RT Journal Article
SR Electronic
T1 Dopamine Transporter Transmembrane Domain Polar Mutants: Δ<em>G</em> and ΔΔ<em>G</em> Values Implicate Regions Important for Transporter Functions
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1093
OP 1103
VO 57
IS 6
A1 Itokawa, Masanari
A1 Lin, Zhicheng
A1 Cai, Ning-Sheng
A1 Wu, Cindy
A1 Kitayama, Shigeo
A1 Wang, Jia-Bei
A1 Uhl, George R.
YR 2000
UL http://molpharm.aspetjournals.org/content/57/6/1093.abstract
AB Polar residues in dopamine transporter (DAT) transmembrane domains (TMs) are likely to act individually and even interactively in recognizing cocaine and dopamine. We initially evaluated the effects of alanine substitution mutants that remove the polar side chains from residues in each of the 12 putative DAT TMs on the recognition of dopamine and the cocaine analog CFT. Eleven combination mutants with multiple substitutions in DAT TMs 4, 5, 7, or 11 were then selected as candidates for more detailed evaluation based on mutation effects on dopamine and cocaine analog affinities. An evaluation of Gibbs free energy changes displayed by single and combined TM mutants (ΔGo and ΔΔGoint) reveals three categories of potential interactions among mutants: 1) independent, noncooperative interactions (five influenced CFT and two influenced dopamine affinities), 2) synergistic influences (two for CFT and four for dopamine), and 3) complementation of influences on CFT recognition (four mutants) or on dopamine affinity (five). Combined mutations in TMs 4 and 5 yield the largest ΔΔGoint values for dopamine uptake. TMs 4 and 11 mutants provide the largest ΔΔGoint for CFT binding. Interactions between residues lying in DAT TMs 4 and 5 support current DAT structural models that suggest the juxtaposition of these two TMs. These data also support contributions of TM 4 and 11 residues to a polar pocket important for cocaine recognition. These candidate interactive DAT polar domains provide larger target sites for compounds that could modulate specific DAT functions than those provided by single mutations alone. The American Society for Pharmacology and Experimental Therapeutics