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The Journal of Neuroscience, August 15, 1998, 18(16):6103-6112
Protein Kinase C Regulates the Interaction between a GABA Transporter and Syntaxin 1A
Matthew L. Beckman1, 2, Eve M. Bernstein1, and Michael W. Quick1 1 Department of Neurobiology and 2 Medical Scientist Training Program, University of Alabama at Birmingham, Birmingham, Alabama 35294-0021
ABSTRACT
Top
Abstract
Introduction
Syntaxin 1A inhibits GABA uptake of an endogenous GABA transporter in neuronal cultures from rat hippocampus and in reconstitution systems expressing the cloned rat brain GABA transporter GAT1. Evidence of interactions between syntaxin 1A and GAT1 comes from three experimental approaches: botulinum toxin cleavage of syntaxin 1A, syntaxin 1A antisense treatments, and coimmunoprecipitation of a complex containing GAT1 and syntaxin 1A. Protein kinase C (PKC), shown previously to modulate GABA transporter function, exerts its modulatory effects by regulating the availability of syntaxin 1A to interact with the transporter, and a transporter mutant that fails to interact with syntaxin 1A is not regulated by PKC. These results suggest a new target for regulation by syntaxin 1A and a novel mechanism for controlling the machinery involved in both neurotransmitter release and reuptake.
Key words: neurotransmitter uptake; synaptic vesicle proteins; second messengers; protein regulation; protein-protein interactions; GABA transport
INTRODUCTION
Neurotransmitter transporters are integral membrane proteins, expressed at or near the synapse on neurons and glia, that function by coupling the uptake of neurotransmitter to the movement of cotransported ions down their electrochemical gradients. The influences of transporters on synaptic signaling are diverse. Pharmacological blockade of specific transporters can prolong and/or increase the amplitude of postsynaptic responses mediated via GABA (Isaacson et al., 1993
), NMDA (Sarantis et al., 1993
Factors that regulate neurotransmitter transporter function will necessarily influence these processes, and such factors include a variety of intracellular second messengers (for review, see Clark and Amara, 1993
Syntaxin 1A is a plasma membrane protein involved both in trafficking and vesicle docking and/or fusion (Bennett et al., 1992
channels (Naren et al., 1997
In the present report, we show that syntaxin 1A and PKC functionally regulate GABA transport in cells that endogenously express these proteins. In mammalian cell reconstitution systems, coimmunoprecipitation experiments demonstrate an association between GAT1 and syntaxin 1A that can be regulated by PKC. Syntaxin 1A is necessary but not sufficient to confer PKC-mediated transporter regulation, and reconstitution experiments suggest that PKC modulation likely occurs via interactions of the syntaxin 1A-GAT1 complex with other syntaxin 1A-binding partners that are substrates for PKC modification (e.g., Munc18). Such functional regulation is lacking in a mutant GAT1 protein that does not interact with syntaxin 1A. These data (1) suggest that a network of protein-protein interactions is responsible for the functional regulation of GABA transport, (2) describe one mechanism for PKC modulation of transporter function, and (3) accumulate additional evidence to suggest that syntaxin 1A is a general regulator of excitability protein activity.
MATERIALS AND METHODS
Cell culture. Primary hippocampal cultures were prepared from postnatal day 0-3 rats by mincing tissue in
-Minimal Essential Medium (
Pheochromocytoma (PC12) cells were maintained in RPMI 1640 supplemented with 10% horse serum, 5% FBS, L-glutamine, and penicillin-streptomycin. 1F9 cells [Chinese hamster ovary cells stably expressing GAT1 (Corey et al., 1994a
[3H]GABA uptake assays. Cells were rinsed twice in 1× HBSS and allowed to equilibrate for 10 min in the final wash. Buffer was then exchanged with control HBSS or drug-containing HBSS; preincubation times were 10-30 min. [3H]GABA was added to initiate the assay. The final [3H]GABA concentration was 30 nM, and assay times were 10-60 min. The assay was terminated by rapidly rinsing the cells three times with 1× HBSS, followed by solubilization in 300 µl of 0.001-0.005% SDS at 37°C for 2 hr. Aliquots were used for scintillation counting and to determine protein concentrations. Statistical analyses of the uptake data were performed using SPSS. Two-sample comparisons were made using t tests; multiple comparisons were made using one-way ANOVAs, followed by Tukey's honest significant difference post hoc test.
