Unveiling the crucial role of betaine: modulation of GABA homeostasis via SLC6A1 transporter (GAT1).

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling.In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Here we demonstrate that betaine at low concentration modulates the GABA uptake by GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. This modulation occurs through the temporal inhibition of the transporter, wherein prolonged occupancy by betaine impedes the swift transition of the transporter to the inward conformation. Importantly, the modulatory effect of betaine on GAT1 is reversible, as the blocking of GAT1 disappears with increased extracellular GABA. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine has a dual role in GAT1: at mM concentration acts as a slow substrate, and at µM as a temporal blocker of GABA, when it is below its K0.5. Given this unique modulatory characteristic and lack of any harmful side effects, betaine emerges as a promising neuromodulator of the inhibitory pathways improving GABA homeostasis via GAT1, thereby conferring neuroprotection against excitotoxicity.

Structure-Activity Relationship of Novel Second-Generation Synthetic Cathinones: Mechanism of Action, Locomotion, Reward, and Immediate-Early Genes.

Several new synthetic cathinones, which mimic the effect of classical psychostimulants such as cocaine or MDMA, have appeared in the global illicit drug market in the last decades. In fact, the illicit drug market is continually evolving by constantly adding small modifications to the common chemical structure of synthetic cathinones. Thus, the aim of this study was to investigate the in vitro and in vivo structure-activity relationship (SAR) of six novel synthetic cathinones currently popular as recreational drugs, pentedrone, pentylone, N-ethyl-pentedrone (NEPD), N-ethyl-pentylone (NEP), 4-methyl-pentedrone (4-MPD), and 4-methyl-ethylaminopentedrone (4-MeAP), which structurally differ in the absence or presence of different aromatic substituents and in their amino terminal group. Human embryonic kidney (HEK293) cells expressing the human isoforms of SERT and DAT were used for the uptake inhibition and release assays. Moreover, Swiss CD-1 mice were used to investigate the psychostimulant effect, rewarding properties (3, 10, and 30 mg/kg, i.p.), and the induction of immediate-early genes (IEGs), such as Arc and c-fos in the dorsal striatum (DS) and ventral striatum (VS) as well as bdnf in the medial prefrontal cortex (mPFC), of the test compounds. Our results demonstrated that all tested synthetic cathinones are potent dopamine (DA) uptake inhibitors, especially the N-ethyl analogs, while the ring-substituted cathinones tested showed higher potency as SERT inhibitors than their no ring-substituted analogs. Moreover, unlike NEP, the remaining test compounds showed clear "hybrid" properties, acting as DAT blockers but SERT substrates. Regarding the locomotion, NEP and NEPD were more efficacious (10 mg/kg) than their N-methyl analogs, which correlates with their higher potency inhibiting the DAT and an overexpression of Arc levels in the DS and VS. Furthermore, all compounds tested induced an increase in c-fos expression in the DS, except for 4-MPD, the least effective compound in inducing hyperlocomotion. Moreover, NEP induced an up-regulation of bdnf in the mPFC that correlates with its 5-HTergic properties. Finally, the present study demonstrated for the first time that NEP, 4-MPD, and 4-MeAP induce reward in mice. Altogether, this study provides valuable information about the mechanism of action and psychostimulant and rewarding properties as well as changes in the expression of IEGs related to addiction induced by novel second-generation synthetic cathinones.

Targeting interleukin-6 to treat neuromyelitis optica spectrum disorders: Implications from immunology, the FcRn pathway and clinical experience.

Neuromyelitis optica spectrum disorder (NMOSD) is a rare disease of the central nervous system (CNS) that is associated with poor outcomes for patients. Until recently, when complement inhibitors were approved, there was no approved therapy. Most recently, clinical trials of interleukin-6 (IL-6) blockade showed a therapeutic benefit for NMOSD. In this review, we introduce the immunological basis of IL-6 blockade in NMOSD and summarize current knowledge about the clinical use of the IL-6 receptor inhibitors tocilizumab and satralizumab. The aim of extending the half-life of monoclonal antibodies (mAbs) has been actualized by successful clinical translation for Satralizumab, achieved via the neonatal Fc receptor (FcRn) pathway. The basic principles of FcRn are highlighted in this review together with the potential therapeutic benefits of this emerging technology.

Insight into the Anticancer Activity of Copper(II) 5-Methylenetrimethylammonium-Thiosemicarbazonates and Their Interaction with Organic Cation Transporters.

