Serotonin Transporter Function and Pharmacology Are Sensitive to Expression Level

Abstract

We express mammalian serotonin transporters (SERTs) in Xenopus oocytes by cRNA injection and measure 5-hydroxytryptamine (5-HT) transport and 5-HT-induced current at varying expression levels. Transport and current both increase sigmoidally with the amount of cRNA injected, but current requires ϳ5-fold more cRNA to elicit a half-maximal response. Western blots of SERT protein demonstrate that current, but not transport, correlates linearly with the amount of SERT on the plasma membrane. In oocytes co-injected with wild-type SERT and an inactive SERT mutant, transport is similar to SERT alone, but current is attenuated. The charge/transport ratio reports the differential sensitivity of transport and current to increasing SERT cRNA injection and mutant co-expression. Manipulations that alter the charge/transport ratio also perturb substrate and inhibitor recognition. 5-HT, d-amphetamine, cocaine, and paroxetine inhibit transport more potently at lower expression levels; however, 5-HT potency for induction of current is similar at high and low expression. Moreover, the apparent potency of cRNA for transport depends on 5-HT concentration. We postulate that SERT interacts allosterically with an endogenous factor of limited abundance to alter substrate and inhibitor potency and the balance of 5-HT transport and channel-like activity.Serotonin transporters accumulate serotonin into cells following 5-HT 1 release, thereby modulating serotonergic signaling and neurotransmission (1). 5-HT is implicated in a variety of behaviors, including mood, sleep, pain, appetite, aggression, and sexual behavior, and serotonin-selective reuptake inhibitors are useful for the treatment of human diseases (depression, obsessive-compulsive disorder, bulimia and eating disorders, alcoholism, anxiety and panic disorders, and premenstrual dysphoric disorder) (2, 3). SERT is a receptor for psychostimulant drugs of abuse such as 3,4-methylenedeoxymethamphetamine, amphetamine, and cocaine (4 -6).SERT (SLC6A4) is a member of the GAT/NET transporter family that encodes carriers for neurotransmitters, solutes, and amino acids that are similar in function (Na ϩ ,Cl Ϫ dependence), structure (12 putative transmembrane-spanning segments), and sequence (1, 7). The three-dimensional structure and relative positions of transmembrane-spanning segments is unknown for any member of the GAT/NET family. The deduced amino acid sequences of mammalian SERT cDNA clones (5, 7, 8) predict a polypeptide of ϳ68 kDa. Protein purification and radiation inactivation studies estimate a similar size for SERT (9, 10). SERT forms oligomeric complexes supported by FRET interactions (11), chemical cross-linking (12), and functional reconstitution following purification of a large protein complex (13,14). Epitope-tagged rSERT proteins can be co-immunoprecipitated and co-expression of (2-aminoethyl)methanethiosulfonate-sensitive or -insensitive mutants alters (2-aminoethyl)methanethiosulfonate inhibition, indicating that intersubunit interactions in the oligomeric complex modula...

A Trimeric Quaternary Structure Is Conserved in Bacterial and Human Glutamate Transporters

“…An alternative explanation might be the existence of multiple homo-oligomerization states with different functions. At low expression levels, serotonin transporters might be formed by a smaller number of subunits than at high levels thus explaining the observed dependence of transporter function and protein expression (43). This hypothesis would also account for the multiple experimentally determined subunit stoichiometries within the SLC6 family.…”

“…Ramsey and DeFelice (43) interpreted this result as evidence for an endogenous regulatory protein that is necessary for the carrier transport mode. At low heterologous expression levels, virtually all serotonin transporters will contain this regulatory protein and thus function as carriers, while the endogenous protein is not sufficiently available at high expression levels resulting in homomultimeric serotonin transporters that function as channels.…”

“…The channel-like activity was reported to become more apparent with increasing expression levels of rat and human serotonin transporters heterologously expressed in Xenopus oocytes (43). Ramsey and DeFelice (43) interpreted this result as evidence for an endogenous regulatory protein that is necessary for the carrier transport mode.…”

Na+, Cl−, and pH Dependence of the Human Choline Transporter (hCHT) inXenopusOocytes: The Proton Inactivation Hypothesis of hCHT in Synaptic Vesicles

“…The isolation of oocytes and cRNA preparation were described previously (Ramsey and DeFelice, 2002;Adams and DeFelice, 2003). Briefly, stage V-VI oocytes were harvested from Xenopus laevis (Nasco, Modesto, CA).…”

“…The full procedures were described previously (Ramsey and DeFelice, 2002). Briefly, equal numbers of oocytes from each cRNA injection (typically 20) were incubated with 1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL) in ice-cold PBS solution.…”