Importance of valine at position 152 for the substrate transport and 2 beta-carbomethoxy-3 beta-(4-fluorophenyl) tropane binding of dopamine transporter

Human and bovine dopamine transporters (DAT) demonstrate discrete functiona l differences in dopamine (DA), 1-methyl-4-phenylpyridium (MPP+) transport, and cocaine analog binding. In a previous study, the functional analyses o n the chimeras of human and bovine DAT have revealed that the region from r esidues 133 through 186 (encompassing the third transmembrane domain) is re sponsible for the substrate transport and cocaine analog binding. The prese nt study has been carried out to determine the specific amino acid(s) confe rring DAT functions by interchanging the amino acid residues in the corresp onding region between human and bovine DAT. As described previously, the DA , MPP+ transport, and 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (C FT) binding almost disappeared in chimera hb3 in which the region from resi dues 133 through 186 of bovine DAT was substituted into human DAT. Replacem ent of isoleucine, residue 152 of chimera hb3 (bovine DAT sequence), with v aline, the human DAT residue at the identical position, remarkably restored the substrate transport and CFT binding to 76% to 98% of the human DAT val ues. Similarly, substitution of isoleucine for valine at position 152 in th e human DAT reduced the substrate transport and CFT binding by 57% to 97%. Among other amino acids tested at position 152 of the chimera hb3, only ala nine resulted in small but significant increases in the DAT functions rangi ng from 16 to 34%. Thus, valine at position 152 plays a crucial role for mo lecular mechanisms underlying the interactions of DA, MPP+, and CFT with hu man DAT.