A pincer-like configuration of TM2 in the human dopamine transporter is responsible for indirect effects on cocaine binding

Abstract

The second transmembrane segment (TM2) of DAT and other neurotransmitter transporters has been proposed to play a role in oligomerization as well as in cocaine binding. In an attempt to determine whether TM2 contributes to the binding site and/or transport pathway of DAT, we mutated to cysteine, one at a time, 25 residues in TM2 – from Phe98 to Gln122 – in an appropriate DAT background construct. Four of the mutants, F98C, G110C, P112C, and E117C, did not express at the cell surface, and G121C was inactive, despite its presence on the cell surface. Of the 21 mutants that expressed, none of the substituted cysteines reacted with MTSEA biotin-CAP, and none of the 20 functional mutants was sensitive to MTSEA or MTSET. Thus, TM2 does not appear to be water-accessible, based both on the lack of functional effects of charged MTS derivatives, and on the biochemical determination of lack of reaction with a biotinylated MTS derivative. This leads to the conclusion that TM2 does not contribute directly to the substrate-binding site or the transport pathway, and suggests that the observed effect of mutations in this region on cocaine binding is indirect. Three mutants, M106C, V107C and I108C, were crosslinked by treatment with HgCl₂. This crosslinking was inhibited by the presence of the cocaine analogue MFZ 2–12, likely due to a conformational rearrangement in TM2 upon inhibitor binding. However, the lack of crosslinking of cysteines substituted for Leu99, Leu113 and Leu120 – three of the residues that along with Met106 form a leucine heptad repeat in TM2 – makes it unlikely that this leucine repeat plays a role in symmetrical TM2 dimerization. Importantly, a high-resolution structure of LeuT, a sodium-dependent leucine transporter that is sufficiently homologous to DAT to suggest a high degree of structural similarity, became available while this manuscript was under review. We have taken advantage of this structure to explore further and interpret our experimental results in a rigorous structural context.

Introduction

The plasma membrane dopamine transporter (DAT) plays an essential role in terminating dopaminergic neurotransmission by reuptake of dopamine into presynaptic neurons (Giros and Caron, 1993). DAT is a member of the neurotransmitter: sodium symporter (NSS) family (2.A.22) (http://www.tcdb.org/tcdb/index.php?tc=2.A.22.1.3), which also includes transporters for serotonin, norepinephrine, GABA, glycine, several other amino acids, and osmolytes. DAT is a principal target for psychostimulant drugs such as cocaine and amphetamine (Kuhar et al., 1991, Ritz et al., 1987), and the serotonin and norepinephrine transporters are primary targets for antidepressant medications. The NSS are secondary active transporters that use electrochemical gradients to drive the translocation of organic substrates across membranes: sodium is co-transported with substrate and the sodium gradient is the major driving force for transport. These proteins are predicted to contain 12 TM domains and to have intracellular amino and carboxy termini (reviewed in Goldberg et al., 2003). Studies using specific antibodies and chemical modification have supported this topology for NSS members (Androutsellis-Theotokis and Rudnick, 2002, Chen et al., 1998, Ferrer and Javitch, 1998, Hersch et al., 1997).

Increasing evidence suggests that neurotransmitter transporters form dimers or oligomers in the plasma membrane (reviewed in Sitte et al., 2004). Mutations in a leucine heptad repeat in TM2 in both DAT and GAT1 disrupt oligomerization and interfere with cell surface expression (Scholze et al., 2002, Torres et al., 2003). A direct interaction of the leucine heptad repeat from TM2 of each protomer in a dimer has been inferred from these studies (Scholze et al., 2002, Torres et al., 2003), and a recent study has also implicated polar residues in TM2 in driving GAT1 oligomerization by intramembrane contacts of adjacent TM2 helices (Korkhov et al., 2004).

The present study investigates a potential role of TM2 in lining the transport pathway and/or binding site of DAT. Based on cysteine accessibility studies (Javitch, 1998, Karlin and Akabas, 1998, Stauffer and Karlin, 1994), we infer that TM2 is not water-accessible and does not contribute directly to the transport pathway or to the binding site for substrate or for cocaine. Although crosslinking of several residues in TM2 was observed in the presence of mercuric chloride, suggesting that TM2 may be located near a symmetric interface, the overall crosslinking pattern does not support a role of the leucine heptad repeat in TM2 in forming these contacts. Crosslinking of residues in TM2 was altered in the presence of a cocaine analog, consistent with state-dependent conformational changes in TM2 and with an indirect role of TM2 in cocaine binding. The high-resolution structure of LeuT, a sodium-dependent leucine transporter from Aquifex aeolicus (Yamashita et al., 2005) that is homologous to DAT, was released while this manuscript was under review. We have taken advantage of this structure to analyze our experimental results in a rigorous structural context.