Adaptor protein-3 produces synaptic vesicles that release phasic dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high-frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a distinct population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Substrate recognition and proton coupling by a bacterial member of solute carrier family 17.

The solute carrier 17 family transports diverse organic anions using two distinct modes of coupling to a source of energy. Transporters that package glutamate and nucleotide into secretory vesicles for regulated release by exocytosis are driven by membrane potential but subject to allosteric regulation by H+ and Cl-. Other solute carrier 17 members including the lysosomal sialic acid exporter couple the flux of organic anion to cotransport of H+. To begin to understand how similar proteins can perform such different functions, we have studied Escherichia coli DgoT, a H+/galactonate cotransporter. A recent structure of DgoT showed many residues contacting D-galactonate, and we now find that they do not tolerate even conservative substitutions. In contrast, the closely related lysosomal H+/sialic acid cotransporter Sialin tolerates similar mutations, consistent with its recognition of diverse substrates with relatively low affinity. We also find that despite coupling to H+, DgoT transports more rapidly but with lower apparent affinity at high pH. Indeed, membrane potential can drive uptake, indicating electrogenic transport and suggesting a H+:galactonate stoichiometry >1. Located in a polar pocket of the N-terminal helical bundle, Asp46 and Glu133 are each required for net flux by DgoT, but the E133Q mutant exhibits robust exchange activity and rescues exchange by D46N, suggesting that these two residues operate in series to translocate protons. E133Q also shifts the pH sensitivity of exchange by DgoT, supporting a central role for the highly conserved TM4 glutamate in H+ coupling by DgoT.

The role of tissue adhesives and sealants in colorectal anastomotic healing-a scoping review.

PURPOSE: Anastomotic leakage (AL) after colorectal resection is a serious postoperative complication with grave consequences for patients. Despite several efforts to reduce its incidence, AL is still seen among 2-20% of colorectal cancer patients receiving an anastomosis. The use of tissue adhesives and sealants as an extra layer of protection around the anastomosis has shown promising results. We conducted a scoping review to provide an overview of the current knowledge on the effect of tissue adhesives and sealants on colorectal anastomosis healing, as well as their effect on the postoperative outcome. METHODS: The databases of PubMed, Embase, and Cochrane Library were systematically searched on 14/10/2022. Studies addressing the use of a tissue adhesive or tissue sealant applied around a colorectal anastomosis, with the goal to prevent AL or to decrease AL-related complications, were included. We presented an overview of the available studies and summarized their results narratively. RESULTS: Seven studies were included out of the 846 screened. All authors reported the rate of AL in their interventions group. Five of the studies found a decreased rate of AL compared to the control group. One study had no incidences of AL, while the last study had a seemingly low rate of AL but no comparison group. Information on secondary outcomes was sparingly reported, but the results hinted at a positive effect. CONCLUSION: Tissue adhesives and sealants might have a beneficial effect on colorectal anastomosis healing. The literature is sparse, and this review has shown the need for further clinical studies.

Resting state structure of the hyperdepolarization activated two-pore channel 3.

Voltage-gated ion channels endow membranes with excitability and the means to propagate action potentials that form the basis of all neuronal signaling. We determined the structure of a voltage-gated sodium channel, two-pore channel 3 (TPC3), which generates ultralong action potentials. TPC3 is distinguished by activation only at extreme membrane depolarization (V50 ∼ +75 mV), in contrast to other TPCs and NaV channels that activate between -20 and 0 mV. We present electrophysiological evidence that TPC3 voltage activation depends only on voltage sensing domain 2 (VSD2) and that each of the three gating arginines in VSD2 reduces the activation threshold. The structure presents a chemical basis for sodium selectivity, and a constricted gate suggests a closed pore consistent with extreme voltage dependence. The structure, confirmed by our electrophysiology, illustrates the configuration of a bona fide resting state voltage sensor, observed without the need for any inhibitory ligand, and independent of any chemical or mutagenic alteration.

Structures suggest a mechanism for energy coupling by a family of organic anion transporters.

Members of the solute carrier 17 (SLC17) family use divergent mechanisms to concentrate organic anions. Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use proton cotransport to drive efflux from the lysosome. To delineate the divergent features of ionic coupling by the SLC17 family, we determined the structure of Escherichia coli D-galactonate/H+ symporter D-galactonate transporter (DgoT) in 2 states: one open to the cytoplasmic side and the other open to the periplasmic side with substrate bound. The structures suggest a mechanism that couples H+ flux to substrate recognition. A transition in the role of H+ from flux coupling to allostery may confer regulation by trafficking to and from the plasma membrane.

