The role of zinc ions in reverse transport mediated by monoamine transporters

TY - JOUR

T1 - The role of zinc ions in reverse transport mediated by monoamine transporters

AU - Scholze, Petra

AU - Nørregaard, Lene

AU - Singer, Ernst A

AU - Freissmuth, Michael

AU - Gether, Ulrik

AU - Sitte, Harald H

PY - 2002/6/14

Y1 - 2002/6/14

N2 - The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.

AB - The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.

KW - Amphetamines

KW - Animals

KW - Binding Sites

KW - Biological Transport

KW - Cell Line

KW - DNA, Complementary

KW - Dopamine

KW - Dose-Response Relationship, Drug

KW - Female

KW - Humans

KW - Ions

KW - Kinetics

KW - Membrane Glycoproteins

KW - Membrane Transport Proteins

KW - Mutation

KW - Neuropeptides

KW - Norepinephrine

KW - Potassium

KW - Rats

KW - Rats, Sprague-Dawley

KW - Temperature

KW - Time Factors

KW - Transfection

KW - Vesicular Biogenic Amine Transport Proteins

KW - Zinc

U2 - 10.1074/jbc.M112265200

DO - 10.1074/jbc.M112265200

M3 - Journal article

C2 - 11940571

SN - 0021-9258

VL - 277

SP - 21505

EP - 21513

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 24

ER -