Neurochem Res (2014) 39:2225–2233 DOI 10.1007/s11064-014-1424-x

ORIGINAL PAPER

Calcium Dependent Interaction of Calmodulin with the GlyT1 C-terminus Andrea Mihalikova • Martina Baliova Frantisek Jursky



Received: 15 May 2014 / Revised: 19 August 2014 / Accepted: 21 August 2014 / Published online: 29 August 2014 Ó Springer Science+Business Media New York 2014

Abstract The cytoplasmic regions of neurotransmitter transporters play an important role in their trafficking. This process is, to a high extent, tuned by calcium and calcium binding proteins, but the exact molecular connection are still not fully understood. In this work we found that the C-terminal region of the mouse glycine transporter GlyT1b is able to specifically interact with calmodulin in the presence of calcium. We found that several GlyT1 C-terminal mutations, including those in the ER retention signal, either eliminate or increase calmodulin interaction in vitro. In tissue-culture-expressed GlyT1 at least two of these mutations altered the sensitivity of GlyT1 surface expression and glycine uptake to calmodulin antagonists. These results suggest the possible involvement of calmodulin or calmodulin-like interactions in the regulation of GlyT1Cmediated transporter trafficking. Keywords

Glycine  Transporter  Calmodulin  Calcium

Introduction Glycine, a proteinogenic amino acid, is present in all tissues including the central nervous system (CNS), where it also functions as a major neurotransmitter [1]. In the CNS glycine concentration is controlled by two high affinity glycine transporters, GlyT1 and GlyT2, members of the larger family of sodium dependent neurotransmitter

A. Mihalikova  M. Baliova  F. Jursky (&) Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovakia e-mail: [email protected]

transporters (SLC6) [2–5]. While both GlyT1 and GlyT2 regulate glycine concentration in the vicinity of inhibitory glycine receptors [6], GlyT1 also participates in excitatory neurotransmission as a part of the NMDA receptor complex [8–11]. Consistent with the transporter’s vital functions, mutations disrupting the GlyT1 function produce a phenotype resembling hereditary human glycine encephalopathy [6, 7] while disruption of GlyT2 is associated with hyperekplexia [12, 13]. Additionally, glycine transporters as drug targets are promising tools for treatment of illnesses such as schizophrenia and neuropathic pain [14]. While the highly conserved core of both transporters has adopted only small changes during the evolution [15, 16], the large, extracellular glycosylated loop and the cytoplasmatically protruding N- and C-terminal regions are not well conserved [17–19]. These regions are not directly involved in transport [20], however it has been found recently that they contain several important regulatory signals [21–29]. Due to their disordered character, information about the 3D structure of these regions is still not available. However, this character probably allows them to adopt several different conformations and to promiscuously interact with numerous partners, most of which still remain to be identified [30]. In the C-terminal region of GlyT1 two important signals for the trafficking and exit of GlyT1 from the endoplasmic reticulum have been identified. First, mutations to the GlyT1 PDZ-interacting motif, -SRI, located at the very C-terminus of GlyT1 delay its delivery to the plasma membrane [31]. Second, a mutational analysis of GlyT1 and several other neurotransmitter transporters revealed an evolutionary conserved RL region, which is important for the exit of transporters from the endoplasmic reticulum [27]. Additionally, it has recently been reported by two groups that this region is important for interaction of GAT1

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[32] and GlyT1 [27] with Sec24D, a component of the COPII (coatomer coat protein II) complex. Although neurotransmitter transporters are generally affected by pathways and proteins regulated by calcium [33–37], no direct interaction of calmodulin with neurotransmitter transporters has been reported so far. Calmodulin (CaM) is an important calcium sensor, which has been previously shown to interact with several membrane receptors [38–41]. Here we identified several amino acids located within or downstream of the SEC24 interaction motif of GlyT1, which are structurally important for the calcium dependent interaction of the transporter C-terminus with calmodulin in vitro. We show that mutations affecting the CaM binding site also alter GlyT1 turnover and it’s trafficking to the plasma membrane.