A series of four water-soluble salicylaldehyde thiosemicarbazones with a positively charged trimethylammonium moiety ([H2LR]Cl, R = H, Me, Et, Ph) and four copper(II) complexes [Cu(HLR)Cl]Cl (1-4) were synthesised with the aim to study (i) their antiproliferative activity in cancer cells and, (ii) for the first time for thiosemicarbazones, the interaction with membrane transport proteins, specifically organic cation transporters OCT1-3. The compounds were comprehensively characterised by analytical, spectroscopic and X-ray diffraction methods. The highest cytotoxic effect was observed in the neuroblastoma cell line SH-5YSY after 24 h exposure and follows the rank order: 3 > 2 > 4 > cisplatin > 1 >>[H2LR]Cl. The copper(II) complexes showed marked interaction with OCT1-3, comparable to that of well-known OCT inhibitors (decynium 22, prazosin and corticosterone) in the cell-based radiotracer uptake assays. The work paves the way for the development of more potent and selective anticancer drugs and/or OCT inhibitors.

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact.

Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

Methamphetamine ("crystal meth") causes induction of DNA damage and chromosomal aberrations in human derived cells.

Methamphetamine (METH) is a widely consumed psychostimulant drug; its acute toxic effects in brain and liver are well known, furthermore, there is some evidence in regard to its DNA damaging properties in humans. Therefore, we studied the impact of the drug on genomic stability in human derived hepatoma (HepG2) cells, which reflect the activation/detoxification of drugs better than other cell lines. Furthermore, experiments with human buccal derived cells (TR146) were conducted as the drug is consumed orally. Induction of DNA damage in both cell types with doses reflecting the exposure in abusers was found in single cell gel electrophoresis (SCGE) assays (which detect single and double strand breaks as well as apurinic sites). Furthermore, induction of micronuclei (formed as a consequence of structural and numerical chromosomal aberrations) and formation of nuclear buds resulting from gene amplifications was detected. Additional experiments with lesion-specific enzymes showed that the drug causes oxidation of purines and pyrimidines, indicating that its genotoxic effects may be due to oxidation of the DNA. Our findings support the assumption that the drug may cause adverse health effects (such as cancer and infertility) in long-term users which are causally related to DNA damage.

Impaired neural stem cell expansion and hypersensitivity to epileptic seizures in mice lacking the EGFR in the brain.

Mice lacking the epidermal growth factor receptor (EGFR) develop an early postnatal degeneration of the frontal cortex and olfactory bulbs and show increased cortical astrocyte apoptosis. The poor health and early lethality of EGFR-/- mice prevented the analysis of mechanisms responsible for the neurodegeneration and function of the EGFR in the adult brain. Here, we show that postnatal EGFR-deficient neural stem cells are impaired in their self-renewal potential and lack clonal expansion capacity in vitro. Mice lacking the EGFR in the brain (EGFRΔbrain ) show low penetrance of cortical degeneration compared to EGFR-/- mice despite genetic recombination of the conditional allele. Adult EGFRΔ mice establish a proper blood-brain barrier and perform reactive astrogliosis in response to mechanical and infectious brain injury, but are more sensitive to Kainic acid-induced epileptic seizures. EGFR-deficient cortical astrocytes, but not midbrain astrocytes, have reduced expression of glutamate transporters Glt1 and Glast, and show reduced glutamate uptake in vitro, illustrating an excitotoxic mechanism to explain the hypersensitivity to Kainic acid and region-specific neurodegeneration observed in EGFR-deficient brains.

The psychostimulant (±)-cis-4,4'-dimethylaminorex (4,4'-DMAR) interacts with human plasmalemmal and vesicular monoamine transporters.

(±)-cis-4,4'-Dimethylaminorex (4,4'-DMAR) is a new psychoactive substance (NPS) that has been associated with 31 fatalities and other adverse events in Europe between June 2013 and February 2014. We used in vitro uptake inhibition and transporter release assays to determine the effects of 4,4'-DMAR on human high-affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). In addition, we assessed its binding affinities to monoamine receptors and transporters. Furthermore, we investigated the interaction of 4,4'-DMAR with the vesicular monoamine transporter 2 (VMAT2) in rat phaeochromocytoma (PC12) cells and synaptic vesicles prepared from human striatum. 4,4'-DMAR inhibited uptake mediated by human DAT, NET or SERT, respectively in the low micromolar range (IC50 values < 2 µM). Release assays identified 4,4'-DMAR as a substrate type releaser, capable of inducing transporter-mediated reverse transport via DAT, NET and SERT. Furthermore, 4,4'-DMAR inhibited both the rat and human isoforms of VMAT2 at a potency similar to 3,4-methylenedioxymethylamphetamine (MDMA). This study identified 4,4'-DMAR as a potent non-selective monoamine releasing agent. In contrast to the known effects of aminorex and 4-methylaminorex, 4,4'-DMAR exerts profound effects on human SERT. The latter finding is consistent with the idea that fatalities associated with its abuse may be linked to monoaminergic toxicity including serotonin syndrome. The activity at VMAT2 suggests that chronic abuse of 4,4'-DMAR may result in long-term neurotoxicity.

Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter.

