scholarly journals Fluorinated Galactoses Inhibit Galactose-1-Phosphate Uridyltransferase and Metabolically Induce Galactosemia-like Phenotypes in HEK-293 Cells

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 607 ◽  
Author(s):  
Verena Janes ◽  
Simona Grabany ◽  
Julien Delbrouck ◽  
Stephane P. Vincent ◽  
Johannes Gottschalk ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Galactose Metabolism ◽  
Galt Activity ◽  
Hek 293 ◽  
Classic Galactosemia ◽  
Developmental Impairment ◽  
The Unfolded Protein Response ◽  
Metabolic Induction

Genetic defects of human galactose-1-phosphate uridyltransferase (hGALT) and the partial loss of enzyme function result in an altered galactose metabolism with serious long-term developmental impairment of organs in classic galactosemia patients. In search for cellular pathomechanisms induced by the stressor galactose, we looked for ways to induce metabolically a galactosemia-like phenotype by hGALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant hGALT with a KI of 0.9 mM. Contrasting with hepatic cells, no alterations of N-glycoprofiles in MIG (metabolic induction of galactosemia)-HEK293 cells were revealed for an inducible secretory netrin-1 probe by MALDI-MS. Differential fluorescence-activated cell sorting demonstrated reduced surface expression of N-glycosylated CD109, EGFR, DPP4, and rhMUC1. Membrane raft proteomes exhibited dramatic alterations pointing to an affection of the unfolded protein response, and of targeted protein traffick. Most prominent, a negative regulation of oxidative stress was revealed presumably as a response to a NADPH pool depletion during reduction of Gal/F-Gal. Cellular perturbations induced by fluorinated galactoses in normal epithelial cells resemble proteomic changes revealed for galactosemic fibroblasts. In conclusion, the metabolic induction of galactosemia-like phenotypes in healthy epithelial/neuronal cells could support studies on the molecular pathomechanisms in classic galactosemia, in particular under conditions of low galactose stress and residual GALT activity.

scholarly journals Current and Future Treatments for Classic Galactosemia

2021 ◽  
Vol 11 (2) ◽  
pp. 75 ◽  
Author(s):  
Britt Delnoy ◽  
Ana I. Coelho ◽  
Maria Estela Rubio-Gozalbo
Keyword(s):  
Standard Of Care ◽  
Type I ◽  
Therapeutic Approaches ◽  
Restricted Diet ◽  
Galactose Metabolism ◽  
Galt Activity ◽  
Classic Galactosemia ◽  
Pathogenic Factors ◽  
Future Treatments ◽  
Novel Therapeutic

Type I (classic) galactosemia, galactose 1-phosphate uridylyltransferase (GALT)-deficiency is a hereditary disorder of galactose metabolism. The current therapeutic standard of care, a galactose-restricted diet, is effective in treating neonatal complications but is inadequate in preventing burdensome complications. The development of several animal models of classic galactosemia that (partly) mimic the biochemical and clinical phenotypes and the resolution of the crystal structure of GALT have provided important insights; however, precise pathophysiology remains to be elucidated. Novel therapeutic approaches currently being explored focus on several of the pathogenic factors that have been described, aiming to (i) restore GALT activity, (ii) influence the cascade of events and (iii) address the clinical picture. This review attempts to provide an overview on the latest advancements in therapy approaches.

scholarly journals Galactose-1-Phosphate Uridyltransferase Activities in Different Genotypes: A Retrospective Analysis of 927 Samples

2018 ◽  
Vol 3 (2) ◽  
pp. 222-230 ◽  
Author(s):  
Tatiana Yuzyuk ◽  
Andrew R Wilson ◽  
Rong Mao ◽  
Marzia Pasquali
Keyword(s):  
Dietary Intervention ◽  
Finite Mixture Models ◽  
Gene Analysis ◽  
Molecular Testing ◽  
Reference Ranges ◽  
Galactose Metabolism ◽  
Galt Activity ◽  
Classic Galactosemia ◽  
Galt Gene ◽  
Life Threatening

