DOI
https://doi.org/10.25772/YFT0-4155
Defense Date
2017
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Physiology and Biophysics
First Advisor
Jose M. Eltit
Abstract
Research in drug development meets many challenges including lengthy, complex and costly procedures to identify novel pharmacotherapies. In our lab, we developed a method for fast screening of small molecules that interact with monoamine transports – dopamine and serotonin (DAT, SERT). These membrane proteins play important roles in brain neurotransmission responsible for cognition, motion and pleasure. Dysfunction in dopaminergic and serotonergic systems result in neurological disorders such as depression, Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and addiction.
DAT and SERT are responsible for uptake of dopamine (DA) or serotonin (5HT) into the synapse and they limit neurotransmitter signaling. Drugs that mimic or antagonize actions of endogenous neurotransmitters (DA and 5HT) increase the concentrations of DA and/or 5HT either by blocking the transporter (blockers) or by competing uptake with neurotransmitter (substrate). The uptake of substrates is associated to an inward current that depolarizes the cell membrane. Voltage-gated calcium channels (CaV) can respond to small changes in membrane potential. In our method, we combined permanent cell line expressing the human dopamine transporter (hDAT) or the human serotonin transporter (hSERT) (FlpIn TREx expression system) with transient transfection of CaV. This system works as a tightly electrically coupled system. Cells challenged with substrate of the transports produce detectable Ca2+ signal while monoamine transporter blockers can inhibit these Ca2+ signals. The novelty of this method relies on the ability to discriminate between substrate and blockers of monoamine transporters.
Preliminary experiments measuring our optimized cell system in a Flex Station 3 plate reader suggest that the co-expression of a voltage-gated Ca2+ channel, a monoamine transporter and a genetically encoded Ca2+ sensor constitute a rapid screening biosensor to identify active drugs at monoamine transporters.
Our novel methodology can rapidly assess drug-effect profile on monoamine transporters and benefit development of new psychotherapeutics for treatment of mental illnesses. It can also be used to characterize mechanism of action of emerging drug of abuse, as well as to discover small molecules with novel drug-effect profile useful in basic neuroscience research.
Rights
Iwona Ruchala
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-10-2017