Materials and Methods Materials Affinity purified primary polyclonal rabbit antibodies (epitopes: 603-626) against the mouse GlyT1 C-terminus were used as previously described [42]. Secondary horseradish peroxidase conjugated antibodies were purchased from Merck Millipore (Darmstadt, Germany). Polyclonal rabbit anti-GST antibodies, ECL reagents, Trifluoperazine dihydrochloride 99 % (TFP), W7 and Calmodulin-agarose saline suspension with the extent of labeling C0.5 mg per mL were from Sigma (St. Louis, MO, USA). Streptavidinagarose and Sulfo-NHS-SS-Biotin were from Thermo Scientific (Rockford, IL, USA). Oligonucleotides were synthesized by Microsynth AG (Balgach, Switzerland). Tris (Hydroxymethyl) aminomethane (Tris) free base, hydroxyethyl piperazineethanesulfonic acid (HEPES) free acid, all molecular biology grade, were from Merck Millipore (Darmstadt, Germany). All other chemicals used were of the per analysis or molecular biology grade. Construction and Overexpression of Wild Type and Mutated GST-mGlyT1C Fusion Proteins To construct the fusion protein GST-mGlyT1C, the region of the mouse glycine transporter GlyT1b containing aminoacids F561–I638 was amplified by PCR using forward EcoRI primer 50 -ttgtacgcagaattccagctctgccgc-30 and reverse SalI primer 50 -ctcatgtcgactagtcctggaagcggctgg-30 and the fragment was inserted into pGEX-5X-1 (GE Healthcare, Freiburg, Germany). The site directed DNA mutants of GST-mGlyT1C used in this work were prepared using the Quick-change mutagenesis kit (Agilent; Stratagene Products, La Jolla, California, USA) following the manufacturer’s suggestions,

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with subsequent verification of introduced mutations via DNA sequencing. The plasmids were transformed into BL21 (DE3) cells Merck Millipore (Darmstadt, Germany); a (20 ml) inoculum of the cells was grown overnight and 6 ml of this culture was transferred into a fresh LB flask containing 250 ml LB. The cells were further grown for 2 h at 37 °C. Afterwards the culture was cooled to 18 °C, and fusion proteins were induced overnight by the addition of IPTG to a final concentration 0.3 mM in a cooled shaker at 180 rpm and 18 °C. The E. coli cells were spun down and resuspended in an ice cold homogenization solution containing 25 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA and 1 % Triton X100. The cells were then opened with sonication on ice and the homogenate was centrifuged at 9,0009g for 15 min at 4 °C. The supernatant was mixed with GST resin (GE Healthcare, Freiburg, Germany) for 30 min at room temperature, washed with homogenization buffer containing Triton X100 reduced to 0.1 %. The resins with the immobilized proteins were stored until use at -20 °C. Interaction of Calmodulin with GST-mGlyT1C Fusion Protein GST, the GST-mGlyT1C fusion protein, and its mutants and truncated forms were isolated on Glutathione Sepharose 4B and eluted with 10 mM glutathione, 50 mM Tris– HCl pH 8.0. Protein determination was performed according to Bradford [43]. Proteins were then diluted with 0.7 ml binding buffer containing 25 mM Tris–HCl pH 7.5 and 50 mM NaCl supplemented either with 1 mM CaCl2 or 5 mM EGTA pH 7.5. The proteins in this diluted eluate were preabsorbed on 0.03 ml of Sepharose 4B-CL beads to remove aggregates and to minimize subsequent, nonspecific binding. Following the addition of 0.03 ml CaMagarose, the sample was mixed for 30 min at room temperature washed 3 times with 1 ml of binding buffer and eluted for 10 min with a minimal amount of binding buffer containing 4 mM EGTA. Eluted samples were then supplemented with SDS sample buffer, boiled, resolved by 12 % PAGE, transferred on immobilon and the proteins were detected with anti-GST antibodies. Construction of GlyT1 Plasmids for Tissue Culture Expression For tissue culture expression, the coding regions of the mouse GlyT1 cDNA and its site directed mutants were inserted downstream of the CMV promoter into the EcoRISalI sites of plasmid pEGFPN1D [44]. The N-terminal region contained an EcoRI site and a Kozak sequence upstream of the methionine-coding triplet (gaattcgccaccatg) [45].

