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Vol. 57, Issue 6, 1093-1103, June 2000
G and G Values Implicate Regions
Important for Transporter Functions
Molecular Neurobiology Branch, National Institute on Drug Abuse,
Intramural Research Program, Baltimore, Maryland
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
(
Go and
Goint) 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
Goint values for dopamine
uptake. TMs 4 and 11 mutants provide the largest
Goint 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.
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