Antisense experiments. The rat syntaxin sense and antisense (Bennett et al., 1992
1 to 18 (sense strand, 5'-GATGAAGGATCGGACTCAG-3'), a region divergent in sequence from other syntaxin isoforms based on the published sequence. For antisense inhibition experiments, 1 ml of the oligonucleotides (10 µM, final concentration) was incubated with cells in 1% serum media with Lipofectamine for 6 hr; 1 ml of 20% serum media was then added to the cells without removing the oligonucleotides.
Mutant GAT1 constructs. Truncation mutants were created using the PCR. The N-terminal truncation mutant (GAT1
N) was produced using the following primer set: (upstream) 5'-TTTCCAAAGCTTTGGATGGCGACTGTAGTCAAGGTGCAGAAG-3'; and (downstream) 5'-TCTGTGAAAGCCCCCAAGGG-3'. This resulted in the production of a GAT1 construct lacking bases 10-91, corresponding to amino acids 4-30. PCR products were cloned into pCR 2.1 (Invitrogen, San Diego, CA), screened by restriction analysis, and subcloned into pRC/cytomegalovirus (Invitrogen) at the HindIII and ApaI sites. A GAT1 consensus phosphorylation site mutant (T236A) was made using Altered Sites I (Promega, Madison, WI). GAT1 T236A was subcloned into pcDNA3 for transfection studies. The GAT1
Immunoprecipitations. Immunoprecipitation was performed by lysing cells in radio-immunoprecipitation assay buffer as described (Naren et al., 1997
RESULTS
Much data demonstrating transporter regulation by PKC and by components of the vesicle docking and fusion apparatus come from experiments involving heterologous expression systems. To establish the biological relevance of these molecules in regulating endogenous transporters in brain, we examined modulation of GABA transport in both neuronal and astrocyte cultures from rat hippocampus. The data from neuronal cultures are shown in Figure 1A. Pretreatment of neurons with phorbol 12-myristate 13-acetate (PMA), a phorbol ester that activates PKC, decreased GABA uptake to 46 ± 4% of control values. A non-PKC-activating phorbol ester, 4
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Figure 1. GABA uptake in hippocampal neurons is regulated by botulinum toxin C1 and protein kinase C. A, Modulation of GABA uptake in neuronal cultures. Drug and toxin concentrations (in µM) are shown below the graph. Pretreatment (30 min) of cultures with PMA, but not 4
To examine the role of components of the docking and fusion apparatus on endogenous GABA transport in hippocampal neurons, we used clostridial toxins to inactivate particular synaptic vesicle and plasma membrane-trafficking proteins. These results are shown in Figure 1A. Pretreatment of neurons with botulinum toxin C1 (BONT/C1), which specifically cleaves syntaxin 1A near its membrane anchor (Blasi et al., 1993
Modulation of GABA transport by PMA and BONT/C1 does not occur in pure astrocyte cultures also prepared from rat hippocampus. These data are shown in Figure 1B. Under the same treatment conditions used for the pure neuronal cultures, PMA (92 ± 11%), BONT/C1 (105 ± 6%), or both compounds together (88 ± 11%) failed to alter GABA uptake significantly when compared with that in untreated control cultures. The lack of effect with BONT/C1 is consistent with the lack of syntaxin 1A in these astrocyte cultures, as assessed by immunoblot using a syntaxin 1A-specific antibody (data not shown). Treatment of astrocytes with SKF89976A did inhibit uptake significantly (57 ± 6%), suggesting that the GAT1 transporter does mediate some GABA uptake in these astrocytes. However, other GABA transporters may also be involved in hippocampal astrocyte uptake because nipecotic acid, a less-selective GABA transporter substrate with higher affinity than GABA, decreased GABA uptake to 8% of control values. These data from hippocampal cultures demonstrate that endogenous GABA transport, specifically in neurons, is regulated by PKC and BONT/C1 and that the PKC-induced inhibition is eliminated by BONT/C1.
