditions (Ferguson et al., 2003; Ribeiro et al., 2003). It is not known whether differences or changes in phosphorylation occur in the plasma membrane resident or intracellular pools of CHTs. It is also possible that other molecular targets that are responsive to PKC and PP1/PP2A may control CHT function, phosphorylation, and surface distribution. Future studies should focus on delineating the serine/threonine in CHT proteins that are targets for PKC and PP1/PP2A. These studies should also examine the possibility that other proteins controlling CHT trafficking, rather than CHT per se, may be direct targets for PKC and PP1/PP2A. Such studies may delineate the relationship among changes in three neurochemical events, namely CHT function, phosphorylation, and surface distribution. Although the mechanisms controlling CHT phosphorylation in vivo remain unknown, the finding that phorbol esters and protein phosphatase inhibitors stimulate CHT phosphorylation indicates the feasibility for involvement of presynaptic somatodendritic and terminal receptors in the regulation of CHT proteins. Coupling of presynaptic receptors to CHT function may provide novel mechanisms for the fine regulation of transport activity and synaptic choline levels. Understanding these pathways may provide insights into regulatory mechanisms controlling CHT function and novel strategies for modulating CHT function.

	Acknowledgements

 
We thank Dr. Alan Levey for providing the monoclonal choline transporter antibody and Jackie Huller for technical assistance.

	Footnotes

 
These studies were supported by the Lyman T. Johnson Fellowship (to J.G.), by the Vanderbilt Brain Institute predoctoral fellowship (to S.F.), and by National Institutes of Health Grants MH58921 (to R.B.), 2T32DA007304 (to J.G.), and P20RR15592 and DA14040-01 (to S.A).

DOI: 10.1124/jpet.104.066795.

ABBREVIATIONS: ACh, acetylcholine; HC-3, hemicholinium-3; CHT, choline transporter; PKC, protein kinase C; PP1/PP2A, protein phosphatase 1/protein phosphatase 2A; OKA, okadaic acid; PMA, phorbol 12-myristate 13-acetate; CL-A, calyculin A; CsA, cyclosporin A; NHS, N-hydroxysuccinimide; KBB, Krebs bicarbonate buffer; PBS, phosphate-buffered saline; RIPA, radioimmunoprecipitation assay; PAGE, polyacrylamide gel electrophoresis; kDa, kilodalton(s); DAT, dopamine transporter.

Address correspondence to: Dr. Subbu Apparsundaram, Anatomy and Neurobiology, 306 Whitney-Hendrickson Building, 800 Rose Street, University of Kentucky Medical Center, Lexington, KY 40536-0098. E-mail: subbu{at}uky.edu

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