TY - JOUR

T1 - Evidence for distinct sodium-, dopamine-, and cocaine-dependent conformational changes in transmembrane segments 7 and 8 of the dopamine transporter

AU - Norregaard, Lene

AU - Loland, Claus Juul

AU - Gether, Ulrik

PY - 2003/8/15

Y1 - 2003/8/15

N2 - Previously we obtained evidence based on engineering of Zn2+ binding sites that the extracellular parts of transmembrane segment 7 (TM7) and TM8 in the human dopamine transporter are important for transporter function. To further evaluate the role of this domain, we have employed the substituted cysteine accessibility method and performed 10 single cysteine substitutions at the extracellular ends of TM7 and TM8. The mutants were made in background mutants of the human dopamine transporter with either two (E2C) or five endogenous cysteines substituted (X5C) that render the transporter largely insensitive to cysteine modification. In two mutants (M371C and A399C), treatment with the sulfhydryl-reactive reagent [2-(trimethylammonium)-ethyl]methanethiosulfonate (MTSET) led to a substantial inhibition of [3H]dopamine uptake. In M371C this inactivation was enhanced by Na+ and blocked by dopamine. Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A399C. The present findings add new functionality to the TM7/8 region by providing evidence for the occurrence of distinct Na+-, substrate-, and perhaps inhibitor-induced conformational changes critical for the proper function of the transporter.

AB - Previously we obtained evidence based on engineering of Zn2+ binding sites that the extracellular parts of transmembrane segment 7 (TM7) and TM8 in the human dopamine transporter are important for transporter function. To further evaluate the role of this domain, we have employed the substituted cysteine accessibility method and performed 10 single cysteine substitutions at the extracellular ends of TM7 and TM8. The mutants were made in background mutants of the human dopamine transporter with either two (E2C) or five endogenous cysteines substituted (X5C) that render the transporter largely insensitive to cysteine modification. In two mutants (M371C and A399C), treatment with the sulfhydryl-reactive reagent [2-(trimethylammonium)-ethyl]methanethiosulfonate (MTSET) led to a substantial inhibition of [3H]dopamine uptake. In M371C this inactivation was enhanced by Na+ and blocked by dopamine. Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A399C. The present findings add new functionality to the TM7/8 region by providing evidence for the occurrence of distinct Na+-, substrate-, and perhaps inhibitor-induced conformational changes critical for the proper function of the transporter.

KW - Amino Acid Sequence

KW - Animals

KW - COS Cells

KW - Cocaine

KW - Dopamine

KW - Dopamine Plasma Membrane Transport Proteins

KW - Dopamine Uptake Inhibitors

KW - Extracellular Space

KW - Humans

KW - Indicators and Reagents

KW - Membrane Glycoproteins

KW - Membrane Transport Proteins

KW - Mesylates

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Nerve Tissue Proteins

KW - Protein Conformation

KW - Protein Structure, Tertiary

KW - Radioligand Assay

KW - Sodium

KW - Tritium

KW - Zinc

U2 - 10.1074/jbc.M303854200

DO - 10.1074/jbc.M303854200

M3 - Journal article

C2 - 12773538

VL - 278

SP - 30587

EP - 30596

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 33

ER -