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Cell Transfection, Surface Biotinylation, Uptake Assays, Fluorescence Localization Studies and Western Blot Analysis N2a from mouse neuroblastoma cell lines obtained from the American Type Culture Collection (Rockville, Maryland, USA) were cultured in high glucose Dulbecco’s modified Eagle’s medium, supplemented with 10 % fetal calf serum and 1 % penicillin–streptomycin (PAA Laboratories GmbH, Pasching, Austria). Cells were maintained under standard cell-culture conditions at 37 °C in a humid environment containing 5 % CO2. For the biotinylation experiments [46], 2.4 9 105 cells were seeded on a 35 mm diameter tissue culture plate and the next day cells were transfected with 8 lg of DNA of mGlyT1b wild type and mutant variants using TurboFectTM Thermo Scientific (Rockford, IL, USA) according to the manufacturer’s instructions. Cells were supplemented with fresh medium 24 and 48 h following transfection. Two hours after the second medium replacement the cells were washed 29 with 2 ml of ice cold PBS pH 7.8 and biotinylated on ice for 30 min with 1 ml of SulfoNHS-SS-biotin, dissolved 1 mg/ml in ice cold PBS. Cells were then washed 39 with 2 ml of ice cold PBS and overlaid with 0.8 ml lysis buffer containing 50 mM Tris– HCl pH 7.5, 150 mM NaCl, 1 % Triton X100, 10 mM EDTA, 0.1 % SDS and lysed on ice for 1 h with shaking. Detached cells were collected into a 1.5 ml tube, centrifuged for 10 min at 13,0009g in a microfuge and all aliquot of supernatant was saved as a sample representing the total protein fraction. The rest of the supernatant was mixed for 1 h at room temperature with 0.05 ml of 50 % v/v streptavidin-agarose. Following washing with lysis buffer (3 9 1.5 ml) proteins were eluted from the agarose with SDS/2-ME sample buffer. Samples representing both total proteins and surface localized proteins isolated on streptavidin agarose were resolved on 7.5 % PAGE and immunobloted with anti-GlyT1C603-626 antibodies. For the uptake experiments 24 well plates transfected 2 lg/well with wild type mGlyT1 and its mutated variants, were used to assay glycine uptake as previously described (44). Uptake was performed for 1 min at 25 °C with 100 lM external glycine. Nonspecific uptake was defined with mock-transfected N2a cells (cells transfected with an empty expression plasmid) and data were normalized by protein concentration. For the investigation of the trifluoperazine and W7 effects on the surface expression of GlyT1, N2a cells transfected with wild type and mutated transporters were treated for 1 h in serum and antibiotic-free high glucose DMEM with various concentration of TFP or W7 before they were subjected to surface biotinylation and uptake assays [47]. Data were measured in duplicates and

