Efecto de un quelante de zinc extracelular sobre el número de celulas ganglionares en retina de rata, y transportadores de taurina y de zinc en estas células

La deficiencia de zinc en humanos produce disminución de antioxidantes, asociados a taurina, en la retina y se relaciona con adaptación anormal a la oscuridad, cataratas, ceguera y degeneración macular. Existe escasa evidencia acerca del efecto del zinc sobre el sistema de taurina en retina de mamíferos, por lo cual estudiamos el efecto del zinc sobre el transportador de taurina (TAUT) y los transportadores de zinc (ZnT-1 y 3) usando el quelante de zinc extracelular, ácido dietileno-triamino-penta-acético (DTPA), mediante inmunocitoquímica e inmunohistoquímica fluorescentes en células ganglionares (CG) y en las capas de la retina de ratas. Tres días después de la administración de DTPA (10 µM) se utilizaron anticuerpos primarios y secundarios conjugados con rodamina o fluoresceína-5-isotiocianato (FITC) según fuera el caso. Para el marcaje inmunocitoquímico se contaron trescientas células por condición y la intensidad de fluorescencia se midió como densidad óptica integrada (DOI) en cuatro áreas por cada capa del tejido. El DTPA produjo una disminución en un 32 % y 29 % de las CG del total de células marcadas con los anticuerpos  contra glicoproteína Thy 1.1 y γ-sinucleína, respectivamente. También produjo una disminución significativa de la distribución de TAUT en un 27 y 28 % respecto a los controles. DTPA disminuyó la localización de ZnT-1 y ZnT-3 en las capas de retina (células ganglionares, CCG) y en las plexiformes externa e interna, CPE y CPI). El estudio de estos transportadores en la retina resulta relevante para entender las interacciones de taurina y de zinc en esta estructura.Zinc deficiency in humans causes decreased antioxidants in the retina and is related with abnormal darkness adaptation, cataracts, blindness, and macular degeneration. There is little information about the effects of zinc on the taurine system in mammalian retinal cells. Therefore, we studied the effect of zinc on the taurine transporter (TAUT) and zinc transporters (ZnT-1 and 3) using the extracellular zinc chelator, diethylenetriaminepentaacetic acid (DTPA) by fluorescence immunocytochemistry and immunohistochemistry in the ganglion cells (CG) and cell layers of the retina of rats. Three days after administration of DTPA (10µM) primary antibodies and secondary antibodies conjugated with rhodamine or fluorescein isothiocyanate (FITC) were used as required. For immunocytochemical labeling approximately three hundred cells per condition were counted. For immunohistochemical labeling, the fluorescence intensity was measured as integrated optical density (DOI) in four areas for each layer of tissue. DTPA produced a decrease of 32 % and 29 % in GC of the total cells labeled with antibody against glycoprotein Thy 1.1 and γ-synuclein, respectively. It also produced a significant decrease in TAUT localization in 27 and 28 % compared to controls. DTPA produced a decrease in the localization of ZnT-1 and ZnT-3 in the retina layers (ganglion cells, GCC and the outer and inner plexiform, CEP and CIP). The study of these molecules in the retina is relevant to understanding the interactions of taurine and zinc in this structure

Monoamine Transporter Photoaffinity Ligands Based On Methylphenidate and Citalopram: Rational Design, Chemical Synthesis, and Biochemical Application

