Abstract
AbstractThe plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.
Funder
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
Austrian Science Fund
The Theodor Körner Prize is a set of annual Austrian awards bestowed by the Theodor Körner Fund in recognition of cultural and/or scientific advances. The prize is awarded at the University of Vienna.
U.S. Department of Health & Human Services | NIH | National Institute on Drug Abuse
EC | Horizon 2020 Framework Programme
Publisher
Springer Science and Business Media LLC
Subject
General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)
Cited by
8 articles.
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