Abstract Background Classic galactosemia is an inherited disorder of galactose metabolism caused by the impaired activity of galactose-1-phosphate uridyltransferase (GALT). Untreated galactosemia is life-threatening; however, early dietary intervention prevents mortality and reduces morbidity associated with this disease. The diagnosis of galactosemia includes the measurement of GALT activity in red blood cells (RBC) and GALT gene analysis. In this study, we evaluate GALT activity in different genotypes using the results of combined biochemical and molecular testing in 927 samples. Methods GALT activity in RBC was measured by LC-MS/MS. The analysis of the GALT gene was performed by targeted gene analysis and/or full gene sequencing. Samples were assigned based on the presence of pathogenic (G) or Duarte 2 (D) variants, or their absence (Neg), to G/G, D/G, G/Neg, D/D, D/Neg, and Neg/Neg genotypes. Finite mixture models were applied to investigate distributions of GALT activities in these genotypes. The reference ranges were determined using the central 95% of values of GALT activities. Results The ranges of GALT activity in G/G, D/G, G/Neg, D/D, D/Neg, and Neg/Neg genotypes are 0.0 to 0.7 μmol·h−1 gHb−1, 3.1 to 7.8 μmol·h−1 gHb−1, 6.5 to 16.2 μmol·h−1 gHb−1, 6.4 to 16.5 μmol·h−1 gHb−1, 12.0 to 24.0 μmol·h−1 gHb−1, and 19.4 to 33.4 μmol·h−1 gHb−1, respectively. Conclusions The GALT activity ranges established in this study are in agreement with the expected impact of the genotype on the enzymatic activity. Molecular findings should be interpreted in view of biochemical results to confirm genotype–phenotype correlation.

scholarly journals The yeast protein Ubx4p contributes to mitochondrial respiration and lithium–galactose–mediated activation of the unfolded protein response

2020 ◽  
Vol 295 (12) ◽  
pp. 3773-3782
Author(s):  
Evandro A. De-Souza ◽  
Felipe S. A. Pimentel ◽  
Ana Luiza F. V. De-Queiroz ◽  
Henrique Camara ◽  
Mikaella L. Felix-Formiga ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Oxygen Consumption ◽  
Unfolded Protein Response ◽  
Mitochondrial Respiration ◽  
Galactose Metabolism ◽  
Classic Galactosemia ◽  
Unfolded Protein ◽  
Yeast Strains ◽  
The Unfolded Protein Response ◽  
Protein Response

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum–associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog–encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium–galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia.

scholarly journals The PDZ-interacting domain of TRPC4 controls its localization and surface expression in HEK293 cells

2002 ◽  
Vol 115 (17) ◽  
pp. 3497-3508
Author(s):  
Laurence Mery ◽  
Bettina Strauß ◽  
Jean F. Dufour ◽  
Karl H. Krause ◽  
Markus Hoth
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Hek 293 ◽  
Protein Insertion ◽  
Cell Surface Biotinylation ◽  
Surface Localization ◽  
Interacting Domain

Mammalian homologs of the Drosophila TRP protein have been shown to form cation-permeable channels in the plasma membrane but very little is known about the mechanisms that control their cell surface localization. Recently it has been demonstrated that the last three C-terminal amino acids(TRL) of TRPC4 comprise a PDZ-interacting domain that binds to the scaffold protein EBP50 [ezrin/moesin/radixin-binding phosphoprotein 50]. In this report, we have examined the influence of the TRL motif on the subcellular distribution of TRPC4 in human embryonic kidney (HEK) 293 cells. We have also analyzed the consequences of the interaction between EBP50 and the membrane-cytoskeletal adaptors of the ezrin/radixin/moesin (ERM) family for the cell surface expression of TRPC4. Using immunofluorescence microscopy, we found that the mutant lacking the TRL motif accumulated into cell outgrowths and exhibited a punctate distribution pattern whereas the wild-type channel was evenly distributed on the cell surface. Deletion of the PDZ-interacting domain also decreased the expression of TRPC4 in the plasma membrane by 2.4-fold, as assessed by cell surface biotinylation experiments. Finally, in a large percentage of cells co-expressing TRPC4 and an EBP50 mutant lacking the ERM-binding site, TRPC4 was not present in the plasma membrane but co-localized with the truncated scaffold in a perinuclear compartment (most probably representing the Golgi apparatus) and in vesicles associated with actin filaments. Our data demonstrate that the PDZ-interacting domain of TRPC4 controls its localization and surface expression in transfected HEK293 cells. They also point to a yet unexplored role of the EBP50-ERM complex in the regulation of protein insertion into the plasma membrane.