The modulatory actions of PKC and BONT/C1 on endogenous GABA transport prompted us to ask whether these molecules also regulate uptake of other transporters. These results, for endogenous glutamate transport in both neuronal and astrocyte cultures, are shown in Figure 1C. In neuronal cultures, application of PMA increased glutamate uptake (167 ± 12%); this increase is consistent with previous reports (Casado et al., 1993
PKC and botulinum toxin regulation of transport can be recapitulated in PC12 cells expressing GAT1
Given the data from hippocampal neurons suggesting that regulation of transport was mediated via GAT1, we sought an expression system that would mimic the endogenous phenomenology and that would allow us to make a detailed characterization of the mechanisms underlying this regulation. We expressed GAT1 in rat PC12 cells. The data in Figure 2A show that this system faithfully reproduces the PKC and BONT/C1 regulation seen in neurons. PMA treatment resulted in a twofold reduction in GABA uptake that did not occur with 4
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Figure 2. PC12 cells expressing GAT1 are regulated by botulinum toxin C1 and protein kinase C. A, GAT1 regulation in PC12 cells mimics endogenous GABA transporter regulation. Cells were treated as described in Figure 1A and the Results. Data are from two to six different experiments, at least three wells per condition per experiment. GABA uptake under control conditions ranged from 2744 to 8297 fmol/min per mg of protein. Experimental conditions that resulted in a significant change (p < 0.05) from control values are denoted by an asterisk. B, PMA decreases transport Vmax (open circles) compared with that in untreated controls (filled circles). Top, Saturation analysis was performed at six different GABA concentrations for cells in the presence or absence of 1 µM PMA. Bottom, Eadie-Hofstee transformations of these data are shown. C, BONT/C1 increases transport Vmax (open circles) compared with that in untreated cells (filled circles). Experiments are described in B. BONT/C1 concentration was 1 µM. Saturation experiments were performed twice; data shown are from one experiment.
Changes in uptake induced by PMA and BONT/C1 could be produced, in general, either by altering the turnover rate of individual transporters or by altering the number of functional transporters. By analogy with receptor-binding experiments, data obtained from saturation curve analysis are often used to distinguish between these two possibilities; changes in the maximum velocity of transport (Vmax) are indicative of changes in the number of transporter binding sites, and changes in affinity (Km) are indicative of changes in the function of individual transporters. Saturation analysis and Eadie-Hofstee transformations were performed on PC12-GAT1 cells assayed in the presence or absence of PMA (Fig. 2B) and in the presence or absence of BONT/C1 (Fig. 2C). The results demonstrate changes in Vmax that reflect the magnitude of the regulation by these molecules. No changes in Km were seen with either treatment. These data are consistent with a mechanism in which these molecules alter the number of functional transporters, as suggested previously (Corey et al., 1994b
Syntaxin 1A interacts with, and functionally regulates, GAT1
Syntaxin 1A is a plasma membrane protein that regulates neurosecretion (Bennett et al., 1993
Antisense oligonucleotides directed against syntaxin 1A inhibit syntaxin 1A expression and upregulate GABA uptake. These results are shown in Figure 3. PC12 cells stably expressing GAT1 were incubated for 48 hr in media containing sense or antisense oligonucleotides comprising the first 18 bases of the syntaxin 1A coding sequence. Compared with either untreated control cultures or cultures incubated with sense strand oligonucleotides, cultures treated with antisense strand oligonucleotides showed significantly increased levels of GABA uptake (155 ± 12%) (Fig. 3A). Incubation of cells with a 19 base scrambled oligonucleotide did not alter GABA uptake (data not shown). Additionally, BONT/C1-mediated increases in GABA uptake were less in cultures treated with antisense oligonucleotides than in cultures treated with sense oligonucleotides. This result is consistent with the inhibition of syntaxin 1A protein synthesis by the antisense oligonucleotides. To examine the time course of this effect, we incubated PC12-GAT1 cells with syntaxin 1A oligonucleotides for 0, 24, and 48 hr. The magnitude of the increase in GABA uptake was dependent on the length of time that PC12-GAT1 cells were incubated with syntaxin 1A oligonucleotides (Fig. 3B, graph). Parallel PC12-GAT1 samples at each time point were assessed by Western blot using anti-syntaxin 1A or anti-GAT1 transporter antibodies. The magnitude of the increase in uptake was inversely correlated with the amount of syntaxin 1A protein present (Fig. 3B, left blot). There was no change in GABA transporter protein levels at any time point (Fig. 3B, right blot).
