Delineating structure-function relationships in the dopamine transporter from natural and engineered Zn2+ binding...

Area of Science:

• Neuroscience
• Structural Biology
• Biochemistry

Background:

• The molecular mechanisms of substrate translocation and tertiary structure of neurotransmitter transporters remain largely unknown.
• The human dopamine transporter (hDAT) plays a crucial role in dopamine signaling.

Purpose of the Study:

• To gain insight into the structural organization of the human dopamine transporter (hDAT).
• To identify key residues involved in the transporter's structure and function.

Main Methods:

• Identification of an endogenous high-affinity Zn2+ binding site on hDAT.
• Engineering of an artificial Zn2+ binding site.
• Site-directed mutagenesis of potential Zn2+ coordinating residues.
• Transient expression of hDAT in COS-7 cells.

Main Results:

• Zn2+ acts as a potent non-competitive inhibitor of dopamine uptake by hDAT.
• Three residues (193His, 375His, 396Glu) were identified as forming the endogenous Zn2+ binding site.
• These residues, though distant in primary sequence, are in close spatial proximity in the tertiary structure.
• An artificial Zn2+ binding site engineered between TM 7 and TM 8 confirmed domain proximity and suggested an alpha-helical structure at the top of TM 8.

Conclusions:

• The study provides the first structural insights into hDAT using Zn2+ as a molecular probe.
• The identified residues and engineered binding site offer a foundation for understanding hDAT structure and developing targeted inhibitors.