Abstract 423: Caveolin-3 Enriches and Dynamically Interacts With Swell1

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Daniel G Turner ◽  
Leonid Tyan ◽  
Sami Stroebel ◽  
Frank Deguire ◽  
Di Lang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Hek293 Cells ◽  
Scaffolding Protein ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Hek 293 ◽  
Dynamic Relationship ◽  
293 Cells ◽  
Fret Efficiency ◽  
Oxide Synthase

Caveolae are small (50-100nm) membrane invaginations formed by caveolin proteins enriched with cholesterol and lipids. Caveolae play a crucial role in mechanoprotection and mechano-electrical transduction by buffering membrane tension and facilitating activation of mechanosensitive ion channels, including a recently discovered swelling-activated chloride channel SWELL1 (also known as LRRC8A). However, the dynamic relationship between the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) and SWELL1 is poorly understood. The objective of this study was to determine how Cav3 interacts with SWELL1 channels and modulates their activity during mechano-electrical transduction. In HEK 293 cells transfected with Cav3, co-immunoprecipitation analysis recapitulated cardiac data showing association between SWELL1 and Cav3. Using transiently expressed Cav3-GFP and SWELL1-mCherry fusion proteins in HEK293 cells, we observed a high FRET efficiency between the two proteins in an isotonic (1T) solution, confirming their close (<5nm) proximity. In a hypotonic solution (0.7T, mimicking cell stretch), FRET efficiency decreased two-fold. Furthermore, FRET efficiency decreased two-fold to control levels when incubated with methyl-beta cyclodextrin, a cholesterol solubilizer. These data suggest that the relationship between Cav3 and SWELL1 is dependent on membrane mechanical tension and caveolae lipid raft integrity. Interestingly, in transfected cells, SWELL1 protein expression and whole cell swelling-activated chloride current ( I Cl,swell ) were increased four-fold and two-fold, respectively, while mRNA expression was reduced two-fold. This may indicate that caveolae formed by Cav3 expression enrich for SWELL1 and increase their half-life, thus requiring lower mRNA availability despite higher protein expression. Our findings indicate a close dynamic interplay between Cav3 and SWELL1, with a strong regulatory action of Cav3 on I Cl,swell activity. Given that I Cl,swell increases and interprotein FRET efficiency decreases in hypotonic solution, it is likely that Cav3 inhibits the activation of SWELL1 similarly to its known inhibition of nitric oxide synthase.

scholarly journals Modulation of Kv4.2/KChIP3 interaction by the ceroid lipofuscinosis neuronal 3 protein CLN3

2020 ◽  
Vol 295 (34) ◽  
pp. 12099-12110
Author(s):  
Carolin Seifert ◽  
Stephan Storch ◽  
Robert Bähring
Keyword(s):  
Neuronal Ceroid Lipofuscinosis ◽  
Surface Expression ◽  
Lower Amount ◽  
Hek 293 ◽  
Current Decay ◽  
Ceroid Lipofuscinosis ◽  
Kv Channels ◽  
Channel Expression ◽  
Lysosomal Membrane Glycoprotein ◽  
And Function

Voltage-gated potassium (Kv) channels of the Kv4 subfamily associate with Kv channel–interacting proteins (KChIPs), which leads to enhanced surface expression and shapes the inactivation gating of these channels. KChIP3 has been reported to also interact with the late endosomal/lysosomal membrane glycoprotein CLN3 (ceroid lipofuscinosis neuronal 3), which is modified because of gene mutation in juvenile neuronal ceroid lipofuscinosis (JNCL). The present study was undertaken to find out whether and how CLN3, by its interaction with KChIP3, may indirectly modulate Kv4.2 channel expression and function. To this end, we expressed KChIP3 and CLN3, either individually or simultaneously, together with Kv4.2 in HEK 293 cells. We performed co-immunoprecipitation experiments and found a lower amount of KChIP3 bound to Kv4.2 in the presence of CLN3. In whole-cell patch-clamp experiments, we examined the effects of CLN3 co-expression on the KChIP3-mediated modulation of Kv4.2 channels. Simultaneous co-expression of CLN3 and KChIP3 with Kv4.2 resulted in a suppression of the typical KChIP3-mediated modulation; i.e. we observed less increase in current density, less slowing of macroscopic current decay, less acceleration of recovery from inactivation, and a less positively shifted voltage dependence of steady-state inactivation. The suppression of the KChIP3-mediated modulation of Kv4.2 channels was weaker for the JNCL-related missense mutant CLN3R334C and for a JNCL-related C-terminal deletion mutant (CLN3ΔC). Our data support the notion that CLN3 is involved in Kv4.2/KChIP3 somatodendritic A-type channel formation, trafficking, and function, a feature that may be lost in JNCL.