Anastomotic leakage following restorative rectal cancer resection: treatment and impact on stoma presence 1 year after surgery-a population-based study.

PURPOSE: Anastomotic leakage (AL) continues to be a challenge after restorative rectal resection (RRR). Various treatment options of AL are available; however, their long-term outcomes are uncertain. We explored the impact of AL on the risk of stoma presence 1 year after RRR for rectal cancer and described treatment of AL after RRR including impact on the probability of receiving adjuvant chemotherapy and stoma presence following different treatment options of AL. METHODS: We included 859 patients undergoing RRR in Central Denmark Region between 2013 and 2019. Stoma presence was calculated as the proportion of patients with stoma 1 year after RRR. Multivariable logistic regression was conducted to estimate the impact of AL on stoma presence adjusting for potential predictors. Descriptive data of outcomes were stratified for various treatment options of AL. RESULTS: The risk of stoma presence 1 year after surgery was 9.8% (95% CI 7.98-12.0). Predictors for having stoma 1 year after RRR were AL (OR 8.43 (95% CI 4.87-14.59)) and low tumour height (OR 3.85 (95% CI 1.22-13.21)). For patients eligible for adjuvant chemotherapy, the probability of receiving it was 42.9% (95% CI 21.8-66.0) if treated with endo-SPONGE and 71.4% (95% CI 47.8-88.7) if treated with other anastomosis preserving treatment options. The risk of having stoma 1 year after RRR was 33.9% (95% CI 21.8-47.8) for patients treated with endo-SPONGE and 13.5% (95% CI 5.6-25.8) for patients treated with other anastomosis preserving treatment options (p = 0.013). CONCLUSION: AL is a strong predictor for stoma presence 1 year after RRR. Patients treated with endo-SPONGE seem to have worse outcomes compared to other anastomosis preserving treatment options.

The impact of multiple firings on the risk of anastomotic leakage after minimally invasive restorative rectal cancer resection and the impact of anastomotic leakage on long-term survival: a population-based study.

PURPOSE: The aim of this study was to evaluate the anastomotic leakage (AL) rate and predictors for AL following minimally invasive restorative rectal resection (RRR) among rectal cancer patients managed according to up-to-date standardized treatment. Furthermore, we explored the impact of symptomatic AL on long-term survival. METHODS: The study cohort was rectal cancer patients undergoing minimally invasive RRR in Central Denmark Region between 2013 and 2017. Data was retrieved from a prospective clinical quality database and supplemented with data from medical records. The AL rate was calculated as the proportion of patients who developed symptomatic AL within 30 days. Predictors for AL were identified through logistic regression. The impact of AL on long-term survival was analyzed using Kaplan-Meier methods and Cox regression. RESULTS: AL occurred in 15.1% of 604 patients. The AL rate for males was 20.1% (95% CI 16.3-24.3) and 5.0% (95% CI 2.4-9.0) for females. Odds ratio (OR) of AL in females vs. males was 0.25 (95% CI 0.12-0.51). The use of at least three firings when transecting the rectum was associated with OR of 2.71 (95% CI 1.17-6.26) for AL. The 5-year survival for patients with vs. those without AL was 76.1% (95%CI 65.1-84.0) and 83.6% (95%CI 79.8-86.7), corresponding to adjusted hazard ratio of 1.43 (95%CI 0.84-2.41). CONCLUSION: Symptomatic AL is still a challenge in a standardized setting using minimally invasive surgery in rectal cancer patients undergoing RRR, especially in men. Multiple firings should be avoided in transection of the rectum with an endoscopic stapler. AL had a statistical non-significant negative impact on survival.

Implementation of a standardized surgical technique in robot-assisted restorative rectal cancer resection: a single center cohort study.

BACKGROUND: Despite increasing focus on the technical performance of total mesorectal excision over recent decades, anastomotic leakage (AL) continues to be a serious complication for many patients, even in the hands of experienced surgical teams. This study describes implementation of standardized surgical technique in an effort to reduce variability, decrease the risk of anastomotic leakage, and improve associated short-term outcomes for rectal cancer patients undergoing robot-assisted restorative rectal resection (RRR). METHODS: We evaluated all rectal cancer patients undergoing robot-assisted RRR at Aarhus University Hospital between 2017 and 2020. Six standardized surgical steps directed to improve anastomotic healing were mandatory for all RRR. Additional changes were made during the period with prohibition of systemic dexamethasone and limiting the use of endoscopic stapling devices. RESULTS: The use of the full standardization, including all six surgical steps, increased from 40.3% (95% CI, 0.28-0.54) to 86.2% (95% CI, 0.68-0.95). The incidence of AL decreased from 21.0% (95% CI, 0.12-0.33) to 6.9% (95% CI, 0.01-0.23). Length of hospital stay (LOS) decreased from 6 days (range 2-50) to 5 days (range 2-26). The rate of patients readmitted within 90 days decreased from 21.0% (95% CI, 0.12-0.33), to 6.9% (95% CI, 0.01-0.23). CONCLUSION: The full standardization was effectively implemented for rectal cancer patients undergoing robot-assisted RRR. The risk of AL, LOS and readmission decreased during the study period. A team focus on high-reliability and peri-operative complications can improve patient outcomes.

Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody.

The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system, and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face, and one to the cytosolic. Two are specific to VGLUT2, whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function, presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.

Diagnostic accuracy of ultrasound-guided core biopsy of peripheral nerve sheath tumors.

PURPOSE: To retrospectively evaluate the diagnostic accuracy of and complications from ultrasound-guided core needle biopsy (UGCNB) of suspected peripheral nerve sheath tumors (PNSTs). METHODS: Patients undergoing UGCNB from January 2004 to December 2016, based on the suspicion of PNST, were included in the study. Age, gender, anatomical location, dates of relevant events, and histopathological reports of the UGCNB cores and the resected tumors were retrieved from the patients' medical records. RESULTS: A total of 154 UGCNBs were identified. One hundred and forty (90.9%) of these resulted in a conclusive histopathological report, while 14 were unsuited for histopathological analysis due to insufficient amount of tissue and/or nonrepresentative tissue. The overall diagnostic accuracy of UGCNB with respect to discriminate malignant from benign tumors was 99.3%, while correct specific UGCNB diagnoses were confirmed in 95.1% of the cases. Sensitivity and specificity were 90.9% (95% CI: 58.7-99.8%) and 100% (95% CI: 97.2-100%), respectively. The positive predictive value was 100%, and the negative predictive value was 99.2%. Except for one patient, who reported mild dysesthesia, which resolved 2 days after the UGCNB, no complications were reported. CONCLUSION: This study suggests that UGCNB is accurate and safe in patients suspected for PNST.

The serotonin transporter undergoes constitutive internalization and is primarily sorted to late endosomes and lysosomal degradation.

The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the "long loop" recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the ß2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation.

A novel dopamine transporter transgenic mouse line for identification and purification of midbrain dopaminergic neurons reveals midbrain heterogeneity.

Midbrain dopaminergic (DAergic) neurons are a heterogeneous cell group, composed of functionally distinct cell populations projecting to the basal ganglia, prefrontal cortex and limbic system. Despite their functional significance, the midbrain population of DAergic neurons is sparse, constituting only 20 000-30 000 neurons in mice, and development of novel tools to identify these cells is warranted. Here, a bacterial artificial chromosome mouse line [Dat1-enhanced green fluorescent protein (eGFP)] from the Gene Expression Nervous System Atlas (GENSAT) that expresses eGFP under control of the dopamine transporter (DAT) promoter was characterized. Confocal microscopy analysis of brain sections showed strong eGFP signal reporter in midbrain regions and striatal terminals that co-localized with the DAergic markers DAT and tyrosine hydroxylase (TH). Thorough quantification of co-localization of the eGFP reporter signal with DAT and TH in the ventral midbrain showed that a vast majority of eGFP-expressing neurons are DAergic. Importantly, expression profiles also revealed DAergic heterogeneity when comparing substantia nigra and ventral tegmental area. Dat1-eGFP mice showed neither change in synaptosomal DA uptake nor altered levels of DAT and TH in both striatum and midbrain. No behavioural difference between Dat1-eGFP and wild-type was found, suggesting that the strain is not aberrant. Finally, cell populations highly enriched in DAergic neurons can be obtained from postnatal mice by fluorescence-activated cell sorting and the sorted neurons can be cultured in vitro. The current investigation demonstrates that eGFP expression in this mouse line is selective for DAergic neurons, suggesting that the Dat1-eGFP mouse strain constitutes a promising tool for delineating new aspects of DA biology.

SLC6 neurotransmitter transporters: structure, function, and regulation.

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.

Protein interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction partners sorted to Rab11-dependent slow recycling pathway.

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the ß(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.

Adaptor Protein-3 Produces Synaptic Vesicles that Release Phasic Dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a novel population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Diversity of function and mechanism in a family of organic anion transporters.

