Tyrosine-533 of rat dopamine transporter: involvement in interactions with 1-methyl-4-phenylpyridinium and cocaine.
To improve our understanding of structure-function relationships for neurotransmitter transporters, we performed site-directed mutagenesis of the rat dopamine transporter (DAT) and assessed the functions of the mutants in transiently-expressing COS cells. Tyrosine-533 of rat DAT lies in the 11th transmembrane region, where the corresponding amino acid of human DAT is phenylalanine. Alanine substitution of tyrosine-533 (Y533A) conferred an increased affinity for 1-methyl-4-phenylpyridinium (MPP+). Phenylalanine substitution of tyrosine-533 (Y533F) increased the velocity of MPP+ uptake but decreased DAT's affinity for MPP+. Cocaine's potency in inhibiting dopamine uptake was unchanged with Y533A, but increased with Y533F. Differences in the uptake kinetics and inhibitory potency of cocaine between rat and human DATs were similar to the differences observed between the wild-type and Y533F mutants DATs. Tyrosine-533 may be important for the DAT function and for species differences in transporter functions, including differential sensitivities to cocaine and 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in humans and rats.
Mitsuhata
C
C
Department of Pharmacology, Hiroshima University School of Dentistry, Kasumi 1-2-3, Minami-ku, Hiroshima 734, Japan.
Kitayama
S
S
Morita
K
K
Vandenbergh
D
D
Uhl
G R
GR
Dohi
T
T
D015655:1-Methyl-4-phenylpyridinium / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D003042:Cocaine / Q000378:metabolism*; D006224:Cricetinae; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D051381:Rats; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism / Q000502:physiology*
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; C493216:Slc6a3 protein, rat; D014443:Tyrosine; D003042:Cocaine; D015655:1-Methyl-4-phenylpyridinium; D004298:Dopamine