DAT Modulation by Protein Kinase C (PKC) |
Rat | Transfected LLC-PK1 cells | β-PMAa | ↓ (Vmax) | ↓ | ↑ | Blocked by staurosporine | Huff et al., 1997 |
↓ | ↓ | ↑ | Blocked by DAT antagonist GBR12909, uptake effects nonadditive decrease induced by DA pretreatment | Gorentla and Vaughan, 2005 |
↓ | ↓ | | Blocked by inhibiting clathrin-mediated endocytosis, disrupting membrane rafts | Foster et al., 2008 |
↓ | ↓ | | Effects still present in syntaxin 1A coexpressing cells, suggesting independent processes for regulating DAT | Cervinski et al., 2010 |
↓ | | | Palmitoylation-deficient DAT (C580A) more sensitive | Foster and Vaughan, 2011 |
| | ↑ | Phosphorylation at Thr53 | Foster et al., 2012 |
| | ↑ | Ser7Ala mutation prevents β-PMA-induced phosphorylation | Moritz et al., 2013 |
BIM-1b | | | | Blocked METH-induced decrease in uptake/increased DAT phosphorylation | Cervinski et al., 2005 |
| | | Blocked decrease in uptake induced by DA pretreatment | Gorentla and Vaughan, 2005 |
Transfected COS cells | β-PMA | ↓ (Vmax) | ↓ | | Blocked by staurosporine | Kitayama et al., 1994 |
↓ (Vmax) | | | Enhanced/reduced by various mutations (see text) | Lin et al., 2003 |
NGF-differentiated PC-12 cells | Ro-32-0432b | | | | Blocked estradiol-induced DAT-dependent DA efflux | (Alyea and Watson, 2009) |
Purified proteins | PKCPKCα/β1/β2/γ | | | ↑ | Phosphorylated DAT N terminus | Gorentla et al., 2009 |
PKCα | | | ↑ | Phosphorylates DAT N-terminal serines (Ser4, Ser7, and Ser14) | Moritz et al., 2013 |
Striatal synaptosomes | β-PMA | ↓ | | | Blocked by staurosporine | Copeland et al., 1996 |
↓ | | | Effect not blocked by BoNT/C (syntaxin 1A protease) | Cervinski et al., 2010 |
↓ | | | Additive with palmitoylation inhibitor 2-BP | Foster and Vaughan, 2011 |
| | ↑ | Phosphorylation at Thr53 | Foster et al., 2012 |
| | ↑ | Induces Ca2+-independent, DAT-dependent DA release, blocked by PKC inhibitors. Phosphorylation blocked by cocaine | Cowell et al., 2000 |
BIM-1 | | | | Blocked (+)-pentazocine (σ2 receptor agonist)-mediated enhancement of AMPH induced DA efflux | Derbez et al., 2002 |
Ro-31-8220b | ↑ | | | Blocked mGluR agonist-induced decrease in DA uptake | Page et al., 2001 |
Striatal slices | Go6976b (PKCα/β/γ) | | | | Blocked AMPH-induced DA efflux, not blocked by PKCγ inhibitor rottlerin | Johnson et al., 2005 |
LY379196b (PKCβ) |
β-PMA | | | | Induces DA efflux, blocked by PKCβ inhibitor LY379196 |
SNr slices | Chelerythrineb | | | | Blocks DAT-dependent DA release induced by glutamate/mGluR agonist | Opazo et al., 2010 |
Prefrontal cortex slices | Chelerythrine | | | | Blocked AMPH-induced DA efflux | Drew and Werling, 2001 |
NAc minced preps | Ro-31-8220 | | | | Blocked AMPH-induced DA efflux | Browman et al., 1998 |
Primary DA neurons | β-PMA | | | | No change in colocalization with early/late endosomal markers (did not look at lysosome) | Rao et al., 2010 |
| ↓ | | DAT increased localization with late endosomal/lysosomal markers | Hong and Amara, 2013 |
Mouse | Striatal synaptosomes | PKCβ Knockout | ↓ | ↓ | | Also reduced AMPH-stimulated efflux, increased locomotion | Chen et al., 2009 |
LY279196b (PKCβ) | | ↓ | | Reversed AMPH effects on surface levels (early decrease, late increase) | Chen et al., 2009 |
| | | Blocked quinpirole-induced increase in DAT surface levels and acceleration of DAT recycling | Chen et al., 2013 |
Primary DA neurons | β-PMA | | ↓ | | Reduced DAT-mediated transient currents, blocked by flotililn-1 siRNA | Cremona et al., 2011 |