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Figure 3. Inhibiting syntaxin 1A expression upregulates GAT1 transport in PC12 cells. A, Syntaxin 1A antisense oligonucleotide treatment of PC12 cells causes an increase in GABA uptake. PC12 cells expressing GAT1 were incubated for 48 hr with 10 µM antisense or sense oligonucleotides as described in Materials and Methods. Some cells were treated 30 min before assay with 1 µM BONT/C1. Data are from three experiments, six wells per condition per experiment. GABA uptake under control conditions ranged from 1865 to 2767 fmol/min per mg of protein. Experimental conditions that resulted in a significant difference (p < 0.05) between the two identified groups are denoted by an asterisk. B, The increase in GABA uptake correlates with a decrease in syntaxin 1A expression. PC12 cells were treated as described in A. [3H]GABA uptake experiments were performed 0, 24, or 48 hr after oligonucleotide application. Parallel samples were harvested for Western blot analysis and probed with antibodies to either syntaxin 1A (lower left blot) or GAT1 (lower right blot).
A specific association between the GAT1 transporter and syntaxin 1A is evident from the data shown in Figure 4. PC12-GAT1 cell lysates were immunoprecipitated using a syntaxin 1A antibody. The presence of GAT1 protein in these immunoprecipitates was assessed by Western blot using GAT1-specific antibodies generated against the C-terminal portion of the GAT1 protein as described previously (Minelli et al., 1995
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Figure 4. Syntaxin 1A interacts with GAT1. A, Syntaxin 1A and GAT1 coimmunoprecipitate. PC12 cells expressing GAT1 were precipitated with anti-syntaxin 1A antibody, and GAT1 was visualized by Western blot using two different anti-GAT1 antibodies (346J and 342J). Wild-type PC12 cells probed with antibody 346J and PC12-GAT1 cells probed with preimmune serum served as controls. Antibody 346J was used in all further immunoblot experiments. B, Protein kinase C regulates the interaction between syntaxin 1A and GAT1. Cells were pretreated for 30 min with PMA or BONT/C1 before precipitation with the syntaxin 1A antibody. C, The interaction between syntaxin 1A and GAT1 is specific. 1F9 cells were cotransfected with either syntaxin 1A (Syn 1A) or syntaxin 3 (Syn 3). Forty-eight hours later, [3H]GABA uptake assays were performed. Data are from three experiments, six wells per condition per experiment. GABA uptake under control conditions ranged from 365 to 923 fmol/min per mg of protein. Experimental conditions that resulted in a significant change (p < 0.05) from control values are denoted by an asterisk. Top blot, Immunoprecipitation with either syntaxin 1A or syntaxin 3 antibodies was performed on parallel samples and Western blotted with the GAT1 antibody. Bottom blots, Specificity of each antibody for syntaxin 1A and syntaxin 3 was determined by immunoblot of membranes prepared from oocytes expressing either syntaxin subtype. For all immunoblot experiments, equal amounts of protein were added in each lane.
As shown above in both hippocampal neurons and PC12-GAT1 cells, BONT/C1 prevented PKC-mediated inhibition of GABA uptake. These data suggested that PKC and syntaxin 1A might be mediating their regulatory actions via a common pathway. To test this hypothesis, we treated PC12-GAT1 cells with PMA for 30 min before immunoprecipitation with the syntaxin 1A antibody. The results of this experiment are shown in Figure 4B. Compared with no treatment of PC12-GAT1 cells, PMA treatment resulted in an increase in the amount of GAT1 protein in complex with syntaxin 1A, as assessed by subsequent Western blotting with antibody 346J. Pretreatment of PC12-GAT1 cells with BONT/C1 greatly reduced the amount of GAT1 in complex with syntaxin 1A. These data show that PKC regulates the interaction between syntaxin 1A and GAT1 and that this interaction requires intact, membrane-anchored syntaxin 1A.