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analyzed using GraphPad Prism 4.00 for Windows GraphPad Software (San Diego, CA, USA). Results represent at least two independent experiments. For fluorescence localization studies, the methionine initiation codon of wild type and mutant mGlyT1b was fused in frame to the 30 end of enhanced green fluorescent protein (EGFP) in plasmid pEGFP-C1 Clontech Laboratories, Inc. A Takara Bio Company (Mountain View, USA). Transfected N2a cells were washed with HBS (25 mM HEPES– NaOH, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,) and fixed with 2 % paraformaldehyde in HBS for 5 min at room temperature [48]. Following washing with HBS, cells were covered with coverslips and observed through a Leica fluorescence filter cube N2.1 (Leica Microsystems, Wetzlar, Germany). Pictures were further processed using ImageJ (http://imagej.nih.gov/ij). Homology Modeling of the mGlyT1b C Terminal Region The protein sequences for the mGlyT1b (SLC6A9 UniProt ID: O35633) and drosophila dopamine dDAT (SLC6A3 UniProt ID: Q7K4Y6) transporter proteins were obtained from the UNIPROT database (http://www.uniprot.org). The structure of the dDAT inward-closed conformation (PDB ID: 4M48) [16] was obtained from the RCSB protein data bank, (www.pdb.org). All water molecules, and detergent molecules and the Fab part were removed from the structure before modeling and the dDAT protein sequence was aligned with mGlyT1b using the program Clustal-W [49] available on the European Bioinformatics Institute’s server (http://www.ebi.ac.uk). Homology model of the GlyT1 in inward-closed conformation was then constructed using this alignment and the dDAT transporter structure 4M48 as a template with help of the program MODELLER9v8 [50]. The default parameters (model-single.py) were used for modeling with the following additions: modeling of the disulphide bridge in the large extracellular loop was included and during the modeling the glycine substrate molecule, chloride and sodium ions were transferred into the new model using the modeller BLK function. The modeller run was set up to produce ten models and the model with the lowest value of the Modeller objective function was then chosen. PyMOL (The PyMOL Molecular Graphics System, Schro¨dinger, LLC; http://www. pymol.org) was used to visualize the structures.

Results To investigate interaction of calmodulin with the mouse GlyT1 C-terminus we incubated GST-mGlyT1C fusion protein, as well as GST as a negative interaction control, with

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calmodulin immobilized on agarose beads in the presence or absence of calcium. Bacterial expression of the fusion proteins was performed at lowered temperature to allow proper protein folding and to prevent the creation of inclusion bodies. Since the calmodulin interaction has a strongly hydrophobic component, we excluded detergents from our assay. To minimize nonspecific interactions, the GSTmGlyT1C fusion protein was eluted via calcium chelation. Eluted proteins were resolved on PAGE, transferred on immobilon and visualized using anti-GST antibodies. Figure1a shows that a GST fusion protein containing in frame mGlyT1 C terminal sequences is able to interact with calmodulin in a calcium dependent manner, while no such interaction is observed with the GST fusion partner alone. The interaction significantly decreases in the presence of the calmodulin antagonist trifluoperazine (TFP) (Fig. 1b) and exhibits sigmoidal dependence (Fig. 1c). To more closely examine the CaM binding region we replaced the tryptophan 586 coding triplet with a TGA stop

Fig. 1 Interaction of the mouse GlyT1 C terminus with CaMagarose. a The control GST protein and the GST-GlyT1C fusion protein were interacted for 30 min at room temperature with calmodulin immobilized on agarose in the presence of calcium. After washing, the proteins were eluted with EGTA and immunobloted with anti-GST antibodies. Load indicates the position and relative amount of the proteins present in the interacting solution. CaM pull-down shows proteins eluted with EGTA. b CaM pull-down shows the amount of GST-GlyT1C eluted with EGTA which previously interacted with CaM-agarose in the absence or presence of the calmodulin antagonist trifluoperazine. c Dose response curve of the GST-GlyT1C interaction with CaM-agarose. Similar results were obtained in two independent experiments

