Dynamic Mechanism of Norepinephrine Reuptake and Antidepressants Blockade Regulated by Membrane Potential

During nerve signaling, changes in membrane potential are key to regulating neuronal activity. The norepinephrine transporter (NET) plays a crucial role in the reuptake of norepinephrine (NE), which is essential for maintaining neurotransmitter homeostasis. However, the impact of membrane potential on NET function has long been understudied. Despite the great biological significance of NET, the dynamic molecular mechanisms of NE transport and the blockade effects of antidepressants on this process remain unclear. Here, we reveal the structural, electrostatic, and dynamic characteristics of the NET-NE/antidepressants systems, indicating the dynamic voltage dependence of the NET function. By analyzing the structure and electrostatic properties of the central binding pocket, we find that a hydrophobic network stabilizes the localization of NE, while the dynamic hydrogen bond and salt bridge network plays a crucial role in facilitating the inward transport of NE. Changes in membrane potential significantly affect the reuptake of NE through an electrostatically driven substrate transport pathway, primarily influencing the substrate entrance, the hydrophilic channel leading to the central site, and the exit region. The hyperpolarized state favors NE reuptake, exhibiting a marked preference for inward movement, which aligns with the physiological need for neurons to regulate neurotransmitter concentration in the synaptic cleft via reuptake. Conversely, in the depolarized state, which corresponds to the generation of nerve impulses, NE reuptake may not peak. Furthermore, antidepressants, with their larger molecular size and longer charged amino groups, initially anchor to the essential residue E382 required for NE reuptake. They subsequently occupy the same binding pathway as NE, creating spatial hindrance that effectively blocks NE binding to the central pocket. Additionally, their binding/dissociation behaviors exhibit significant voltage dependence. Under the hyperpolarized state, antidepressants can better block NE entry through more flexible electrostatic and hydrophobic interactions with NET, while the depolarized state raises the binding barrier for antidepressants, facilitating their dissociation. And with this work, a computational strategy for membrane protein-ligand is proposed to emphasize that considering the effects of electric fields in the calculations can reveal more underlying mechanisms and key interactions.