The role of phosphorylation in organic cation transporter 3

Organic cation transporter 3 (OCT3) is one of the neurotransmitter-removing transporters that actively terminate synaptic transmission by removing neurotransmitters from the synapse. In the periphery, OCT3 plays a role in cardiac contractility, thermogenesis, liver fibrosis and tumorigenesis. In the central nervous system, OCT3 participates in monoaminergic transmission, mood- and stress-related modulation, and vulnerability in neurodegeneration. Although phosphorylation has been extensively studied in neurotransmitter transporters, little is known about the phosphorylation of OCT3. Direct phosphorylation of OCT3 has not been shown and evidence for the importance of its regulatory function still remains enigmatic. The aim of the project is to establish knowledge about the structure-function relationship in OCT3 in relation to its phosphorylation status. Overall, we attempt to improve our understanding of the function of OCT3 and how it is controlled by phosphorylation by aiming to i) identify kinase/phosphatase-specific phosphorylation sites in OCT3 and their implication in regulating OCT3 transport function, ii) study the effects of phosphorylation on the pharmacodynamics and pharmacokinetics of OCT3 from human genetic variants and tyrosine kinase inhibitors (TKIs), respectively, and iii) to establish a plausible hypothesis for the observed effects in relationship to the structural and dynamic context. The methods build on a combination of experimental in vitro, microscopic and computational approaches, which are iteratively used for hypothesis generation, refinement and testing. Experimental in vitro approaches will mainly use biochemistry, tracer flux experiments and mass spectrometry. Cryo-electron microscopy and molecular dynamics (MD) simulations will be employed to elucidate the structural and dynamic effects of phosphorylation in OCT3.