{"id":{"repo_id":"toronto-retro","oai_identifier":"oai:utoronto.scholaris.ca:1807/78039"},"canonical_url":"https://search.dev.ndltd.org/etd/toronto-retro/oai:utoronto.scholaris.ca:1807/78039","repository":{"repo_id":"toronto-retro","name":"University of Toronto","base_url":"https://utoronto.scholaris.ca/server/oai/request"},"display":{"title":"Pharmacological Chaperones of the Dopamine Transporter: A Strategy for Increasing Function of Wild Type and Mutant Transporter","abstract":"The dopamine transporter (DAT) is a membrane protein that is essential for regulating signaling and intracellular stores of the neurotransmitter dopamine. An array of pathological conditions have been linked to mutations in the dopamine transporter gene, including ADHD, bipolar disorder, autism, and dopamine transporter deficiency syndrome (DTDS). DTDS is of particular interest, because it is exclusively caused by autosomal recessive loss-of-function mutations in the dopamine transporter (DAT), resulting in a debilitating pediatric movement disorder that is eventually fatal. The majority of DTDS DAT mutations disrupt DAT function by affecting folding and maturation of the transporter. Pharmacological chaperones have previously been used to rescue mutations in other membrane proteins, and we therefore examined the possibility of using pharmacological chaperones to rescue DTDS mutations. We developed a DAT surface expression assay in order to screen candidate compounds for potential pharmacological chaperone activity. After screening a set of known DAT ligands, we found that the DAT inhibitors bupropion and ibogaine increased DAT surface expression, whereas other inhibitors, including cocaine and methylphenidate, had no effect. The effect appears to be associated with stabilization of an inward- facing or occluded, as opposed to an outward-facing conformation of the DAT. Bupropion and ibogaine increased wild type DAT protein levels, demonstrating that drug effects were not merely due to protein trafficking. Bupropion and ibogaine also promoted maturation of the endoplasmic reticulum (ER)-retained DAT mutant K590A, which could be blocked by inhibiting ER to Golgi transport. Furthermore, bupropion decreased the half-life of immature ER-retained K590A DAT in cycloheximide chase assays. The data suggest that bupropion and ibogaine promote maturation of DAT by acting as pharmacological chaperones in the ER. Importantly, both drugs rescue DAT maturation and functional activity of a subset of DTDS-associated DAT mutants. Tests of pharmacological chaperoning of DAT in mice were not conclusive, and future studies will have to assess in vivo activity of bupropion in a true DTDS mouse model. Together, these results are the first demonstration of pharmacological chaperoning of DAT, and suggest this may be a viable approach to increase DAT levels in DTDS and other conditions associated with reduced DAT function.","abstract_html":"The dopamine transporter (DAT) is a membrane protein that is essential for regulating signaling and intracellular stores of the neurotransmitter dopamine. An array of pathological conditions have been linked to mutations in the dopamine transporter gene, including ADHD, bipolar disorder, autism, and dopamine transporter deficiency syndrome (DTDS). DTDS is of particular interest, because it is exclusively caused by autosomal recessive loss-of-function mutations in the dopamine transporter (DAT), resulting in a debilitating pediatric movement disorder that is eventually fatal. The majority of DTDS DAT mutations disrupt DAT function by affecting folding and maturation of the transporter. Pharmacological chaperones have previously been used to rescue mutations in other membrane proteins, and we therefore examined the possibility of using pharmacological chaperones to rescue DTDS mutations. We developed a DAT surface expression assay in order to screen candidate compounds for potential pharmacological chaperone activity. After screening a set of known DAT ligands, we found that the DAT inhibitors bupropion and ibogaine increased DAT surface expression, whereas other inhibitors, including cocaine and methylphenidate, had no effect. The effect appears to be associated with stabilization of an inward- facing or occluded, as opposed to an outward-facing conformation of the DAT. Bupropion and ibogaine increased wild type DAT protein levels, demonstrating that drug effects were not merely due to protein trafficking. Bupropion and ibogaine also promoted maturation of the endoplasmic reticulum (ER)-retained DAT mutant K590A, which could be blocked by inhibiting ER to Golgi transport. Furthermore, bupropion decreased the half-life of immature ER-retained K590A DAT in cycloheximide chase assays. The data suggest that bupropion and ibogaine promote maturation of DAT by acting as pharmacological chaperones in the ER. Importantly, both drugs rescue DAT maturation and functional activity of a subset of DTDS-associated DAT mutants. Tests of pharmacological chaperoning of DAT in mice were not conclusive, and future studies will have to assess in vivo activity of bupropion in a true DTDS mouse model. Together, these results are the first demonstration of pharmacological chaperoning of DAT, and suggest this may be a viable approach to increase DAT levels in DTDS and other conditions associated with reduced DAT function.","abstract_has_math":false,"creators":["Beerepoot, Pieter Claus"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Pharmacology","school":null,"contributors":[],"advisors":["Salahpour, Ali"],"committee_chairs":[],"committee_members":[],"year":2017,"date_issued":"2017-06","date_published":"2017-06","updated_at":"2026-07-27T21:28:22Z","subjects":["Dopamine","Dopamine transporter deficiency syndrome","Pharmacological chaperones","Protein folding"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1807/78039","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Salahpour, Ali"]},{"key":"dc:contributor.department","label":"Department","values":["Pharmacology"]},{"key":"dc:creator","label":"Author","values":["Beerepoot, Pieter