The region of dopamine transporter encompassing the 3rd transmembrane domain is crucial for function

Bovine dopamine transporter (bDAT) shows relatively low dopamine (DA) uptake efficacy and scant MPP+ transport and cocaine binding, while all of these activities are avid in human dopamine transporter (hDAT). In this study, we constructed a series of chimeras between human and bovine DAT to find out the structure(s) responsible for each DAT functions. DA uptake, MPP+ uptake, and cocaine binding in hDAT nearly disappeared only by the substitution of the 3rd transmembrane domain (TMD) with that of bDAT. On the contrary, the substitution of an identical region of hDAT into bDAT produced a complete shift toward the value of WT hDAT in the DA uptake, even though the MPP+ uptake and CFT binding maintained the values of WT bDAT. Collectively, these results suggest that the 3rd TMD in DAT is crucial for the function, especially for DA uptake.