Human | Transfected PC12 cells | β-PMA | ↓ | ↓ | | Increased movement of DAT to TfR-positive endosomes | Melikian and Buckley, 1999 |
| ↓ | | Internalization rates increase, recycling rates decrease | Loder and Melikian, 2003 |
| ↓ | | Tac fusion protein with DAT C terminus, 587–591(5Ala) mutant does not internalize in response to β-PMA | Holton et al., 2005 |
| ↓ | | DAT 587-590(4A) mutant does not internalize | Boudanova et al., 2008 |
| ↓ | | Dominant-negative Rin blocks effect. β-PMA increases association between DAT and Rin | Navaroli et al., 2011 |
GF109203Xb | ↑ | ↑ | | Blocks EGCG induced decrease in uptake, surface levels | Li et al., 2006 |
Transfected HEK 293 cells | β-PMA | ↓ | ↓ | ↑ | Deletion of first 22 N-terminal residues does not block effect, despite loss of phosphorylation. Mutation of putative phosphorylation sites also does not block downregulation. Substance P has similar effect | Granas et al., 2003 |
| ↓ | | Deletion of first 22 N-terminal residues does not block effect | Khoshbouei et al., 2004 |
| ↓ | | DAT also ubiquitinated, both effects blocked by siRNA knockdown of Nedd4-2, Ube2D1-3, Ube2L3 | Vina-Vilaseca and Sorkin, 2010 |
↓ | ↓ | | Prevented by knockdown of flotillin-1, which also reduces raft-associated DAT | Cremona et al., 2011 |
↓ | ↓ | | Effect absent in Arg615Cys ADHD-associated DAT variant | Sakrikar et al., 2012 |
| ↓ | | DAT also ubiquitinated, colocalizes with early/late endosomal and lysosomal markers | Hong and Amara, 2013 |
| ↓ | | Not blocked by flotillin-1 siRNA | Sorkina et al., 2013 |
PKCβ overexpression | | | | Potentiation of AMPH-induced DA efflux | Johnson et al., 2005 |
Transfected HeLa cells | β-PMA | | | | Increased ubiquitination/degradation of DAT | Miranda et al., 2005 |
| ↓ | | DAT also ubiquitinated, blocked by siRNA knockdown of Nedd4-2 | Sorkina et al., 2006 |
| ↓ | | Blocked by mutation of N-terminal lysine residues (ubiquitination sites) | Miranda et al., 2007 |
| ↓ | | Not blocked by siRNA of flotillin-1 | Sorkina et al., 2013 |
Transfected COS 7 cells | β-PMA | ↓ (Vmax) | ↓ | ↑ | Mutation of PKC consensus sites in DAT abolishes β-PMA-induced DAT phosphorylation, but uptake and surface levels still decrease | Chang et al., 2001 |
Injected Xenopus oocytes | β-PMA | ↓ | | | Partially blocked by BIM-1. Also observed: Decreased substrate-dependent and -independent currents, membrane capacitance, [3H]mazindol binding | Zhu et al., 1997 |
| | | Decreased DAT-mediated currents, blocked by PKC inhibitors BIS I, chelerythrine, Go6979 (cPKC), Rottlerin (δPKC), EGTA (Ca2+ chelator) | Doolen and Zahniser, 2002 |
Transfected C6 Glioma cells | β-PMA/PDBua | ↓ | ↓ | | Decreased surface DAT measured by [3H]WIN35,428 binding. Decreased uptake blocked by staurosporine | Zhang et al., 1997 |
Transfected MDCK cells | β-PMA | ↓ | ↓ | | DAT colocalizes with TfR early after treatment. Increased degradation of DAT. Dominant-negative dynamin prevents internalization. | Daniels and Amara, 1999 |
Transfected PAE cells | β-PMA | | ↓ | | DAT colocalizes with TfR-/EEA.1-positive endosomes. Internalization blocked by siRNA of clathrin heavy chain/dynamin II. No effect of raft disruptors, truncation of N terminus. | Sorkina et al., 2005 |
| | | Increased FRET signal between CFP-DAT and YFP-ubiquitin, indicating ubiquitination of DAT | Miranda et al., 2005 |
| | ↑ | Blocked by Go6976 (PKC inhibitor) and PKCβ specific inhibitor | Vargas-Medrano et al., 2011 |
