Abstract
The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.
Highlights
The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space
To address the possible risk of perturbing the structure and dynamics of dopamine transporter (DAT) by the use of detergent micelles as membrane mimic, we carried out a comparison of all-atom MD simulations of DAT from Drosophila melanogaster (dDAT) embedded in a n-dodecyl β-Dmaltoside (DDM) micelle and in a mixed lipid bilayer consisting of phosphatidylcholine (POPC), phosphatidylethanolamine (POPE), phosphatidylglycerol (POPG), and cholesterol (CHOL) in a 3:1:1:1 ratio
We retained the crystallographically-resolved cholesterol molecule bordered between transmembrane domains (TMs) 1a, 5, and 7 as well as a cholesteryl hemisuccinate molecule (CHS; which we replaced by cholesterol) at the interface between TM 2, 7, and 11, which have been suggested to be important for transporter activity[13,32]
Summary
The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the statedependent conformational changes associated with the alternating access model of the dopamine transporter. NSS proteins are believed to follow an alternating access transport mechanism[16,17] suggesting that the central binding site is alternatingly accessible to the intracellular and the extracellular side of the membrane. This requires the existence of external and internal ‘gates’, i.e., dynamic structural units that are capable of occluding access to the substrate binding site from the externalor internal environment, respectively.
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