Claus"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2017-06"]},{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2017-07-21T20:00:10Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2017-07-21T20:00:10Z"]},{"key":"dc:date.issued","label":"Date","values":["2017-06"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Dopamine","Dopamine transporter deficiency syndrome","Pharmacological chaperones","Protein folding"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1807/78039"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The dopamine transporter (DAT) is a membrane protein that is essential for regulating signaling and intracellular stores of the neurotransmitter dopamine. An array of pathological conditions have been linked to mutations in the dopamine transporter gene, including ADHD, bipolar disorder, autism, and dopamine transporter deficiency syndrome (DTDS). DTDS is of particular interest, because it is exclusively caused by autosomal recessive loss-of-function mutations in the dopamine transporter (DAT), resulting in a debilitating pediatric movement disorder that is eventually fatal. The majority of DTDS DAT mutations disrupt DAT function by affecting folding and maturation of the transporter. Pharmacological chaperones have previously been used to rescue mutations in other membrane proteins, and we therefore examined the possibility of using pharmacological chaperones to rescue DTDS mutations. We developed a DAT surface expression assay in order to screen candidate compounds for potential pharmacological chaperone activity. After screening a set of known DAT ligands, we found that the DAT inhibitors bupropion and ibogaine increased DAT surface expression, whereas other inhibitors, including cocaine and methylphenidate, had no effect. The effect appears to be associated with stabilization of an inward- facing or occluded, as opposed to an outward-facing conformation of the DAT. Bupropion and ibogaine increased wild type DAT protein levels, demonstrating that drug effects were not merely due to protein trafficking. Bupropion and ibogaine also promoted maturation of the endoplasmic reticulum (ER)-retained DAT mutant K590A, which could be blocked by inhibiting ER to Golgi transport. Furthermore, bupropion decreased the half-life of immature ER-retained K590A DAT in cycloheximide chase assays. The data suggest that bupropion and ibogaine promote maturation of DAT by acting as pharmacological chaperones in the ER. Importantly, both drugs rescue DAT maturation and functional activity of a subset of DTDS-associated DAT mutants. Tests of pharmacological chaperoning of DAT in mice were not conclusive, and future studies will have to assess in vivo activity of bupropion in a true DTDS mouse model. Together, these results are the first demonstration of pharmacological chaperoning of DAT, and suggest this may be a viable approach to increase DAT levels in DTDS and other conditions associated with reduced DAT function."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["Pharmacological Chaperones of the Dopamine Transporter: A Strategy for Increasing Function of Wild Type and Mutant Transporter"]}]}],"canonical_facts":{"dc:contributor.advisor":["Salahpour, Ali"],"dc:contributor.department":["Pharmacology"],"dc:creator":["Beerepoot, Pieter Claus"],"dc:date":["2017-06"],"dc:date.accessioned":["2017-07-21T20:00:10Z"],"dc:date.available":["2017-07-21T20:00:10Z"],"dc:date.issued":["2017-06"],"dc:description.abstract":["The dopamine transporter (DAT) is a membrane protein that is essential for regulating signaling and intracellular stores of the neurotransmitter dopamine. An array of pathological conditions have been linked to mutations in the dopamine transporter gene, including ADHD, bipolar disorder, autism, and dopamine transporter deficiency syndrome (DTDS). DTDS is of particular interest, because it is exclusively caused by autosomal recessive loss-of-function mutations in the dopamine transporter (DAT), resulting in a debilitating pediatric movement disorder that is eventually fatal. The majority of DTDS DAT mutations disrupt DAT function by affecting folding and maturation of the transporter. Pharmacological chaperones have previously been used to rescue mutations in other membrane proteins, and we therefore examined the possibility of using pharmacological chaperones to rescue DTDS mutations. We developed a DAT surface expression assay in order to screen candidate compounds for potential pharmacological chaperone activity. After screening a set of known DAT ligands, we found that the DAT inhibitors bupropion and ibogaine increased DAT surface expression, whereas other inhibitors, including cocaine and methylphenidate, had no effect. The effect appears to be associated with stabilization of an inward- facing or occluded, as opposed to an outward-facing conformation of the DAT. Bupropion and ibogaine increased wild type DAT protein levels, demonstrating that drug effects were not merely due to protein trafficking. Bupropion and ibogaine also promoted maturation of the endoplasmic reticulum (ER)-retained DAT mutant K590A, which could be blocked by inhibiting ER to Golgi transport. Furthermore, bupropion decreased the half-life of immature ER-retained K590A DAT in cycloheximide chase assays. The data suggest that bupropion and ibogaine promote maturation of DAT by acting as pharmacological chaperones in the ER. Importantly, both drugs rescue DAT maturation and functional activity of a subset of DTDS-associated DAT mutants. Tests of pharmacological chaperoning of DAT in mice were not conclusive, and future studies will have to assess in vivo activity of bupropion in a true DTDS mouse model. Together, these results are the first demonstration of pharmacological chaperoning of DAT, and suggest this may be a viable approach to increase DAT levels in DTDS and other conditions associated with reduced DAT function."],"dc:description.degree":["Ph.D."],"dc:identifier.uri":["http://hdl.handle.net/1807/78039"],"dc:subject":["Dopamine","Dopamine transporter deficiency syndrome","Pharmacological chaperones","Protein folding"],"dc:title":["Pharmacological Chaperones of the Dopamine Transporter: A Strategy for Increasing Function of Wild Type and Mutant Transporter"],"dc:type":["Thesis"]},"updated_at":"2026-07-27T21:28:22Z"}