The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by single-molecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization. After trafficking to the plasma membrane, however, the SERT stoichiometry is fixed. Stabilization of the oligomeric SERT complexes is mediated by the direct binding to phosphoinositide phosphatidylinositol-4,5-biphosphate (PIP2). The observed spatial decoupling of oligomer formation from the site of oligomer operation provides cells with the ability to define protein quaternary structures independent of protein density at the cell surface.

Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter.

Neurotransmitter/sodium symporters (NSSs) are responsible for Na+-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K+ inhibits Na+-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K+-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni2+ bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K+-induced closure of the transporter to the outside, which was counteracted by Na+ and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K+-effect. The K+-effect depended on an intact Na1 site and mutating the Na2 site potentiated K+ binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K+ to regulate the LeuT transport cycle.

[Guidelines for vaccination of immunocompromised individuals]. / Impfungen bei Immundefekten/Immunsuppression - Expertenstatement und Empfehlungen.

Immunosuppression of various origins is associated with an increased risk of infection; therefore the prevention of infectious diseases by vaccination is especially important in immunocompromised patients. However, the response to vaccinations is often reduced in these risk groups and the application of live vaccines is contraindicated during immunosuppression.In the following expert statement, recommendations for vaccination were created on the basis of current evidence and theoretical/immunological considerations. A first, general part elaborates on efficacy and safety of vaccinations during immunosuppression, modes of action of immunosuppressive medications and recommended time intervals between immunosuppressive treatments and vaccinations. A core piece of this part is a graduation of immunosuppression into three stages, i. e. no relevant immunosuppression, mild to moderate and severe immunosuppression and the assignment of various medications (including biologicals) to one of those stages; this is followed by an overview of possible and necessary vaccinations in each of those stages.The second part gives detailed vaccination guidelines for common diseases and therapies associated with immunosuppression. Primary immune deficiencies, chronic kidney disease, diabetes mellitus, solid and hematological tumors, hematopoetic stem cell transplantation, transplantation of solid organs, aspenia, rheumatological-, gastroenterologic-, dermatologic-, neurologic diseases, biologicals during pregnancy and HIV infection are dealt with.These vaccination guidelines, compiled for the first time in Austria, aim to be of practical help for physicians to facilitate and improve vaccination coverage in immunocompromised patients and their household members and contact persons.

A cytosolic relay of heat shock proteins HSP70-1A and HSP90ß monitors the folding trajectory of the serotonin transporter.

Mutations in the C terminus of the serotonin transporter (SERT) disrupt folding and export from the endoplasmic reticulum. Here we examined the hypothesis that a cytosolic heat shock protein relay was recruited to the C terminus to assist folding of SERT. This conjecture was verified by the following observations. (i) The proximal portion of the SERT C terminus conforms to a canonical binding site for DnaK/heat shock protein of 70 kDa (HSP70). A peptide covering this segment stimulated ATPase activity of purified HSP70-1A. (ii) A GST fusion protein comprising the C terminus of SERT pulled down HSP70-1A. The interaction between HSP70-1A and SERT was visualized in live cells by Förster resonance energy transfer: it was restricted to endoplasmic reticulum-resident transporters and enhanced by an inhibitor that traps HSP70-1A in its closed state. (iv) Co-immunoprecipitation confirmed complex formation of SERT with HSP70-1A and HSP90ß. Consistent with an HSP relay, co-chaperones (e.g. HSC70-HSP90-organizing protein) were co-immunoprecipitated with the stalled mutants SERT-R607A/I608A and SERT-P601A/G602A. (v) Depletion of HSP90ß by siRNA or its inhibition increased the cell surface expression of wild type SERT and SERT-F604Q. In contrast, SERT-R607A/I608A and SERT-P601A/G602A were only rendered susceptible to inhibition of HSP70 and HSP90 by concomitant pharmacochaperoning with noribogaine. (vi) In JAR cells, inhibition of HSP90 also increased the levels of SERT, indicating that endogenously expressed transporter was also susceptible to control by HSP90ß. These findings support the concept that the folding trajectory of SERT is sampled by a cytoplasmic chaperone relay.

Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation.

Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.

Axonal targeting of the serotonin transporter in cultured rat dorsal raphe neurons is specified by SEC24C-dependent export from the endoplasmic reticulum.

Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). Here, we visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. We conclude that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT-(607)RI(608)-AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C-D(796)V/D(797)N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein-SERT from axons to the somatodendritic region. (4) SERT-K(610)Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes.

Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

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.

The cysteines of the extracellular loop are crucial for trafficking of human organic cation transporter 2 to the plasma membrane and are involved in oligomerization.

Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2-hybrid mating-based split-ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, cross-linking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization.

The large extracellular loop of organic cation transporter 1 influences substrate affinity and is pivotal for oligomerization.