Transfected SK-N-MC cells | β-PMA | ↓ | ↓ | | Blocked by siRNA of Rin | Navaroli et al., 2011 |
| ↓ | | Nonadditive with Cdc42 inhibitors Pirl1 and casin and Ack1 siRNA knockdown. Effect lost in cells overexpressing constitutively-active or kinase-dead Ack1. | Wu et al., 2015 |
Transfected N2A cells | β-PMA | ↓ | | | Deletion of first 22 N-terminal residues does not block effect, nor does mutation of various Ser/Thr residues | Granas et al., 2003 |
LY279196 (PKCβ) | | | | Blocked quinpirole-induced increase in DAT surface levels | Chen et al., 2013 |
Transfected Sf9 cells | β-PMA | ↓(Vmax) | ↓ | | Blocked by staurosporine | Pristupa et al., 1998 |
| Purified proteins | PKCα | | | ↑ | Phosphorylated hDAT N-terminal peptide (residues 1–27) | Fog et al., 2006 |
DAT Modulation by cAMP-Dependent Protein Kinase/Protein Kinase A (PKA) |
Rat | Purified proteins | PKA | | | ↑ | Phosphorylated DAT N terminus | Gorentla et al., 2009 |
Striatal suspensions | 8-Br-cAMPa | ↑(Vmax) | | | Effect seen at 1 min, not 12 min. Also seen with forskolin and blocked by PKA inhibitor H-89 | Batchelor and Schenk, 1998 |
H-89b | ↓(Vmax) | | | Blocked quinpirole-induced increase in DA uptake | Batchelor and Schenk, 1998 |
Striatal synaptosomes | 8-Br-cAMP | ↑(Vmax) | | | Blocked by PKA inhibitor H-89 as well as CaMKII inhibitor KN-62 and okadaic acid (phosphatase inhibitor) | Page et al., 2004 |
Mouse | Primary DA neurons | db-cAMPa | | ↑ | | Blocked Rho-dependent AMPH stimulated reduction in DAT surface levels. Stimulation of Gαs-coupled D5 receptors also blocked effect. | Wheeler et al., 2015 |
Human | Transfected Sf9 cells | Rp-cAMPSb | ↑(Vmax) | ↑ | | Inhibition of PKA by Rp-cAMPS increased Vmax of DA uptake | Pristupa et al., 1998 |
Transfected SK-N-SH cells | db-cAMP | ↓ | | | Blocked Rho-dependent AMPH stimulated reduction in DA uptake. Stimulation of Gαs-coupled D5 and β2 adrenergic receptors also blocked effect. | Wheeler et al., 2015 |
DAT Modulation by cGMP-Dependent Protein Kinase/ Protein Kinase G (PKG) |
Rat | Purified proteins | PKG | | | ↑ | Phosphorylated DAT N terminus on serine residues | Gorentla et al., 2009 |
DAT Modulation by Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) |
Rat | PC12 cells | KN-62b, KN-93b | | | | Blocked (+)pentazocine (σ receptor agonist) enhancement of AMPH-evoked DA release | Weatherspoon and Werling, 1999 |
Purified proteins | CaMKII | | | ↑ | Phosphorylated DAT N terminus on serine residues | Gorentla et al., 2009 |
Striatal synaptosomes | KN-62 | ↓(Vmax) | | | Blocked stimulation of DA uptake induced by increased external Ca2+ | Uchikawa et al., 1995 |
↓ | | | Blocked 8-Br-cAMP-induced increase in DA uptake | Page et al., 2004 |
Striatal slices | KN-93 | | | | Blocked enhancement of AMPH-induced DA efflux by repeated AMPH pre-exposure, no effect on non-pretreated rats | Kantor et al., 1999 |
| | | Blocked AMPH-evoked DA release | Fog et al., 2006 |
Cultured midbrain DA neurons | CaMKIIα overexpression (through patch clamp) | | | | Enhanced AMPH-evoked DA release | Fog et al., 2006 |
KN-93 | ↓ | | | Blocked increased DA uptake seen with chronic stimulation by 4-AP. 4-AP increases pCaMKII and Ser19 pTH (CaMKII target) | Padmanabhan et al., 2008 |
Mouse | Striatal synaptosomes | KN-93, AIPb | | | | Blunted AMPH induced MPP+ efflux | Steinkellner et al., 2012 |
αCamKII KO mice | | | | Blunted AMPH induced MPP+ efflux | Steinkellner et al., 2012 |
