Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter: IMPLICATIONS FOR ANGELMAN SYNDROME

The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP(+)) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII(T305D)), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP(+) efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.     

N-terminal phosphorylation of the dopamine transporter is required for amphetamine-induced efflux

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake.     

Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-stimulated Efflux

In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate efflux, and other DAT functions through currently unknown mechanisms. DAT is phosphorylated by protein kinase C within a serine cluster at the distal end of the cytoplasmic N terminus, whereas recent work in model cells revealed proline-directed phosphorylation of rat DAT at membrane-proximal residue Thr(53). In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr(53) in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr(53) occurred with a stoichiometry of ～50% and was strongly increased by phorbol esters and protein phosphatase inhibitors, demonstrating modulation of the site by signaling pathways that impact DAT activity. Mutations of Thr(53) to prevent phosphorylation led to reduced dopamine transport V(max) and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism.     

Multiple molecular determinants in the carboxyl terminus regulate dopamine transporter export from endoplasmic reticulum

The plasma membrane dopamine transporter (DAT) has an essential role in terminating dopaminergic neurotransmission by reuptake of dopamine into the presynaptic neurons. Therefore, the amount of DAT at the cell surface is a critical determinant of DAT function. In this study, we examined the role of the carboxyl terminus of DAT in trafficking of the transporter through the biosynthetic pathway to the plasma membrane. Live cell fluorescence microscopy and cell surface biotinylation were used to study the effects of systematic deletions and alanine substitutions in the carboxyl terminus on DAT localization. It was found that alanine substitutions of Lys-590 and Asp-600 significantly delayed the delivery of DAT to the plasma membrane because of retention of DAT in the endoplasmic reticulum (ER). Most surprising, mutation of Gly-585 to alanine completely blocked the exit of DAT from the ER and surface expression of the transporter. The effect of these three mutations on ER export of DAT was demonstrated in porcine aortic endothelial cells and the immortalized neuronal cell line 1RB3AN27. In primary cultures of rat embryonic midbrain neurons, DAT G585A, K590A, and D600A mutants were restricted to the cell soma and did not traffic to the dendrites or axonal processes. These data are consistent with the model whereby the local conformation and/or intramolecular interactions of the sequences of the DAT carboxyl terminus proximal to the last transmembrane domain are essential for the ER export of the transporter.     

Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1

PDZ domain-containing proteins play an important role in the targeting and localization of synaptic membrane proteins. Here, we report an interaction between the PDZ domain-containing protein PICK1 and monoamine neurotransmitter transporters in vitro and in vivo. In dopaminergic neurons, PICK1 colocalizes with the dopamine transporter (DAT) and forms a stable protein complex. Coexpression of PICK1 with DAT in mammalian cells and neurons in culture results in colocalization of the two proteins in a cluster pattern and an enhancement of DAT uptake activity through an increase in the number of plasma membrane DAT. Deletion of the PDZ binding site at the carboxyl terminus of DAT abolishes its association with PICK1 and impairs the localization of the transporter in neurons. These findings indicate a role for PDZ-mediated protein interactions in the localization, expression, and function of monoamine transporters.     

Phosphatidylinositol 3-kinase,protein kinase C,and MEK1/2 kinase regulation of dopamine transporters (DAT) require N-terminal DAT phosphoacceptor sites