To examine the specificity of the association between the GAT1 transporter and syntaxin 1A, we coexpressed either syntaxin 1A or syntaxin 3 with the stably expressing GAT1 cell line 1F9 (Corey et al., 1994a
To support the hypothesis that syntaxin 1A specifically and functionally associates with GAT1, several additional control experiments were performed. First, potential cross-reactivity for the syntaxin 1A and syntaxin 3 antibodies was evaluated by injecting Xenopus oocytes with cRNA encoding syntaxin 1A, 3, or 4, and by subjecting the oocytes to immunoblot analysis. The bottom two blots in Figure 4C show that the antibodies are subtype-specific. Second, to eliminate the possibility that the syntaxin 3 antibody was nonfunctional in immunoprecipitation experiments, we used GAT1 to precipitate the complex and then probed the precipitate with the syntaxin antibodies. Results similar to those shown in Figure 4C were obtained (data not shown).
Reconstitution of PKC-mediated regulation of GAT1
Although PKC regulation of transporters is well described (see Beckman and Quick, 1998
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Table 1. Reconstitution of syntaxin inhibition and PKC sensitivity in 1F9 cells. aData are from 2-4 experiments, 4-6 wells/experiment. bAll data in this Table are expressed relative to this control condition. Control uptake values range from 514 to 1160 fmol/min/mg protein. cExperiment not performed. dp<0.05 compared to untreated GAT1. ep<0.05 compared to untreated GAT1/Syntaxin 1A. fp<0.05 compared to untreated GAT1/Syntaxin 1A. gp<0.05 compared to PMA-treated GAT1 and compared to untreated GAT1/Syntaxin 1A/Munc 18.
We next examined regulation of GABA transport in 1F9 cells expressing GAT1 and syntaxin 1A (Table 1, second row) and compared these results with 1F9 cells expressing GAT1 alone. In untreated GAT1- and syntaxin 1A-expressing cells, GABA transport was reduced approximately twofold. BONT/C1 reversed this inhibition. However, PMA failed to regulate the syntaxin 1A inhibition of uptake. Taken together with the PMA data from hippocampal neurons and PC12 cells, these data suggest that syntaxin 1A is not sufficient to confer PKC regulation of GAT1.
In neurons and PC12 cells, syntaxin 1A interacts with several proteins involved in neurosecretion, including Ca2+ channels, synaptosomal associated protein-25, and Munc18 (Hata et al., 1993
A GAT1 mutant that does not interact with syntaxin 1A is not regulated by PKC
The 1F9 cell reconstitution experiments and the PC12 coimmunoprecipitation experiments suggested that PKC regulates the availability of syntaxin to interact with, and functionally inhibit, the transporter. To test this hypothesis, we generated a number of GABA transporter mutants and examined their ability to both (1) interact with syntaxin 1A and (2) be modulated by PKC. We reasoned that mutants in which syntaxin 1A and GAT1 could not be coimmunoprecipitated would be insensitive to modulation by PKC. Data from two of these mutants are shown in Figure 5. GAT1
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Figure 5. GAT1 mutants that fail to interact with syntaxin 1A are not regulated by PKC. A, A GAT1 mutant lacking a leucine heptad repeat sequence does not interact with syntaxin 1A. Mutant GABA transporters lacking the cytoplasmic N-terminal tail (
DISCUSSION
The data from the present experiments are consistent with the model of GABA transporter regulation shown in Figure 6. The principal findings are the following: (1) GABA transport in cells that endogenously express a GABA transporter and syntaxin 1A can be regulated by PKC. (2) Syntaxin 1A associates with GAT1. This result is revealed via coimmunoprecipitation of a complex containing these two proteins. (3) Syntaxin 1A inhibits GAT1 transporter function. This result is supported by four lines of evidence. Botulinum toxin cleavage of syntaxin 1A upregulates transport, antisense oligonucleotide inactivation of syntaxin 1A upregulates GABA transport, coexpression of syntaxin 1A and GAT1 in reconstitution experiments downregulates GABA transport, and a GAT1 transporter mutant that fails to interact with syntaxin 1A is not regulated. (4) PKC, via a network of protein interactions, inhibits GABA uptake by regulating the availability of syntaxin 1A to interact with the transporter. This result is supported by four lines of evidence. PKC inhibition of GABA uptake is eliminated in cells treated with botulinum toxin; more syntaxin 1A is found associated with GAT1 in PKC-treated immunoprecipitates; reconstitution of PKC inhibition of GAT1 requires both syntaxin 1A and Munc18, a high-affinity syntaxin 1A-binding partner that is a substrate for PKC phosphorylation; and a GAT1 transporter mutant that fails to interact with syntaxin 1A is not regulated by PKC.