In vivo striatum | KN-93 | | | | Blocked AMPH-evoked DA release (high-speed chronoamperometry) | Fog et al., 2006 |
αCamKII KO mice | | | | Blunted AMPH-evoked DA release (microdialysis), AMPH-induced locomotor activation | Steinkellner et al., 2014 |
Human | Transfected HEK 293 cells | CaMKIIα overexpression (through patch clamp) | | | | Enhanced AMPH-evoked DA release, not seen in DAT with N-terminal serine mutations | Fog et al., 2006 |
CaMKIIα overexpression (induction in TRex cells) | | | | Enhanced AMPH-evoked DA release, not seen in DAT with C-terminal residues 612–614(4A) mutation | Fog et al., 2006 |
KN-93 | | ↑ | | Blocked AMPH-induced decrease in surface DAT, also blocked reduction in p-GSK3α (Akt activity) | Wei et al., 2007 |
| | | Blocked AMPH-evoked DA release, increase in DAT/Syntaxin 1A Co-IP | Binda et al., 2008 |
↓(Vmax) | No | | In cells with 615C variant, Vmax of uptake reduced with no change in surface DAT. No effect on WT | Sakrikar et al., 2012 |
CaMKII Inhibitory Peptide | | | | Blocked basal anomalous DA efflux in cells expressing A559V DAT (which have higher p-CaMKII) | Bowton et al., 2010 |
Transfected EM4 cells | KN-93 | | | | Blocked AMPH-evoked DA release | Fog et al., 2006 |
Purified proteins | CaMKIIα | | | ↑ | Phosphorylated DAT N-terminal peptide in vitro | Fog et al., 2006 |
DAT Modulation by ERK1/2 |
Rat | Transfected COS cells | U0126b | ↓(Vmax) | ↓ | | Effects blocked by a number of Ser/Thr mutations | Lin et al., 2003 |
Purified proteins | ERK1 | | | ↑ | Phosphorylated Thr53 in DAT N-terminus | Gorentla et al., 2009 |
| | ↑ | Phosphorylated Thr53 in DAT N terminus (α-pThr53 antibody) | Foster et al., 2012 |
Striatal Synaptosomes | PD90859b | | ↓ | | Decreased [125I]RTI-55 binding | Rothman et al., 2002 |
PD90859/U0126 | ↓(Vmax) | | | Synaptosomes showed constitutive activation of ERK1/2 that was significantly reduced by MEK inhibitors | Moron et al., 2003 |
PD90859 | ↓ | ↓ | | Blocked Salvinorin A (KOR agoinst)-induced increase in DA uptake and DAT surface levels in cells co-expressing KOR and DAT. No basal effects with PD90859 alone. | Kivell et al., 2014 |
In vivo striatum | SL327b | ↓ | | | Decreased clearance of exogenous DA when drug is applied intrastriatally (HSCA). Insulin increases rate of clearance and increases striatal pERK1/2 | Owens et al., 2012 |
Human | Transfected HEK 293 cells | PD90859/U0126 | ↓(Vmax) | ↓ | | Increased colocalization with clathrin light chain. ConA (clathrin inhibitor) blocks reduction in surface DAT | Moron et al., 2003 |
Transfected EM4 cells | PD90859 | ↓ | | | Blocked quinpirole-induced increase in ASP+ (DAT substrate) uptake in cells co-expressing D2 and DAT | Bolan et al., 2007 |
↓ | | | Blocked quinpirole-induced increase in ASP+ (DAT substrate) uptake in cells coexpressing D2 and DAT. Effect also blocked by LY294002 (PI3K inhibitor) | Zapata et al., 2007 |
↓ | ↓ | | Blocked salvinorin A (KOR agoinst)-induced increase in ASP+ (DAT substrate) uptake and DAT surface levels in cells coexpressing KOR and DAT. No basal effects with PD90859 alone. | Kivell et al., 2014 |
DAT Modulation by p38 MAP Kinase |
Rat | Transfected COS cells | SB202190b | ↑ | | | Effects only seen with N-terminal alanine substitutions (Val14, Ser2, Ser4, Ser12, Ser13, Ser21, Ser45) | Lin et al., 2003 |
Purified proteins | p38 | | | ↑ | Phosphorylated DAT N terminus on threonine residues | Gorentla et al., 2009 |