The dopamine transporter (DAT) modulates dopamine neurotransmission and is a primary target for psychostimulant influences on locomotion and reward. Selective DAT expression by dopaminergic neurons has led to use of cocaine analog DAT radioligands to assess rates of progression of dopamine neuronal degeneration in Parkinson's disease. We have documented that DAT is a phosphoprotein that is regulated by phosphorylation through pathways that include protein kinase C cascades. We now extend this work using drugs selective for phosphatidylinositol 3-kinase (PI3K), protein kinase C, MEK1/2, p38 kinase, and Ca2+/calmodulin kinase II. We compare the drug effects on wild type DAT to the effects on 20 DAT mutants and a DAT deletion. PI3K and MEK1/2 modulators exert strong effects on DAT expression patterns and dopamine uptake Vmax. PKC principally modulates Vmax. Neither p38 nor Ca2+/calmodulin kinase II agents exert significant influences on wild type DAT. Several mutants and a DAT with an N-terminal deletion display alterations that interact with the effects of kinase modulators, especially S7A for PKC effects; T62A, S581A, and T612A for PI3K effects; and S12A and T595A mutants for MEK1/2 effects. 32P-Labeling studies confirm several of these effects of kinase pathway modulators on DAT phosphorylation. DAT expression and activities can be regulated by kinase cascades that require phosphoacceptor sites most concentrated in its N terminus. These results have a number of implications for DAT regulation and mandate caution in using DAT radioligand binding to infer changes in dopaminergic neuronal integrity after treatments that alter activities of these kinase pathways.     

Membrane-permeable C-terminal Dopamine Transporter Peptides Attenuate Amphetamine-evoked Dopamine Release

The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate the role of the DAT C terminus in AMPH-evoked DA efflux using cell-permeant dominant-negative peptides. A peptide, which corresponded to the last 24 C-terminal residues of DAT (TAT-C24 DAT) and thereby contained the Ca²⁺-calmodulin-dependent protein kinase IIα (CaMKIIα) binding domain and the PSD-95/Discs-large/ZO-1 (PDZ)-binding sequence of DAT, was made membrane-permeable by fusing it to the cell membrane transduction domain of the HIV-1 Tat protein (TAT-C24WT). The ability of TAT-C24WT but not a scrambled peptide (TAT-C24Scr) to block the CaMKIIα-DAT interaction was supported by co-immunoprecipitation experiments in heterologous cells. In heterologous cells, we also found that TAT-C24WT, but not TAT-C24Scr, decreased AMPH-evoked 1-methyl-4-phenylpyridinium efflux. Moreover, chronoamperometric recordings in striatum revealed diminished AMPH-evoked DA efflux in mice preinjected with TAT-C24WT. Both in heterologous cells and in striatum, the peptide did not further inhibit efflux upon KN-93-mediated inhibition of CaMKIIα activity, consistent with a dominant-negative action preventing binding of CaMKIIα to the DAT C terminus. This was further supported by the ability of a peptide with perturbed PDZ-binding sequence, but preserved CaMKIIα binding (TAT-C24AAA), to diminish AMPH-evoked DA efflux in vivo to the same extent as TAT-C24WT. Finally, AMPH-induced locomotor hyperactivity was attenuated following systemic administration of TAT-C24WT but not TAT-C24Scr. Summarized, our findings substantiate that DAT C-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects.     

Dopamine transporters are phosphorylated on N-terminal serines in rat striatum

Dopamine transporters (DATs) are neuronal phosphoproteins that clear dopamine from the synaptic cleft. Activation of protein kinase C (PKC) and inhibition of protein phosphatases by okadaic acid (OA) increase phosphorylation of DAT and lead to concomitant reduction in DAT activity and cell surface expression. Numerous potential sites for phosphorylation are present on DAT, but the sites utilized and their relationship to transport regulation are currently unknown. We used peptide mapping and epitope-specific immunoprecipitation to identify the region of DAT that undergoes phosphorylation in rat striatal tissue. Phosphoamino acid analysis revealed that basal and stimulated samples were phosphorylated primarily on serine. Digestion of (32)PO(4)-labeled DAT with trypsin and immunoprecipitation with N- or C-terminal specific antisera failed to isolate phosphopeptide fragments corresponding to photoaffinity-labeled fragments that contain all internal interhelical loops. However, digestion of (32)PO(4)-labeled DAT with endoproteinase asp-N and immunoprecipitation with an N-terminal antiserum extracted two phosphopeptide fragments from both basal and PKC/OA-stimulated samples, demonstrating that the N-terminal cytoplasmic tail is a major site of phosphorylation. Aminopeptidase treatment of PKC- and/or OA-stimulated DAT cleaved essentially all (32)PO(4) label without proteolysis extending past transmembrane domains 1 and 2, providing further evidence that most phosphorylation sites are near the N terminus and not in intracellular loops or C-terminal domains. In situ proteolysis of the N-terminal tail indicates that the majority of stimulated phosphorylation sites are N-terminal to an antibody epitope at residues 42-59. Two-dimensional analysis of purified protein produced three tryptic phosphopeptides that may result from phosphorylation of multiple sites, but the fragments did not co-migrate with synthetic tryptic peptides phosphorylated at serines 2 and 4. These results indicate that most or all of the basal and stimulated phosphorylation of DAT in striatal tissue occurs on one or more residues in a group of six serines clustered near the distal end of the cytoplasmic N terminus.     