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Figure 6. A model for the regulation of GAT1 by syntaxin 1A and protein kinase C. In the absence of PKC, syntaxin 1A can bind to many partners including GAT1. Phosphorylation of these binding partners, e.g., Munc18 or n-Sec1, results in a greater number of syntaxin 1A and GAT1 interactions.
The twofold modulation of GABA transport by syntaxin 1A and PKC places constraints on the likely physiological role of transporter regulation in synaptic signaling. Transporter turnover rates are slow (Mager et al., 1993
The evidence of a role for syntaxin 1A in regulating the GAT1 transporter is consistent with the evolving concept of syntaxin 1A as a general regulator of excitability protein function. Although originally characterized as a component of the machinery involved in the trafficking, docking, and fusion of small synaptic vesicles at the plasma membrane (Bennett et al., 1992
For calcium channels and CFTR, the interaction with syntaxin 1A occurs via direct protein-protein interactions. Calcium channels that are regulated by syntaxin 1A have a "synprint," a conserved region of amino acids that directly interact with synaptic proteins (Sheng et al., 1994
Also to be determined is the mechanism by which syntaxin 1A alters the number of functional transporter molecules. Interactions with syntaxin 1A may put plasma membrane transporters in an inactive conformation; alternatively, syntaxin 1A could control the trafficking of transporters to and from the plasma membrane. This latter hypothesis is supported by the evidence of regulated redistribution of expressed GAT1 transporters to and from subcellular compartments in oocytes (Corey et al., 1994b
PKC-mediated regulation of neurotransmitter transport is well described (for review, see Beckman and Quick, 1998
Our data on modulation of endogenous glutamate transport lends support to the idea of multiple pathways for PKC-mediated regulation. In both neurons and glia, PMA induces an increase in glutamate transport that occurs in the presence of BONT/C1. This syntaxin 1A-independent increase in transport may be attributable to direct phosphorylation of the transporter by PKC (Casado et al., 1993
In summary, the present data show that syntaxin 1A regulates transporter function and that PKC exerts its effects on neurotransmitter uptake by controlling the interaction between these two neuronal proteins. These data also imply the converse, namely, that the transporter could control the availability of syntaxin 1A to interact with its other binding partners: calcium channels and components of the docking and fusion apparatus. If the availability of syntaxin 1A is limiting, then the participation of syntaxin 1A in vesicle release might preclude it from downregulating transporter function. Thus, transporter function will be positively correlated with neurotransmitter release; such a mechanism would provide one route by which neurons could exert greater control over transmitter-mediated synaptic signaling.
FOOTNOTES Received Feb. 19, 1998; revised May 26, 1998; accepted May 28, 1998.
These experiments were supported by a United States Public Health Service Grant (DA-10509), the Epilepsy Foundation of America, and the W. M. Keck Foundation (931360). We thank Nick Brecha for providing the anti-GAT1 antibodies, Zucheng Ye for instructions on preparing primary astrocytes and for providing initial cultures, Kevin Kirk for providing PC12 cells and syntaxin and Munc18 cDNAs and antibodies, and Smith-Kline Beecham for a sample of SKF89976A.
Correspondence should be addressed to Dr. Michael W. Quick, Department of Neurobiology, CIRC 446, 1719 Sixth Avenue South, Birmingham, AL 35294-0021.
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