Human | Transfected CHO cells | anisomycina | ↓ | | | Blocked by p38 inhibitor SB203580 | Zhu et al., 2005 |
Phosphatidylinositol 3-kinase (PI3K)/Akt |
Rat | Transfected COS cells | LY294002b | ↓(Vmax) | ↓ | ↓ | Not present in T62A, S581A, and T612A mutants | Lin et al., 2003 |
Striatal synaptosomes | LY294002 | ↓(Vmax) | | | Opposite effect seen with insulin treatment | Carvelli et al., 2002 |
Striatal tissue | Akt inhibitors (Akt2 and dual inhibitors) | | ↓ | | No effect of Akt1 inhibitor | Speed et al., 2010 |
Viral overexpression of IRS2 (Akt activator) in SN | ↑ | ↑ | | Resored lower pAkt in high-fat fed rats, restores deficit in DAT surface levels, striatal DA uptake, AMPH-stimulated locomotor activity | Speed et al., 2011 |
In vivo striatum | LY294002 | | | | Decreased AMPH-evoked DA release in striatum (HSCA) | Williams et al., 2007 |
Insulin (intrastriatal) | ↑ | | | Restored DA uptake/AMPH-evoked DA release deficit in hypoinsulinemic rats | Williams et al., 2007 |
Akt1/2 inhibitor | | | | Decreased AMPH-evoked DA release in striatum (HSCA) | Speed et al., 2011 |
Mouse | DA neuron cultures | LY294002 | | ↓ | | Reduced AMPH-induced DA efflux and DAT-mediated transient currents (surface DAT) | Lute et al., 2008 |
VTA slices | Insulin | ↑ | | | Reduced electrically evoked somatodendritic DA release (effect absent in DAT KO, blocked by GBR12909). Suppression blocked by Wortmannin (PI3K inhibitor) and rapamycin (mTor inhibitor) | Mebel et al., 2012 |
Human | Transfected HEK 293 cells | LY294002 | ↓ | ↓ | | Blocked insulin-induced increase in DA uptake. Internalization blocked by ConA (clathrin inhibitor) | Carvelli et al., 2002 |
| ↓ | | Reduced AMPH-induced DA efflux and DAT-mediated transient currents (surface DAT) | Lute et al., 2008 |
Constitutively-active PI3K overexpression | ↑ | ↑ | | Blocked AMPH-induced internalization of DAT as assessed by immunofluorescence microscopy | Carvelli et al., 2002 |
Akt inhibitors (Akt2 and dual inhibitors) | | ↓ | | Akt1 inhibitor showed no effect | Speed et al., 2010 |
Transfected EM4 cells | Insulin | | ↑ | | Blocked AMPH-induced decrease in surface DAT, depends on Akt (prevented by siRNA knockdown) | Garcia et al., 2005 |
ML-9b | ↓ | ↓ | | Prevented insulin block of AMPH-induced decrease in surface DAT | Garcia et al., 2005 |
DAT Modulation by Tyrosine Kinases |
Rat | Striatal synaptosomes | Genisteinb, Tyrphostin 23b | ↓(Vmax) | ↓ | | Treatments also reduced phospho-ERK1/2 | Hoover et al., 2007 |
Mesencephalic cultures | Genistein, Tyrphostin 23 | ↓ | | | Inhibitors decreased levels of an unidentified 155 kDa tyrosine-phosphorylated protein | Hoover et al., 2007 |
BDNF | ↑ | | | Blocked by TrkB antagonist K252a, MEK1/2 inhibitor U0126, and PI3K inhibitor LY294002 | Hoover et al., 2007 |
Mouse | Striatal tissue | Genistein, Tyrphostin 23 | ↓ | | | TK inhibitors Tyrphostin 25 and lavendustin A had no effect on DA uptake | Simon et al., 1997 |
Human | Injected Xenopus oocytes | Genistein | ↓(Vmax) | ↓ | | Also observed: decreased substrate- (tyramine) induced, DAT leak currents. Decreased surface levels measured by [3H]WIN35,428 binding | Doolen and Zahniser, 2001 |
Transfected HTR cells | Src overexpression | ↑ | | | Effects also seen with SERT and NET, not the taurine transporter (TauT) | Annamalai et al., 2012 |
Transfected HEK 293 cells | GDNF | ↓ | ↓ | | Dependent on RhoGEF Vav2, which interacts with DAT and the RTK Ret. Vav2−/− and Ret+/− mice have increased DAT activity. | Zhu et al., 2015 |