Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringer Strasse 13A, 1090, Vienna, Austria.
Institute of Applied Physics, TU Wien, Lehargasse 6, 1060, Vienna, Austria.
Commun Biol. 2022 Nov 17;5(1):1259. doi: 10.1038/s42003-022-04210-1.
The 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 (PIP) on NET oligomerization and efflux. NET forms monomers (60%) and dimers (40%) at the plasma membrane. PIP 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 PIP binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP depletion; this indicates that PIP interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP directly regulates NET organization and function.
质膜去甲肾上腺素转运体（NET）通过清除突触间隙中的细胞外去甲肾上腺素来调节心血管交感神经活动。在这里，我们使用单分子荧光显微镜和流量测定法研究 NET 的亚基组成和功能。特别是，我们展示了磷脂酰肌醇 4,5-二磷酸（PIP）对 NET 寡聚化和外排的影响。NET 在质膜上形成单体（60%）和二聚体（40%）。PIP 耗竭导致平均寡聚状态降低，并减少 NET 介导的底物外排，而不影响底物摄取。将假定的 PIP 结合残基 R121、K334 和 R440 突变为丙氨酸不会影响 NET 二聚体化，但会导致底物外排减少，而 PIP 耗竭不会改变这种情况；这表明 PIP 与这些残基的相互作用会影响 NET 介导的外排。去甲肾上腺素和 PIP 信号的失调都与神经精神和心血管疾病有关。这项研究提供了证据表明 PIP 直接调节 NET 的组织和功能。
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