Serotonin (5HT ) transporters (SERTs) couple to existing ion gradients to transport 5HT into presynaptic terminals. In mammalian SERTs, the transport cycle is reported as electroneutral, with a translocation of zero net charge, and 5HT uptake is independent of membrane voltage. Yet mammalian SERTs exhibit 5HT-induced currents, and Drosophila SERTs (dSERTs) show voltage-dependent uptake. Thus, the relationship between uptake and current remains controversial; furthermore, the number of 5HT molecules translocated per ion channel event is unknown. To investigate this, we have used heterologous expression of cloned dSERTs to measure 5HT flux and dSERT currents concurrently under voltage clamp, and we have used fluctuation analysis to measure the size of the elementary ionic events in the same cells. RNA-injected Xenopus oocytes accumulate 5HT, and paroxetine or desipramine inhibit this uptake. RNA-injected oocytes also display paroxetinesensitive 5HT-induced currents and 5HT-independent leak currents. Na replacement decreases the uptake and the induced currents. 5HT-induced current and 5HT uptake both increase at negative potentials, where 5HT carries ϳ5% of the induced current. Recently, several groups have reported similar phenomena for other transporters, in which transmitter-induced currents exceed the predictions of coupled transport. We now provide evidence that in dSERT, ϳ500 5HT molecules are translocated per channel opening, which, at Ϫ20 mV, carries ϳ10,000 electronic charges. These data support a model in which 500 SERT cycles occur for each 5HT-induced channel opening or a model in which 500 5HT molecules and 10,000 electronic charges pass through a common pore. Key words: serotonin; transporter; uptake; antidepressants; channels; Xenopus oocytesNeurotransmitter transporters couple the gradients of ions to the flux of the transmitter (Amara and Kuhar, 1993;. Na-and Cl-coupled transporters constitute a large family of related proteins that take up GABA, catecholamines, and serotonin (5HT). Na-coupled transporters make up a separate gene family for the uptake of glutamate and aspartate. Recently, several groups have described transporter-associated currents that exceed the predictions of stoichiometric transport Sonders and Amara, 1996). Excess currents have been observed in heterologous expression systems for 5HT Mager et al., 1994), glutamate (Vandenberg et al., 1995; Wadiche et al., 1995a,b), norepinephrine (Galli et al., 1995(Galli et al., , 1996, GABA (Cammack et al., 1994(Cammack et al., , 1996, and dopamine (Sonders et al., 1997) transporters. Similar phenomena appear in native preparations for 5HT (Bruns et al., 1993), GABA (Cammack and Schwartz, 1993), and glutamate (Larson et al., 1996) transporters. Here we report the first data to correlate in the same cells the movement of transmitter with the channel activity underlying the transmitter-induced current. We propose two models that relate this information to voltage-dependent uptake.Serotonin uptake has been characterized by radiolabeled 5HT flu...

Serotonin transporters (SERTs) are targets for drugs such as Prozac that increase serotonin (5HT) levels by blocking 5HT reuptake. Although SERTs saturate in the micromolar range, synaptic 5HT may exceed 1 mM. To examine SERT's response to high 5HT concentrations, we expressed Drosophila SERT (dSERT) in Xenopus oocytes and found that transport continued to increase with concentration up to 0.3 mM 5HT. As 5HT is a monovalent cation, its entry through an ion channel in SERT might explain uptake at high concentrations. We therefore investigated dSERT using traditional ion channel methods, including mole-fraction experiments under voltage clamp. We propose that SERTs may function as 5HT-permeable channels, and that this mechanism may be important for clearance of the neurotransmitter at high concentrations.

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The cardiac potassium channel encoded by the human ether-à-go-go related gene (HERG) is blocked by a diverse array of common therapeutic compounds. Even transient exposure to such agents may provoke the life-threatening cardiac arrhythmia torsades de pointes in some, but not all, individuals. Although the molecular and genetic factors predicting such wide variability in drug response remain unclear, known sequence variations within the coding region of HERG do not explain the adverse drug response in many cases. Although other proteins can modulate HERG function, no studies have identified protein partners capable of limiting the pharmacological sensitivity of HERG. Here we show that KCR1, a protein identified previously in rat cerebellum, is a plasma membrane-associated protein expressed at the RNA level in the human heart and can be immunoprecipitated with HERG. Functionally, KCR1 reduces the sensitivity of HERG to classic proarrhythmic HERG blockers (sotalol, quinidine, dofetilide) in both cardiac and noncardiac cell lines. We propose that KCR1, when coupled to HERG, may limit the sensitivity of HERG to proarrhythmic drug blockade and may be a rational target for modifying the proarrhythmic effects of otherwise clinically useful compounds.

Human ether-a-go-go-related gene (HERG) encodes the pore-forming subunit of IKr, a cardiac K ؉ channel. Although many commonly used drugs block IKr, in certain individuals, this action evokes a paradoxical life-threatening cardiac rhythm disturbance, known as the acquired long QT syndrome (aLQTS). Although aLQTS has become the leading cause of drug withdrawal by the U.S. Food and Drug Administration, DNA sequencing in aLQTS patients has revealed HERG mutations only in rare cases, suggesting that unknown HERG modulators are often responsible. By using the worm Caenorhabditis elegans, we have developed in vivo behavioral assays that identify candidate modulators of unc-103, the worm HERG orthologue. By using RNA-interference methods, we have shown that worm homologues of two HERG-interacting proteins, Hyperkinetic and K channel regulator 1 (KCR1), modify unc-103 function. Examination of the human KCR1 sequence in patients with drug-induced cardiac repolarization defects revealed a sequence variation (the substitution of isoleucine 447 by valine, I447V) that occurs at a reduced frequency (1.1%) relative to a matched control population (7.0%), suggesting that I447V may be an allele for reduced aLQTS susceptibility. This clinical result is supported by in vitro studies of HERG dofetilide sensitivity by using coexpression of HERG with wild-type and I447V KCR1 cDNAs. Our studies demonstrate the feasibility of using C. elegans to assay and potentially identify aLQTS candidate genes.C aenorhabditis elegans unc-103 shares 70% amino acid identity with HERG in the conserved transmembrane and pore regions of the protein (see Scheme 1).Studies using unc-103 promoter GFP reporter constructs reveal expression of unc-103 in body-wall muscle, egg-laying muscles, pharyngeal muscles, and neurons that innervate these tissues (D.J.R, J.H.T., and R. Garcia, unpublished data). Worm strains carrying mutations in this gene, unc-103 (n500) and unc-103 (e1597), were isolated in screens for locomotion-defective mutants (1). Analysis of both of these neomorphic mutant strains revealed the same mutation: conversion of a conserved alanine in the S6 transmembrane domain to a threonine (A334T, indicated in bold above). Our studies reveal that these mutant worms exhibit profound neuromuscular defects, and the severity of these defects is sensitive to modulators that decrease the level of mutant channel activity. Further, the human homologues of these modulators may have physiologically relevant interactions with human ether-a-go-go-related gene (HERG) and represent acquired long QT syndrome (aLQTS) candidate genes. MethodsMolecular Biology. The human K channel regulator 1 (KCR1) cDNA, an IMAGE clone (no. 650823) was purchased from Research Genetics (Huntsville, AL). Site-directed mutagenesis was performed as described (2). For in vitro cRNA transcription, we used the SP6 mMessage mMachine high-yield capped RNA transcription kit (Ambion, Austin, TX). For RNA interference (RNAi) vectors, we PCR-amplified fragments of 0.9-1.5 kb of the target gene f...