CaMKII translocation requires local NMDA receptor-mediated Ca2+ signaling

Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca(2+) affecting several downstream signaling pathways that involve Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca(2+) responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIalpha translocation was observed. In cells solely expressing Ca(2+)-impermeable NMDARs containing NR1(N598R)-mutant subunits, prolonged NMDA application elevated internal Ca(2+) to the same degree as in wild-type controls, yet failed to translocate CaMKIIalpha. Brief local NMDA application evoked smaller Ca(2+) transients in dendritic spines of mutant compared to wild-type cells. CaMKIIalpha mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1(N598R) cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca(2+) entry directly through NMDARs, rather than other Ca(2+) sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event.     

Apomorphine Does Not Alter Amphetamine-Induced Dopamine Release Measured in Striatal Dialysates

Amphetamine facilitates the release of dopamine from nerve terminals, but the mechanisms underlying this effect have not been fully delineated. The present experiments were designed to test the extent to which amphetamine-induced dopamine release is dependent on impulse flow and autoreceptor function in dopaminergic neurons. Rats were pretreated with a low dose of apomorphine (0.05 mg/kg) to inhibit dopamine neuronal activity, and the striatal dopaminergic response to amphetamine (0.5 mg/kg) was assessed by in vivo dialysis in freely moving animals. Consistent with previous results, apomorphine alone substantially decreased, whereas amphetamine increased, striatal dialysate dopamine concentrations. However, whereas apomorphine pretreatment decreased the locomotor response to amphetamine, the amphetamine-induced increase in dialysate dopamine was unaffected. These results indicate that amphetamine-facilitated dopamine release is independent of neuronal firing and autoreceptor regulation, consistent with the putative accelerative exchange-diffusion mechanism of amphetamine-induced dopamine release. Other possible mechanisms underlying the inhibitory effects of apomorphine on amphetamine locomotor activation are discussed.     

Temporal expression of mutant LRRK2 in adult rats impairs dopamine reuptake

Parkinson's disease (PD) results from progressive degeneration of dopaminergic neurons. Most PD cases are sporadic, but some have pathogenic mutation in the individual genes. Mutation of the leucine-rich repeat kinase-2 (LRRK2) gene is associated with familial and sporadic PD, as exemplified by G2019S substitution. While constitutive expression of mutant LRRK2 in transgenic mice fails to induce neuron death, transient expression of the disease gene by viral delivery causes a substantial loss of dopaminergic neurons in mice. To further assess LRRK2 pathogenesis, we created inducible transgenic rats expressing human LRRK2 with G2019S substitution. Temporal overexpression of LRRK2(G2019S) in adult rats impaired dopamine reuptake by dopamine transporter (DAT) and thus enhanced locomotor activity, the phenotypes that were not observed in transgenic rats constitutively expressing the gene throughout life time. Reduced DAT binding activity is an early sign of dopaminergic dysfunction in asymptomatic subjects carrying pathogenic mutation in LRRK2. Our transgenic rats recapitulated the initiation process of dopaminergic dysfunction caused by pathogenic mutation in LRRK2. Inducible transgenic approach uncovered phenotypes that may be obscured by developmental compensation in constitutive transgenic rats. Finding in inducible LRRK2 transgenic rats would guide developing effective strategy in transgenic studies: Inducible expression of transgene may induce greater phenotypes than constitutive gene expression, particularly in rodents with short life time.     