Polyspecific organic anion transporters (OATs) and organic cation transporters (OCTs) of the SLC22 transporter family play a pivotal role in absorption, distribution, and excretion of drugs. Polymorphisms in these transporters influence therapeutic effects. On the basis of functional characterizations, homology modeling, and mutagenesis, hypotheses for how OCTs bind and translocate structurally different cations were raised, assuming functionally competent monomers. However, homo-oligomerization has been described for OATs and OCTs. In the present study, evidence is provided that the large extracellular loops (EL) of rat Oct1 (rOct1) and rat Oat1 (rOat1) mediate homo- but not hetero-oligomerization. Replacement of the cysteine residues in the EL of rOct1 by serine residues (rOct1(6ΔC-l)) or breaking disulfide bonds with dithiothreitol prevented oligomerization. rOct1 chimera containing the EL of rOat1 (rOct1(rOat1-l)) showed oligomerization but reduced transporter amount in the plasma membrane. For rOct1(6ΔC-l) and rOct1(rOat1-l), similar K(m) values for 1-methyl-4-phenylpyridinium(+) (MPP(+)) and tetraethylammonium(+) (TEA(+)) were obtained that were higher compared with rOct1 wild type. The increased K(m) of rOct1(rOat1-l) indicates an allosteric effect of EL on the cation binding region. The similar substrate affinity of the oligomerizing and non-oligomerizing loop mutants suggests that oligomerization does not influence transport function. Independent transport function of rOct1 monomers was also demonstrated by showing that K(m) values for MPP(+) and TEA(+) were not changed after treatment with dithiothreitol and that a tandem protein with two rOct1 monomers showed about 50% activity with unchanged K(m) values for MPP(+) and TEA(+) when one monomer was blocked. The data help to understand how OCTs work and how mutations in patients may affect their functions.

The plasma membrane-associated GTPase Rin interacts with the dopamine transporter and is required for protein kinase C-regulated dopamine transporter trafficking.

Dopaminergic signaling and plasticity are essential to numerous CNS functions and pathologies, including movement, cognition, and addiction. The amphetamine- and cocaine-sensitive dopamine (DA) transporter (DAT) tightly controls extracellular DA concentrations and half-life. DAT function and surface expression are not static but are dynamically modulated by membrane trafficking. We recently demonstrated that the DAT C terminus encodes a PKC-sensitive internalization signal that also suppresses basal DAT endocytosis. However, the cellular machinery governing regulated DAT trafficking is not well defined. In work presented here, we identified the Ras-like GTPase, Rin (for Ras-like in neurons) (Rit2), as a protein that interacts with the DAT C-terminal endocytic signal. Yeast two-hybrid, GST pull down and FRET studies establish that DAT and Rin directly interact, and colocalization studies reveal that DAT/Rin associations occur primarily in lipid raft microdomains. Coimmunoprecipitations demonstrate that PKC activation regulates Rin association with DAT. Perturbation of Rin function with GTPase mutants and shRNA-mediated Rin knockdown reveals that Rin is critical for PKC-mediated DAT internalization and functional downregulation. These results establish that Rin is a DAT-interacting protein that is required for PKC-regulated DAT trafficking. Moreover, this work suggests that Rin participates in regulated endocytosis.

Subtype-specific differences in corticotropin-releasing factor receptor complexes detected by fluorescence spectroscopy.

G protein-coupled receptors have been proposed to exist in signalosomes subject to agonist-driven shifts in the assembly disassembly equilibrium, affected by stabilizing membrane lipids and/or cortical actin restricting mobility. We investigated the highly homologous corticotropin-releasing factor receptors (CRFRs), CRFR1 and -2, which are different within their hydrophobic core. Agonist stimulation of CRFR1 and CRFR2 gave rise to similar concentration-response curves for cAMP accumulation, but CRFR2 underwent restricted collision coupling. Both CRFR1 and CRFR2 formed constitutive oligomers at the cell surface and recruited beta-arrestin upon agonist activation (as assessed by fluorescence resonance energy transfer microscopy in living cells). However, CRFR2, but not CRFR1, failed to undergo agonist-induced internalization. Likewise, agonist binding accelerated the diffusion rate of CRFR2 only (detected by fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) but reduced the mobile fraction, which is indicative of local confinement. Fluorescence intensity distribution analysis demonstrated that the size of CRFR complexes was not changed. Disruption of the actin cytoskeleton abolished the agonist-dependent increase in CRFR2 mobility, shifted the agonist concentration curve for CRFR2 to the left, and promoted agonist-induced internalization of CRFR2. Our observations are incompatible with an agonist-induced change in monomer-oligomer equilibrium, but they suggest an agonist-induced redistribution of CRFR2 into a membrane microdomain that affords rapid diffusion but restricted mobility and that is stabilized by the actin cytoskeleton. Our data show that membrane anisotropy can determine the shape and duration of receptor-generated signals in a subtype-specific manner.