Oligomerization of serotonin transporter and its functional consequences

Abstract

Two forms of serotonin transporter (SERT) were prepared with different epitope tags. When co-expressed in HeLa cells, the form containing a FLAG tag (Res-FLAG) was associated with the form containing a c-myc tag (Sens-myc). Antibody against c-myc precipitated Res-FLAG from detergent extracts of cells expressing both forms, but not when Res-FLAG was expressed alone. The specificity of the interaction was demonstrated by the observation that anti-myc antibodies did not precipitate the unrelated vesicular stomatitis virus coat glycoprotein when it was co-expressed with Sens-myc. Sens-myc contained a reactive cysteine at position 172, which reacted with both (2-aminoethyl)methanethiosulfonate and N-biotinylaminoethyl methanethiosulfonate on the surface of intact cells. Sens-myc, but not Res-FLAG, was inactivated by these reagents. When co-expressed with Sens-myc, functionally active Res-FLAG was precipitated by immobilized streptavidin from digitonin-solubilized cells that had been treated with N-biotinylaminoethyl methanethiosulfonate. In cells co-expressing mixtures of Sens-myc and Res-FLAG, the amount of inactivation by (2-aminoethyl)methanethiosulfonate was less than expected if the two forms were independent. The results are consistent with a dimeric form of SERT with functional interactions between subunits, and with association of dimers into a higher order complex, possibly a tetramer. Serotonin transporter (SERT) is responsible for the accumulation of serotonin by neurons and peripheral cells. It is a target for antidepressant drugs and also for psychostimulants such as cocaine and amphetamine derivatives. SERT is part of a larger family of Na ϩ -and Cl Ϫ -dependent solute carriers including transporters for the biogenic amine neurotransmitters norepinephrine and dopamine, and for the amino acid transmitters ␥-aminobutyric acid and glycine, as well as other substrates. SERT is a polytopic membrane protein predicted to contain 12 transmembrane (TM) domains. The high degree of sequence identity between members of this gene family suggests that the structures of all these proteins are very similar.As transport proteins, carriers like SERT share many structural features with channels. One of the characteristics that distinguishes carriers from channels is that, in channels, the pore through which ions flow is believed to be formed by multiple similar subunits or domains, each of which donates a TM domain to form the channel (1). In contrast, carriers are believed to exist in two distinct states that bind internal and external substrate, respectively, and that are interconverted by conformational changes (2). These carriers contain approximately 12 TM domains that are believed to form the binding sites and the permeation pathway within each monomer. Judging by the size of particles in freeze-fracture images, Eskandari et al. (3) estimated that Na ϩ -glucose symporter was a monomer in the membrane. In lac permease of Escherichia coli, interaction between monomers was not observed, even when the monomers were co...

RTI-76, an isothiocyanate derivative of a phenyltropane cocaine analog, as a tool for irreversibly inactivating dopamine transporter function in vitro

“…Combined with the lack of disparity in potency of WIN 35,428 in uptake and binding measures, the data suggests that oligomerization phenomena cannot be detected, with the current pharmacological approaches, and in the present cell line expressing the dopamine transporter. A more direct biochemical determination of dopamine transporter oligomers may be needed, such as the recent molecular approaches applied to serotonin transporters (Jess et al 1996;Kilic and Rudnick 2000). Based on the present results, the possibility should be considered that dopamine transporter oligomers are not formed in heterologously expressing cell lines even though they may exist in native tissue (however, see below results for COS-7 cells by Lee et al 1996).…”

“…Complications relating to this simple view have recently emerged because IC 50 values of various uptake blockers for inhibiting cocaine analog binding to the transporter in rat striatum were approximately twofold greater than those for inhibiting dopamine uptake when measured under identical and kinetically relevant conditions . In addition, preliminary evidence suggests the possibility of dopamine transporter dimers (Xu et al 1997b) or more complex oligomers (Berger et al 1994;Milner et al 1994), analogous to indications for serotonin transporter oligomers (Jess et al 1996;Kilic and Rudnick 2000).…”

Functionally independent subunits in the oligomeric structure of the GABA cotransporter rGAT1

“…In contrast, transporters, although forming structural oligomers, often appear to function as individual monomers. It may be appropriate to recall an interesting consideration by Kilic and Rudnick [12] stated in a paper in which they actually proposed functional interaction between subunits of the serotonin transporter: 'Small ions present a symmetrical surface to any protein with which they interact whereas carriers for more complex molecules must recognize an asymmetric substrate and in some cases discriminate between stereoisomers. An asymmetric binding site formed from many different TM domains would be more suited to binding complex substrates than a symmetrical binding site formed from identical TM domains.…”

“…This secondary structure has been confi rmed by the recently published crystal structure of LeuT Aa , a bacterial homologue of the Na + /Cl --dependent neurotransmitter transporters [8], also showing an oligomeric quaternary structure as previously described in almost all the transporters of this family, with the only exception of glycine transporters 1 and 2, which were shown to exist as monomers at least in the plasma membrane [9]. Biochemical evidence indicated the existence of oligomeric complexes in the norepinephrine (NET) [10], serotonin (SERT) [11,12] and dopamine (DAT) transporters [13]. This latter has been investigated in more detail using a cross-linking assay [14][15][16]: DAT has been shown to form dimers of dimers with two symmetrical interfaces in transmembrane domains 4 and 6.…”

“…The formation of oligomeric complexes appears to be important for the correct targeting of the transporters to the membrane, although additional functional roles could not be excluded. In fact, experimental evidence indicates a functional interaction between the subunits of hSERT dimers [12], and the functional oligomerization of hNET and hDAT [13,25]. Furthermore, a very interesting countertransport model for monoamine transporters has recently been proposed [26].…”