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codon. This resulted in the production of a GST fusion protein during translation containing only the first quarter of the GlyT1 C-terminal protein. This truncated version of the fusion protein encompassed the GST-mGlyT1C region F561–D585 and had lost more than 90 % of its calmodulin binding ability (Fig. 2a, b). Similarly we used site-directed DNA mutagenesis to introduce a second EcoRI restriction site into GlyT1C region W586–G587–P588. Cleavage of mutated DNA with EcoRI and subsequent religation of the plasmid resulted in the loss of the DNA coding sequence corresponding to the GlyT1C fragment F561–P588. Here again the truncated version of the fusion protein containing residues A589–I638 did not interact with calmodulin (Fig. 2a, b). To examine whether the multiple W586– G587–P588 to G586–E587–F588 mutations associated with the introduction of EcoRI itself affect CaM binding, we overexpressed the corresponding fusion protein and interacted it with CaM-agarose. As shown in Fig. 2a, b the mutations eliminated CaM binding, indicating that the W586–G587–P588 region is somehow important for CaM binding. Finally, mutation of the individual WGP aminoacids showed that calcium dependent CaM binding is largely not affected by G587A, it seems to be further increased by P588A, but is totally eliminated by W586A (Fig. 2c, d). In the GlyT1 C-terminus, a conserved region R575– L576–X8–D585 containing an endoplasmic retention/ SEC24 binding motif has been previously identified [27]. In this region mutations to R575 and D585 have a significant effect on GlyT1 function. In our experiments mutation R575A in the GST-mGlyT1C fusion protein resulted in the elimination of its binding to CaM-agarose while D585A, on the other hand significantly increased it (Fig. 3). To investigate the effect of these mutations on GlyT1 trafficking we introduced them into the mouse GlyT1b sequence and compared total versus surface expression in mouse neuroblastoma cell line N2a using surface biotinylation, glycine uptake and GFP fluorescence. Since the trafficking of mutants R575A and D585A has been investigated in detail elsewhere [27], we focused on mutations in the GlyT1 WGP region. As shown in Fig. 4a, mutations W586A and P588A but not G587A significantly decreased both total and surface expression of the transporter. Consistent with this, expression of GFP tagged GlyT1 constructs revealed that a large amount of the W586A and P588A mutated transporters accumulate intracellularly and in the perinuclear region while the major fluorescence signals of wild type GlyT1 and the G587A mutant localized on the cell surface (Fig. 4b). To investigate effect of calmodulin antagonists on GlyT1 trafficking we exposed cells which had been expressing GlyT1 for 1 h to various concentrations of trifluoperazine

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Fig. 2 The effect of various deletions and single amino-acid substitutions in GlyT1C on its binding to CaM-agarose. The upper panels of A and C indicate the position and relative amount of proteins present in the interaction solution (load). The lower A and C panels show those proteins eluted from CaM agarose with EGTA. Panels B and D depict the deletions and mutations corresponding to the interactions shown in panels A and C. Similar results were obtained in two independent experiments

Fig. 3 Calmodulin binding of the GST-mGlyT1C fusion proteins mutated in the SEC24 interaction motif. Control GST, GSTmGlyT1C, GST-mGlyT1C-R575A and GST-mGlyT1C-D585A fusion proteins were interacted with calmodulin agarose in the presence of calcium, and, following washing, the interacting proteins were eluted with EGTA. Similar results were obtained in two independent experiments

(TFP) and W7 in serum-free DMEM (to avoid possible interference from serum components [47]). Subsequently, the amount of surface localized GlyT1 was investigated using surface biotinylation (Fig. 5a) and glycine uptake (Fig. 5b, c, d). The CaM antagonist TFP downregulated both GlyT1 surface expression and GlyT1 uptake. These results were corroborated by a similar decrease in GlyT1 uptake induced by another calmodulin antagonist W7. Surprisingly,

Fig. 4 Total and surface expression of single amino-acid mutants in the C-terminal WGP region of mouse GlyT1. a N2a cells expressing the indicated GlyT1 constructs were biotinylated on the surface with a membrane impermeable biotinylation reagent and surface localized GlyT1 was isolated using streptavidin agarose. Both total and surface localized GlyT1 were resolved in 7.5 % PAGE and immunobloted with anti-GlyT1C603-626 antibodies. Filled and open arrows indicate glycosylated and nonglycosylated forms of GlyT1. Similar results were obtained in two independent experiments. b Localization of wild type mGlyT1b (WT) and its W586A, G587A and P588A mutants in N2a cells. All transporters were fused with enhanced green fluorescence protein and transfected into N2a cells; the localization of GFP fluorescence was examined under a fluorescence microscope. Note the preferential cell surface localization of the wild type and G587A mutant and the significant intracellular accumulation of the W586A and P588A mutants. To reduce background noise the images were processed with a minimum ImageJ filter using pixel radius 0.1. Processing did not substantially change the figure appearance. Scale bar 20 lm. Similar results were obtained in two independent experiments