Originally identified as transporters for inorganic phosphate, solute carrier 17 (SLC17) family proteins subserve diverse physiological roles. The vesicular glutamate transporters (VGLUTs) package the principal excitatory neurotransmitter glutamate into synaptic vesicles (SVs). In contrast, the closely related sialic acid transporter sialin mediates the flux of sialic acid in the opposite direction, from lysosomes to the cytoplasm. The two proteins couple in different ways to the H+ electrochemical gradient driving force, and high-resolution structures of the Escherichia coli homolog d-galactonate transporter (DgoT) and more recently rat VGLUT2 now begin to suggest the mechanisms involved as well as the basis for substrate specificity.

Postendocytic sorting of constitutively internalized dopamine transporter in cell lines and dopaminergic neurons.

The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cells lines, we fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular accumulation was increased by the lysosomal protease inhibitor leupeptin and by monensin, an inhibitor of lysosomal degradation and recycling. Monensin also reduced TacDAT surface expression consistent with partial recycling. In both HEK293 cells and in the dopaminergic cell line 1Rb3An27, constitutively internalized TacDAT displayed primary co-localization with the late endosomal marker Rab7, less co-localization with the "short loop" recycling marker Rab4, and little co-localization with the marker of "long loop" recycling endosomes, Rab11. Removal by mutation of N-terminal ubiquitination sites did not affect this sorting pattern. The sorting pattern was distinct from a bona fide recycling membrane protein, the beta(2)-adrenergic receptor, that co-localized primarily with Rab11 and Rab4. Constitutively internalized wild type DAT probed with the fluorescently tagged cocaine analogue JHC 1-64, exhibited the same co-localization pattern as TacDAT in 1Rb3An27 cells and in cultured midbrain dopaminergic neurons. We conclude that DAT is constitutively internalized and sorted in a ubiquitination-independent manner to late endosomes/lysosomes and in part to a Rab4 positive short loop recycling pathway.

Membrane localization is critical for activation of the PICK1 BAR domain.

The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N-terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain-dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative alpha-helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit, the GluR2 C-terminus transferred to the single transmembrane protein Tac or the dopamine transporter C-terminus transferred to Tac. In contrast, transfer of the GluR2 C-terminus to cyan fluorescent protein, a cytosolic protein, did not elicit BAR domain-dependent clustering. Instead, localizing PICK1 to the membrane by introducing an N-terminal myristoylation site produced BAR domain-dependent, but ligand-independent, PICK1 clustering. The data support that in the absence of PDZ ligand, the PICK1 BAR domain is inhibited through a PDZ domain-dependent and linker-dependent mechanism. Moreover, they suggest that unmasking of the BAR domain's membrane-binding capacity is not a consequence of ligand binding to the PDZ domain per se but results from, and coincides with, recruitment of PICK1 to a membrane compartment.

Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs.

The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. Here we use novel fluorescently tagged cocaine analogs to visualize DAT and DAT trafficking in cultured live midbrain dopaminergic neurons. The fluorescent tags were extended from the tropane N-position of 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane using an ethylamino-linker. The rhodamine-, OR Green-, or Cy3-labeled ligands had high binding affinity for DAT and enabled specific labeling of DAT in live neurons and visualization by confocal imaging. In the dopaminergic neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. FRAP (fluorescence recovery after photobleaching) experiments demonstrated bidirectional movement of DAT in the extensions and indicated that DAT is highly mobile both in the extensions and in the varicosities (immobile fraction less than approximately 30%). DAT was constitutively internalized into vesicular structures likely representing intracellular transporter pools. The internalization was blocked by lentiviral-mediated expression of dominant-negative dynamin and internalized DAT displayed partial colocalization with the early endosomal marker EGFP-Rab5 and with the transferrin receptor. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC.

Regulation of dopamine transporter function by protein-protein interactions: new discoveries and methodological challenges.

The dopamine transporter (DAT) plays a key role in regulating dopaminergic signalling in the brain by mediating rapid clearance of dopamine from the synaptic clefts. The psychostimulatory actions of cocaine and amphetamine are primarily the result of a direct interaction of these compounds with DAT leading to attenuated dopamine clearance and for amphetamine even increased dopamine release. In the last decade, intensive efforts have been directed towards understanding the molecular and cellular mechanisms governing the activity and availability of DAT in the plasma membrane of the pre-synaptic neurons. This has led to the identification of a plethora of different kinases, receptors and scaffolding proteins that interact with DAT and hereby either modulate the catalytic activity of the transporter or regulate its trafficking and degradation. Several new tools for studying DAT regulation in live cells have also recently become available such as fluorescently tagged cocaine analogues and fluorescent substrates. Here we review the current knowledge about the role of protein-protein interactions in DAT regulation as well as we describe the most recent methodological developments that have been established to overcome the challenges associated with the study of DAT in endogenous systems.