| Summary: | Cellular membranes were once considered static and passive structures, but are now appreciated as a fluidic and dynamic assembly of macromolecules that play an active role in cellular function. Membrane composition has been proposed to play a critical role in modulating protein function by affecting everything from post-translational modifications to conformation, but the physiologic relevance of the relationship between protein and membrane has been difficult to establish. For example, membrane-associated proteins such as Flotillin-1 (Flot1) have been implicated to scaffold proteins into cholesterol-rich membranes, as well as play a role in a wide array of functions such as endocytosis and axon pathfinding; however, genetic elimination of Flot1 expression had little to no reported consequence, leaving to question the physiologic importance of scaffolding proteins to membrane microdomains. Using genetic and biochemical approaches, I sought to understand how the immediate lipid environment can influence the function of a transmembrane protein, and how this might impact brain function. Specifically, I examined how a cholesterol-rich environment can affect the function of the cell surface neurotransmitter transporter for dopamine, the dopamine transporter (DAT), and how this interaction may influence the ability of an organism to respond to the psychostimulant amphetamine (AMPH).
Although neurotransmitter transporters (NTTs) such as DAT and the serotonin transporter (SERT), have been predicted to reside in membrane rafts, it has been difficult to establish the role of microdomain localization in transporter function. DAT localizes to the plasma membrane, where it modulates the strength and duration of neurotransmission by clearing dopamine (DA) from the perisynaptic space. Defects in DAT have been implicated in a range of psychiatric and neurological disorders, from schizophrenia to Parkinson’s disease. Additionally, as a target of psychostimulants, such as AMPH and cocaine (COC), the role of DAT in addiction is of societal interest.
Given that Flot1 was required for scaffolding heterologously expressed DAT to cholesterol-rich membranes in cell-based systems, and was selectively necessary for the non-exocytic release of DA through DAT in response to AMPH, I sought to test the hypothesis that the Flot1-mediated membrane localization of DAT was significant for the ability of mice to respond to AMPH. To this end, I created a series of genetic models to determine how the presence of Flot1 impacts DAT function in the brain. I found that Flot1 is not only important for scaffolding DAT into cholesterol-rich membranes, but that the ability of DAT to partition into these membranes was necessary for DAergic neurons, DAT, and ultimately mice, to respond to AMPH. Given that the other parameters of DA neuron function, as well as the ability of the animals to respond to COC was unaffected by DAT partitioning, my findings demonstrate that AMPH and COC exert different mechanisms of action in vivo. Moreover, I found that the cholesterol-rich membrane environment promoted a conformation of DAT that was favorable for reverse transport of DA through DAT, namely increasing the ability of its N-terminus to bind to the phospholipid, PIP2. This dissertation provides the first glimpse into not only how membrane localization can affect protein conformation and function but also the physiologic relevance of these Flot1-dependent membrane microdomains in brain.
|
|---|