Monoamine transporters (MATs) are a family of proteins that include the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). Specifically, dysregulation of MAT function is associated with a host of disease states including drug abuse, major depressive disorder, and anxiety. Additionally, several drugs acting as MAT inhibitors are clinically available to treat multiple disorders. However, details regarding the transport inhibition mechanism created by these drugs, as well as their discrete ligand-binding pockets within their target MAT proteins, remains poorly understood. This knowledge gap in turn hinders rational development of novel therapeutics for numerous MAT-associated disorders. The objective of this research dissertation was to develop irreversible chemical probes based on methylphenidate (MP) and citalopram (CIT), two therapeutically significant MAT inhibitors, in order to map their binding sites and poses within their major MAT target protein. The central hypothesis was that MP and CIT could be rationally derivatized, without significant loss in pharmacological activity, to contain a tag moiety and a photoreactive group capable of forming a covalent bond to their target MAT protein, thus allowing application of a Binding Ensemble Profiling with (f)Photoaffinity Labeling (BEProFL) experimental approach. Specifically, BEProFL rationally couples photoaffinity labeling, chemical proteomics, and computational molecular modeling in order to map the binding sites and poses of ligands within their target proteins. This central hypothesis was tested by pursuing three specific aims: 1) identification of non-tropane photoprobes based on MP suitable for DAT structure-function studies, 2) identification of photoprobes based on CIT and ( S )-CIT suitable for SERT structure-function studies, and 3) development of a tandem photoaffinity labeling-bioorthogonal conjugation protocol for SERT structure-function studies. In the first aim, MP was structurally modified to contain an aryl azide photoreactive group and a 125 I radioisotope tag. The compounds were then subjected to DAT pharmacological evaluation in order to identify suitable candidates for DAT structure-function studies. In the second aim, CIT and (S )-CIT were structurally modified to contain an aryl azide or benzophenone photoreactive group and 125 I, a terminal alkyne, or an aliphatic azide as a tag. Likewise, these compounds were subjected to SERT pharmacological evaluation in order to identify suitable candidates for SERT structure-function studies. Finally, under the third aim, a tandem photoaffinity labeling-bioorthogonal conjugation protocol was developed to label purified hSERT expressed in HEK-293 cells using a ( S )-CIT-based benzophenone-alkyne clickable photoprobe. Probe-labeled hSERT samples from this protocol are currently being analyzed by high resolution mass spectrometry in order to map the ( S )-CIT-binding site(s) within the hSERT

The Role of Human Dopamine Transporter in NeuroAIDS

HIV-associated neurocognitive disorder (HAND) remains highly prevalent in HIV infected individuals and represents a special group of neuropathological disorders, which are associated with HIV-1 viral proteins, such as transactivator of transcription (Tat) protein. Cocaine abuse increases the incidence of HAND and exacerbates its severity by enhancing viral replication. Perturbation of dopaminergic transmission has been implicated as a risk factor of HAND. The presynaptic dopamine (DA) transporter (DAT) is essential for DA homeostasis and dopaminergic modulation of the brain function including cognition. Tat and cocaine synergistically elevate synaptic DA levels by acting directly on human DAT (hDAT), ultimately leading to dysregulation of DA transmission. Through integrated computational modeling and experimental validation, key residues have been identified in hDAT that play a critical role in Tat-induced inhibition of DAT and induce transporter conformational transitions. This review presents current information regarding neurological changes in DAT-mediated dopaminergic system associated with HIV infection, DAT-mediated adaptive responses to Tat as well as allosteric modulatory effects of novel compounds on hDAT. Understanding the molecular mechanisms by which Tat induces DAT-mediated dysregulation of DA system is of great clinical interest for identifying new targets for an early therapeutic intervention for HAND

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

Mutational Analysis of the High-Affinity Zinc Binding Site Validates a Refined Human Dopamine Transporter Homology Model