Intracellular Methamphetamine Prevents the Dopamine-induced Enhancement of Neuronal Firing

The dysregulation of the dopaminergic system is implicated in multiple neurological and neuropsychiatric disorders such as Parkinson disease and drug addiction. The primary target of psychostimulants such as amphetamine and methamphetamine is the dopamine transporter (DAT), the major regulator of extracellular dopamine levels in the brain. However, the behavioral and neurophysiological correlates of methamphetamine and amphetamine administration are unique from one another, thereby suggesting these two compounds impact dopaminergic neurotransmission differentially. We further examined the unique mechanisms by which amphetamine and methamphetamine regulate DAT function and dopamine neurotransmission; in the present study we examined the impact of extracellular and intracellular amphetamine and methamphetamine on the spontaneous firing of cultured midbrain dopaminergic neurons and isolated DAT-mediated current. In dopaminergic neurons the spontaneous firing rate was enhanced by extracellular application of amphetamine > dopamine > methamphetamine and was DAT-dependent. Amphetamine > methamphetamine similarly enhanced DAT-mediated inward current, which was sensitive to isosmotic substitution of Na⁺ or Cl⁻ ion. Although isosmotic substitution of extracellular Na⁺ ions blocked amphetamine and methamphetamine-induced DAT-mediated inward current similarly, the removal of extracellular Cl⁻ ions preferentially blocked amphetamine-induced inward current. The intracellular application of methamphetamine, but not amphetamine, prevented the dopamine-induced increase in the spontaneous firing of dopaminergic neurons and the corresponding DAT-mediated inward current. The results reveal a new mechanism for methamphetamine-induced dysregulation of dopaminergic neurons.     

Serine mutations in transmembrane V of the dopamine D1 receptor affect ligand interactions and receptor activation.

Several serines present in transmembrane domain V are conserved among members of the G-protein-coupled receptor family that bind catecholamines. Two of these serines that are present in the beta-adrenergic receptor were previously shown by site-directed mutagenesis to affect agonist binding and receptor activation (Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S., and Dixon, R. A. F. (1989) J. Biol. Chem. 264, 13572-13578). We investigated the role of the serines present in transmembrane V of another catecholamine receptor, the dopamine D1 receptor, by site-directed mutagenesis, and the results show that mutations at serines 198, 199, and 202 affect dopamine binding. The substitution of serine 198 or serine 199 by an alanine also affects the binding of several other agonist and antagonist dopaminergic compounds while an alanine substitution at serine 202 has no effect on the binding of these compounds. Moreover, each single serine mutation decreased the maximal cAMP accumulation elicited by a dopamine D1 partial agonist. These results suggest that serines present in transmembrane V of the D1 receptor affect ligand interactions and receptor signal transduction, but not entirely in the manner that would be predicted from the model proposed for the beta-adrenergic receptor.     

Ethanol potentiates the function of the human dopamine transporter expressed in Xenopus oocytes

Ethanol alters a variety of properties of brain dopaminergic neurons including firing rate, synthesis, release, and metabolism. Recent studies suggest that ethanol's action on central dopamine systems may also involve modulation of dopamine transporter (DAT) activity. The human DAT was expressed in Xenopus oocytes to examine directly the effects of ethanol on transporter function. [3H]Dopamine (100 nM) accumulation into DAT-expressing oocytes increased significantly in response to ethanol (10 min; 10-100 mM). In two-electrode voltage-clamp experiments, DAT-mediated currents were also enhanced significantly by ethanol (10-100 mM). The magnitude of the ethanol-induced potentiation of DAT function depended on ethanol exposure time and substrate concentration. Cell surface DAT binding ([3H]WIN 35,428; 4 nM) also increased as a function of ethanol exposure time. Thus, the increase in dopamine uptake was associated with a parallel increase in the number of DAT molecules expressed at the cell surface. These experiments demonstrate that DAT-mediated substrate translocation and substrate-associated ionic conductances are sensitive to intoxicating concentrations of ethanol and suggest that DAT may represent an important site of action for ethanol's effects on central dopaminergic transmission. A potential mechanism by which ethanol acts to enhance DAT function may involve regulation of DAT expression on the cell surface.     

