DrosophilaSerotonin Transporters Have Voltage-Dependent Uptake Coupled to a Serotonin-Gated Ion Channel

Abstract

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...

The Family of Na+/Cl− Neurotransmitter Transporters

“…Monoamine transporters, as well as other family members, are regulated by phosphorylation and dephosphorylation by protein kinases and phosphatases. This subject was discussed recently in an excellent review and will not be covered in this review (see Blakely et a!., 1997).…”

“…It was demonstrated that transporters may be involved in the action of numerous drugs and specific toxins that affect the nervous system (Snyder and D'Amato, 1986;Bergman et al, 1989;Kuhar et al, 1991). Monoamine transporters, for example, are targeted by major classes of antidepressants, psychostimulants, and antihypertensive drugs (Blakely et al, 1997). They are also necessary for the actions of several classes of neurotoxins that selectively poison specific neuronal targets (Snyder and D'Amato, 1986).…”

“…The drug also possesses a reasonable affinity to the other Na + -and Cl -dependent plasma membrane monoamine transporters, as well as to some Na~channels (Bergman et al, 1989;Kuhar et al, 1991). Effective antidepressants share the ability to inhibit the serotonin transporter (Blakely et al, 1997). Transporter-mediated accumulation and/or sequestration of neurotoxins plays a key role in several current experimental models of the specific dopaminergic neurodegenerative processes found in Parkinson's disease (Snyder and D'Amato, 1986).…”

“…As the uncoupled current is influenced by substrate binding or transport, it is difficult to estimate the contribution of each of these processes to the steady-state current during transport. Fluctuation analysis with Drosophila serotonin transporter, expressed in Xenopus oocytes, showed that =500 serotonm molecules are transported per channel opening (Galli et al, 1996(Galli et al, , 1997. At the same time, at -2() mV, 10,000 electronic charges were translocated.…”

The Chicken Serotonin Transporter Discriminates between Serotonin-selective Reuptake Inhibitors

“…An internal K ϩ ion is bound and countertransported, after dissociation of 5-HT, Na ϩ , and Cl Ϫ , as SERT returns to its initial state (8). Although the stoichiometry of 5-HT transport predicts an electroneutral process, electrophysiological studies have revealed a net inward current, which probably arises from an uncoupled excess inward flux of Na ϩ ions (9,10).…”