<div><p>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.</p> </div

Attachment Sites Of Irreversible Cocaine Analogs On The Dopamine Transporter

The dopamine transporter (DAT) is an integral membrane protein that reuptakes dopamine (DA) from the extracellular space into the presynaptic neuron. DAT regulates dopaminergic neurotransmission as it maintains homeostatic synaptic DA levels in the brain. Psychostimulants such as cocaine disrupt DA homeostasis as it binds to DAT and prevents reuptake of DA. Excess DA in the synapse leads to prolonged dopaminergic neurotransmission, which is associated with cocaine related euphoria often leading to addiction. The DAT consists of 12 transmembrane domains (TMs) with N- and C-termini facing the cytoplasm. TMs 1, 3, 6, and 8 make up the core of the protein and the residues from these domains are involved in binding substrates and inhibitors. Although the effect of cocaine binding to DAT is known, the mechanism of DAT-cocaine interaction at the molecular level is still unknown. Therefore, to elucidate how cocaine binds to the DAT and how it is positioned in the binding pocket, we mapped the attachment site of the irreversible binding cocaine analogs, [125I]MFZ 2-24, [125I]RTI 82, and [125I]JHC 2-48. These compounds share a similar cocaine-based core structure, but have analog specific photo activatable side chains that extend from different regions of the cocaine core structure. Upon ultraviolet light activation, the photo activatable phenyl N3 (azido) group forms phenyl nitrene that becomes covalently attached to a residue on the protein, hence irreversible binding cocaine analogs. Previous studies narrowed the [125I]RTI 82 adduction site to the region surrounding TM6, between Ile291 and Arg344 on human DAT and between Met290 and Lys336 on rat DAT. The [125I]MFZ 2-24 attachment site was localized between residues Ile67 and Leu80 in TM1. To identify the specific amino acid attachment site of these analogs we created several methionine substitution mutants across TMs 1 and 6. This resulted in generation of custom cyanogen bromide (CNBr) cleavage sites. The results from peptide maps of photoaffinity labeled mutants proteolyzed with CNBr narrowed the adduction of [125I]MFZ 2-24 to Asp79 or Leu80 in TM1 and the adduction of [125I]RTI 82 to Phe320 in TM6. Trypsin and CNBr proteolysis of [125I]JHC 2-48 labeled rat DAT indicated a ligand attachment site C-terminal to TM6. Incorporation of three structural analogs to three distinct TM domains demonstrates that the appended azido groups on these analogs identify different faces of the ligand binding pocket. Thus, allowing for triangulation of cocaine orientation in its binding site via computational modeling

Exploring the Dopamine Transporter Utilizing a Two-Pronged Approach with Novel Cathinone Analogs and Mutant Dopamine Transporters

The dopamine transporter (DAT) is responsible for the removal of the neurotransmitter from the synaptic gap and a therapeutic target for a multitude of drugs. While the ortholog Drosophila melanogaster dopamine transporter (dDAT) and human serotonin transporter (hSERT) have resolved structures, the human dopamine transporter (hDAT) does not. A 3-D computational homology model of hDAT was constructed for the study of molecular interactions with agents within the central binding site (S1) of the transporter. Synthetic cathiones are a class of abused stimulant drugs that primarily target DAT. Greater than 150 cathinones have been identified on the clandestine market but there is not much known about the structure-activity relationship (SAR) of these abused compounds. A dichloro substituted benzoylpiperdine compound, part of novel series of benzoylpiperdine cathinones, was found to be a potent DAT inhibitor. Two new disubstituted compounds were computationally modelled, synthesized, and biologically evaluated to investigate the effect of these substituents in DAT inhibitor potency. It was found that all compounds were active and that the hybrids with electron donating substituents were weaker compared to the hybrids with electron withdrawing substituents tested. Cathinones based on the pyrrolidinophenone scaffold, such as 3,4-methylenedioxypyrovalerone (MDPV), a-pyrrolidinopentiophenone (a-PVP), and 3,4-methylenedioxy-a-pyrrolidinopropiophenone (MDPPP), are established illicit psychostimulants. These compounds are potent inhibitors of hDAT but have little to no activity at dDAT. In collaboration with Dr. Eltit’s lab (VCU) gain-of-function studies and in silico modeling using mutant DAT transporters were performed. Four non-conserved amino acid residues critical for MDPV’s high potency activity as a reuptake inhibitor at hDAT were identified. These residues can drive MDPV selectivity not only by stabilizing binding, but also by controlling access to its binding site