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Fig. 5 The effect of calmodulin antagonists TFP and W7 on the surface expression of wild type, W586A, and P588A mutated GlyT1. a Total and surface expression of control and TFP treated cells expressing wild type, W586A and P588A mutated GlyT1. b, c, d Glycine uptake of control, TFP and W7 treated cells expressing wild type, W586A, and P588A mutated GlyT1. Similar results were obtained in two independent experiments. 100 % represents 0.6 nM of glycine/mg/min for the wild type 0.45 nM of glycine/mg/min for the W586A mutant and 0.43 nM of glycine/mg/min for the P588A mutant

Fig. 6 A model of the GlyT1 C-terminal endoplasmic reticulum retention motif. The primary amino acid sequences of mGlyT1b and drosophila dopamine transporter dDAT were aligned using ClustalW. This alignment was then used as the input for homology modeling of mGlyT1 with dDAT (PDB 4M48) using MODELLER9v8. Positive amino acids (R, K) are marked in blue, negative amino acids (D) are in red. The last transmembrane region (TM12) of GlyT1 is in orange (Color figure online)

both W586A and P588A mutations further significantly increased the sensitivity of GlyT1 to TFP and W7 compared to the wild type (Fig. 5b, c, d). The crystal structure of a homologous protein, the drosophila dopamine transporter (dDAT), has recently been solved, however [16]. The C-terminal region of this protein is homologous to the GlyT1 C-terminal region upstream of tryptophan-586. Analysis of a GlyT1C homology model build using this structure as a template revealed that five of the basic positively charged residues, which are part of the R575–L576–X8–D585 region containing the endoplasmic retention/SEC24b binding motif, [27] are in position to potentially interact with the acidic, negatively charged calmodulin (Fig. 6).

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Discussion We have recently found several calpain cleavage sites in the glycine transporter GlyT1 C-terminus [42]. Calpain contains calmodulin-like domains, which makes its substrates potential calmodulin binding proteins [51]. To verify the hypothesis that the GlyT1 C terminus is such a protein, we set up a simple interaction pull-down assay using immobilized calmodulin and a GST-GlyT1 C terminal fusion protein. We found that the C-terminal region of GlyT1 does indeed interact with calmodulin in a calcium-dependent manner and moreover that this interaction can be eliminated by the calmodulin antagonist trifluoperazine (TFP).

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We further found that the calmodulin inhibitors TFP and W7 also downregulate the surface expression of GlyT1 in N2a cells as determined by a glycine uptake assay and surface biotinylation. Although the GlyT1 W586A mutant does not interact with CaM in vitro and the interaction of P588A with calmodulin has increased, in cell lines they both appear to be much more sensitive to calmodulin antagonists than the wild type transporter. Similarly, the R575A mutant eliminated CaM binding in vitro while the D585A mutant increased it. In these two mutants, however, the sensitivity of glycine uptake to TFP remained identical to the wild type after their expression in cell lines (not shown). These results suggest that potential in vivo regulation of GlyT1 by CaM is probably complex. One of the explanations for these apparently contradictory results could be the existence of a larger, tight regulatory complex in which CaM is simultaneously attached to both the GlyT1 C terminus and other interacting partners. In some cases, mutations which either disrupt or enhance calmodulin binding to the GlyT1 C terminus might alter the functionality exhibited by the complex in similar ways by increasing the accessibility of calmodulin within the partially disrupted complex to TFP or W7. In any case, the fact that certain mutations affecting CaM binding to GlyT1C in vitro also affect the sensitivity of GlyT1 trafficking to two calmodulin antagonists suggests that the GlyT1 C-terminus and CaM are involved in the regulation of GlyT1 function. The differential sensitivity of the GlyT1 wild type and mutants in cell lines suggests that this effect is not mediated through the previously reported nonspecific effects of calmodulin antagonists [52]. How calmodulin interacts with the GlyT1 C terminus is presently not clear. Our mutational analysis revealed the crucial role of tryptophan 586 and proline 588 for CaM binding, but residues R575 and D585, which were previously identified as important structural determinants of the ER retention signal, also either eliminated or increased the calmodulin interaction. A previous investigation of R575 and D585 mutations revealed that they have significant effect on GlyT1 trafficking, and prevent the transporter from reaching the plasma membrane [27]. Here we show that mutations W586A and P588A have a similar effect on surface immunoreactivity and GFP fluorescence. Since all these mutations affect the ability of calmodulin to bind the GlyT1 C-terminus, it suggests that the WGP region identified here might be part of the ER retention signal. Calmodulin interactions usually have both ionic and hydrophobic components. The hydrophobic component frequently involves tryptophan [53], which is consistent with the sensitivity of CaM binding to the W586A mutation. Due to its highly acidic character under physiological pH calmodulin adopts a significant negative charge, which is subsequently neutralized via interactions with basic, positively charged interaction partners.