scholarly journals Effects of GABAA Receptor α3 Subunit Epilepsy Mutations on Inhibitory Synaptic Signaling

2020 ◽  
Vol 13 ◽  
Author(s):  
Parnayan Syed ◽  
Nela Durisic ◽  
Robert J. Harvey ◽  
Pankaj Sah ◽  
Joseph W. Lynch
Keyword(s):  
Expression System ◽  
Epileptic Seizures ◽  
Surface Expression ◽  
Decay Rates ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Decay Kinetics ◽  
Neuronal Synapses ◽  
Α3 Subunit

Missense mutations T166M, Q242L, T336M, and Y474C in the GABAA receptor (GABAAR) α3 subunit gene are associated with epileptic seizures, dysmorphic features, intellectual disability, and developmental delay. When incorporated into GABAARs expressed in oocytes, all mutations are known to reduce GABA-evoked whole-cell currents. However, their impact on the properties of inhibitory synaptic currents (IPSCs) is unknown, largely because it is difficult to establish, much less control, the stoichiometry of GABAAR expressed in native neuronal synapses. To circumvent this problem, we employed a HEK293 cell-neuron co-culture expression system that permits the recording of IPSCs mediated by a pure population of GABAARs with a defined stoichiometry. We first demonstrated that IPSCs mediated by α3-containing GABAARs (α3β3γ2) decay significantly slower than those mediated by α1-containing isoforms (α1β2γ2 or α1β3γ2). GABAAR α3 mutations did not affect IPSC peak amplitudes or 10–90% rise times, but three of the mutations affected IPSC decay. T336M significantly accelerated the IPSC decay rate whereas T166M and Y474C had the opposite effect. The acceleration of IPSC decay kinetics caused by the T366M mutation was returned to wild-type-like values by the anti-epileptic medication, midazolam. Quantification experiments in HEK293 cells revealed a significant reduction in cell-surface expression for all mutants, in agreement with previous oocyte data. Taken together, our results show that impaired surface expression and altered IPSC decay rates could both be significant factors underlying the pathologies associated with these mutations.

scholarly journals Cellular cholesterol controls TRPC3 function: evidence from a novel dominant-negative knockdown strategy

2006 ◽  
Vol 396 (1) ◽  
pp. 147-155 ◽  
Author(s):  
Annarita Graziani ◽  
Christian Rosker ◽  
Sepp D. Kohlwein ◽  
Michael X. Zhu ◽  
Christoph Romanin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transient Receptor Potential ◽  
Lipid Membrane ◽  
Membrane Conductance ◽  
Receptor Potential ◽  
Surface Expression ◽  
Dominant Negative ◽  
Membrane Microdomains ◽  
Membrane Expression ◽  
Hek 293

TRPC3 (canonical transient receptor potential protein 3) has been suggested to be a component of cation channel complexes that are targeted to cholesterol-rich lipid membrane microdomains. In the present study, we investigated the potential role of membrane cholesterol as a regulator of cellular TRPC3 conductances. Functional experiments demonstrated that cholesterol loading activates a non-selective cation conductance and a Ca2+ entry pathway in TRPC3-overexpressing cells but not in wild-type HEK-293 (human embryonic kidney 293) cells. The cholesterol-induced membrane conductance exhibited a current-to-voltage relationship similar to that observed upon PLC (phospholipase C)-dependent activation of TRPC3 channels. Nonetheless, the cholesterol-activated conductance lacked negative modulation by extracellular Ca2+, a typical feature of agonist-activated TRPC3 currents. Involvement of TRPC3 in the cholesterol-dependent membrane conductance was further corroborated by a novel dominant-negative strategy for selective blockade of TRPC3 channel activity. Expression of a TRPC3 mutant, which contained a haemagglutinin epitope tag in the second extracellular loop, conferred antibody sensitivity to both the classical PLC-activated as well as the cholesterol-activated conductance in TRPC3-expressing cells. Moreover, cholesterol loading as well as PLC stimulation was found to increase surface expression of TRPC3. Promotion of TRPC3 membrane expression by cholesterol was persistent over 30 min, while PLC-mediated enhancement of plasma membrane expression of TRPC3 was transient in nature. We suggest the cholesterol content of the plasma membrane as a determinant of cellular TRPC3 activity and provide evidence for cholesterol dependence of TRPC3 surface expression.

