The Norepinephrine Transporter - Gβγ Subunit Interaction Promotes Norepinephrine Efflux through the Transporter: In silico and Functional Studies

Norepinephrine (NE) uptake through the NE transporter (NET) is the primary mechanism to ter-minate the action of this neurotransmitter. Dysregulation of brain NE levels is associated with various conditions, including attention deficits, depression, and Alzheimer's disease. Recently, we identified an interaction between the related dopamine (DA) transporter (DAT) and G protein βγ subunits. Activation of Gβγ in cells and brain slices leads to DA efflux through DAT. However, the potential interaction between Gβγ and NET has not been reported. Here, we used a combina-tion of molecular modeling approaches to elucidate the structural details of the NET/Gβγ inter-action, supported by experimental evidence. Through 1.5µs of molecular dynamic simulation, we observed a favorable NET/Gβγ interaction mediated by the intracellular carboxy terminus of NET. As a consequence, the binding energy of NE for NET also changes resulting in alterations of the electrostatic profile of NET. We propose that these changes mediate the NE efflux effect. Fi-nally, we provide experimental evidence demonstrating that Gβγ physically interacts with NET and that the Gβγ activator mSIRK induces NE efflux through the transporter. These findings identified a novel interaction between Gβγ and NET and highlighted its potential implications in the regulation of NE neurotransmission and associated disorders.