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To see if such a potentially basic region can be found in the GlyT1 C-terminus we constructed a 3D homology model using the crystal structure of the drosophila dopamine transporter dDAT as a template. Significant primary amino-acid homology and identical function suggests that structural alignment of GlyT1 with dDAT protein sequences may provide reliable 3D model of GlyT1C terminal region upstream of tryptophan 586. Our model revealed that the region R575–L576–X8– D585 which contains five basic, positively charged aminoacids encompasses a a-helix. Positively charged R575 and negatively charged D585 lie at the ends of this helix. Consistent with this the elimination of positively charged R575 also eliminated CaM binding, while the elimination negatively charged D585 further increased it. This indicates that R575 not only has an important role for ER retention signaling but is also crucial for the interaction with calmodulin. Possibly, the negative charge of D585 might moderate the strength of the interaction, which could be important for subsequent dissociation during biological signaling. While charge and hydrophobicity are known factors influencing CaM interactions, proline 588 is neither charged nor hydrophobic, therefore its effect on CaM binding likely arises from introducing a bend in the protein chain. Calmodulin interactions do not depend on precisely conserved amino-acid sequences and do take place through multiple contacts [54]. This indicates that in addition to R575, D585, W586, and P588, several other amino acid residues might also contribute to the CaM interaction, especially when considering that a number of dynamic charges may occur during the putative calmodulin signaling. There are several cases where calmodulin has been found to regulate the surface expression of membrane proteins by interacting with their C-terminus. For example the L-type Ca2? channel, a-actinin interacts with the C-terminal region of the channel to stabilize it in the membrane. Displacement of a-actinin by competition with calmodulin triggers Ca2? induced endocytosis and provides a negative feedback for Ca2? influx [41]. Similarly CaM bound on KCNQ2 has also been reported to act as a Ca2? sensor, conferring Ca2? dependence to the trafficking of the channels to the plasma membrane [39]. Calmodulin antagonists downregulate GlyT1 in Muller glial cells [55]. Here we found that CaM interacts with the GlyT1 C terminus in a calcium dependent manner and that mutations affecting the CaM binding site alter the trafficking of GlyT1. These results indicate that calmodulin might be directly involved in a calmodulin-regulated GlyT1 trafficking pathway. Alternatively, the interaction site might be part of the protein complexes associated with calmodulin such as calcineurin [38] or calmodulin kinase II [36].

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Acknowledgments This work was supported by the Slovak Grant agency VEGA, Grants 2/0084/13 and 2/0086/13. We thank Dr. G. Bukovska for the access to fluorescent microscope and Dr. J. Bauer for careful reading of the manuscript. 18.

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Calcium dependent interaction of calmodulin with the GlyT1 C-terminus.

The cytoplasmic regions of neurotransmitter transporters play an important role in their trafficking. This process is, to a high extent, tuned by calc...
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