Proteomic Analysis of the alphaIIbbeta3 Interactome Reveals Novel Chaperone and Trafficking Proteins, Including An HSP40 Chaperone, DNAJC10, That Regulates alphaIIbbeta3 Surface Expression

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2863-2863
Author(s):  
Amanda Chen ◽  
Mahmoud Yazdani-Abyaneh ◽  
W. Beau Mitchell
Keyword(s):  
Protein Interactions ◽  
Protein Trafficking ◽  
Protein Delivery ◽  
Uv Light ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Physical Interaction ◽  
Granule Formation ◽  
Angiogenic Proteins ◽  
Proplatelet Formation

Abstract Platelet alpha granules, which contain both membrane-bound and secreted proteins, are formed in the megakaryocyte and then delivered along proplatelet elaborations to the newly forming platelets. A critical but poorly understood process is the post-translational processing, sorting and delivery of proteins to alpha granules prior to their delivery to the proplatelet. Defects in the processing and trafficking of platelet proteins can result in reduced platelet formation. In addition, platelet alpha granules may be differentially packaged with pro- or anti-angiogenic proteins, suggesting that protein sorting events that occur before alpha granule formation may significantly impact platelet end-point function. Thus, research at the intersection of protein trafficking and thrombopoiesis may lead to clearer understanding of the mechanisms of proplatelet formation, the mechanisms of disease in inherited platelet disorders, and the mechanisms of platelet function in inflammation, tumor metastasis and angiogenesis. We have used the platelet integrin aIIbb3 as a model protein to investigate the mechanisms of these processes. In this study we identified proteins that interacted with aIIb in either HEK293 cells or stem cell derived megakaryocytes. The purpose of expanding the “interactome” of aIIb is to identify novel protein-protein interactions that are important for protein delivery to the megakaryocyte surface, and thus could be important for alpha granule and proplatelet formation. We used two methods of capturing interacting proteins: 1) a two-cell pull-down assay using Histidine-tagged aIIb and b3 as bait for umbilical cord blood derived megakaryocyte lysate, followed by nickel bead extraction, 2) a crosslinking assay in which photoreactive, crosslinking amino acids are incorporated into growing megakaryocytes, then crosslinked by exposure to UV light. To enrich the population of aIIb residing in the ER and Golgi, a mutant aIIb subunit containing a R858G mutation, which prevents cleavage of pro-aIIb to mature aIIb resulting in intracellular retention, was used as bait in some assays. The captured proteins from both methods were separated by SDS-PAGE and analyzed by mass spectroscopy. Two or more unique peptides were identified for 93 proteins, and 33 proteins were identified in two or more separate experiments. Of these proteins, 45 were potential protein-trafficking proteins, known to interact with aIIb, or of unknown function. Further analysis of one of these proteins, DNAJC10, suggested that it plays a role in aIIbb3 biogenesis and trafficking. DNAJC10 is an HSP40 type protein with a BiP binding domain and a second domain containing two disulfide isomerase motifs. It has been shown to be induced during ER stress, and may assist in delivering misfolded ER proteins to the proteasome for degradation. Immunoprecipitation of aIIb and b3 followed by immunoblot with anti-DNAJC10 mAb revealed protein bands corresponding to the molecular mass of DNAJC10, indicating direct or indirect physical interaction of aIIb and b3 with DNAJC10. siRNA mediated knockdown of DNAJC10 increased aIIbb3 surface expression on human megakaryocytes by 12%. Together these findings indicate that DNAJC10 interacts with aIIbb3 and may play a role in regulating aIIbb3 surface expression. This study offers new insights into the control of aIIbb3 surface expression and further studies may reveal new targets for anti- or pro-integrin therapies.

scholarly journals Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

2013 ◽  
Vol 24 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Susumu Hara ◽  
Shigeki Arawaka ◽  
Hiroyasu Sato ◽  
Youhei Machiya ◽  
Can Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Dopamine Transporter ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Receptor Kinase ◽  
Wild Type ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

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