Analysis of the functional maturation of olfactory neurons in chicks before and after birth

There has been indirect evidence that the olfactory system of mammals could be functional shortly before birth. Taking advantage of the accessibility of bird embryos, we studied the functional maturation of the olfactory mucosa during embryonic development in birds. Using the combination of electrophysiological EOG recordings and immunohistochemical studies, it was possible to directly demonstrate for the first time that the olfactory system is functional during embryogenesis from embryonic day (ED) 13 and that the beginning of olfactory function coincides with the first localization of the calcium dependent calmodulin kinase II (CaMKIIalpha) in the dendrites of the olfactory receptor neurons. CaMKII and olfactory receptor genes are expressed much earlier in olfactory neurons, both involved in the sensory transduction, but the pattern of expression of CaMKIIalpha changes during the ontogenesis. The increase of EOG amplitude between ED13 and ED15 also coincides with the increase of the number of neurons presenting the dendritic localization of CaMKIIalpha. These results suggest that the enzyme CaMKII might play a role in the functional maturation of the olfactory mucosa.     

Amphetamine Stimulates Endocytosis of the Norepinephrine and Neuronal Glutamate Transporters in Cultured Locus Coeruleus Neurons

Amphetamines and amphetamine-derivatives elevate neurotransmitter concentrations by competing with endogenous biogenic amines for reuptake. In addition, AMPHs have been shown to activate endocytosis of the dopamine transporter (DAT) which further elevates extracellular dopamine (DA). We previously found that the biochemical cascade leading to this cellular process involves entry of AMPH into the cell through the DAT, stimulation of an intracellular trace amine-associated receptor, TAAR1, and activation of the small GTPase, RhoA. We also showed that the neuronal glutamate transporter, EAAT3, undergoes endocytosis via the same cascade in DA neurons, leading to potentiation of glutamatergic inputs. Since AMPH is a transported inhibitor of both DAT and the norepinephrine transporter (NET), and EAAT3 is also expressed in norepinephrine (NE) neurons, we explored the possibility that this signaling cascade occurs in NE neurons. We found that AMPH can cause endocytosis of NET as well as EAAT3 in NE neurons. NET endocytosis is dependent on TAAR1, RhoA, intracellular calcium and CaMKII activation, similar to DAT. However, EAAT3 endocytosis is similar in all regards except its dependence upon CaMKII activation. RhoA activation is dependent on calcium, but not CaMKII, explaining a divergence in AMPH-mediated endocytosis of DAT and NET from that of EAAT3. These data indicate that AMPHs and other TAAR1 agonists can affect glutamate signaling through internalization of EAAT3 in NE as well as DA neurons.

     

Regional distribution of protein kinases in normal and odor-deprived mouse olfactory bulbs

Unilateral naris closure produced dramatic down-regulation of tyrosine hydroxylase (TH) gene expression in periglomerular dopaminergic neurons in the olfactory bulb. To explore molecular mechanisms of TH gene regulation, the present study investigated the regional distribution of protein kinase A (PKAalpha), protein kinase C (PKCalpha), and CaM kinases II (CaMKIIalpha, beta) and IV (CaMKIV) in the normal olfactory bulb and in response to odor deprivation. Strong PKAalpha immunostaining was found in the glomerular, granule cell, external plexiform and olfactory nerve layers. PKCalpha staining was strong in granule cell and external plexiform layers but weak in the glomerular layer. Whereas CaMKIV was primarily found in granule cells, CaMKII was present in the glomerular, external plexiform, mitral cell and granule cell layers. No change in immunoreactivities of these kinases occurred in the olfactory bulb ipsilateral to naris closure. The expression of PKAalpha, PKCalpha and CaMKII, but not CaMKIV, in periglomerular cells suggests that these three kinases may play a role in TH gene regulation in the olfactory bulb. The lack of change in kinase protein levels after naris closure also suggests that any involvement of these kinases in TH gene expression in the olfactory bulb must be through altered kinase activity and not protein levels.