Unifying Concept of Serotonin Transporter-associated Currents

Schicker, Klaus; Uzelac, Zeljko; Gesmonde, Joan; Bulling, Simon; Stockner, Thomas; Freissmuth, Michael; Boehm, Stefan; Rudnick, Gary; Sitte, Harald H.; Sandtner, Walter

doi:10.1074/jbc.m111.304261

Cited by 103 publications

(206 citation statements)

References 46 publications

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“…The direct interaction of PIP 2 with ion channels and exchange proteins is required to permit ion fluxes in those proteins (8,16). To test for analogous phenomena in SERT, currents through this transporter were triggered by the application of a substrate such as para-chloroamphetamine (pCA) (17). To disrupt PIP 2 interactions, a palmitoylated peptide was used (Pal-HRQKHFEKRR; 10 μM), which is known to inhibit the effects of PIP 2 on ion channels by binding to its polar head groups (18).…”

Section: Resultsmentioning

confidence: 99%

Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate

Buchmayer

Schicker

Steinkellner

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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116

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Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP 2 ) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP 2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP 2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP 2 -synthesis. We tested the effects of the verified scarcity of PIP 2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP 2 in pull-down assays. On decreased PIP 2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamineinduced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP 2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP 2 . Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP 2 -dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP 2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.phosphoinositide | reuptake | release | mass spectrometry | amperometry

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Section: Resultsmentioning

confidence: 99%

Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate

Buchmayer

Schicker

Steinkellner

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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116

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“…The presence of extracellular Na + stabilizes outward-facing states of SERT (24), and the subsequent addition of 5-HT leads to a synchronous conformational change associated with a transient current (28). By analogy with prokaryotic SERT homologs, this substrate-dependent change represents opening of a cytoplasmic substrate permeation pathway (21,29) that also has been observed with SERT (19) and leads to a release of substrate to the cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

“…By analogy with prokaryotic SERT homologs, this substrate-dependent change represents opening of a cytoplasmic substrate permeation pathway (21,29) that also has been observed with SERT (19) and leads to a release of substrate to the cytoplasm. Subsequently, SERT returns to an outward-facing conformation in a K + -stimulated step that is rate-limiting for transport (28,30). Because the transition from inward-facing to outward-facing conformations is the slow step in the transport cycle, SERT will accumulate in an inward-facing conformation when transporting 5-HT.…”

Section: Discussionmentioning

confidence: 99%

“…3). 5-HT, the substrate for SERT, stimulated conversion PKG activity against the peptide substrate (RKRSRAE) was determined in the presence of the indicated concentrations of various agents as described previously (15) of the transporter to inward-open conformations (19,27,28) and also stimulated phosphorylation, although not as strongly as ibogaine (Fig. 3).…”

Section: -Brmentioning

confidence: 99%

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Control of serotonin transporter phosphorylation by conformational state

Zhang

Turk

Rudnick

2016

Proc. Natl. Acad. Sci. U.S.A.

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Serotonin transporter (SERT) is responsible for reuptake and recycling of 5-hydroxytryptamine (5-HT; serotonin) after its exocytotic release during neurotransmission. Mutations in human SERT are associated with psychiatric disorders and autism. Some of these mutations affect the regulation of SERT activity by cGMPdependent phosphorylation. Here we provide direct evidence that this phosphorylation occurs at Thr276, predicted to lie near the cytoplasmic end of transmembrane helix 5 (TM5). Using membranes from HeLa cells expressing SERT and intact rat basophilic leukemia cells, we show that agents such as Na + and cocaine that stabilize outward-open conformations of SERT decreased phosphorylation and agents that stabilize inward-open conformations (e.g., 5-HT, ibogaine) increased phosphorylation. The opposing effects of the inhibitors cocaine and ibogaine were each reversed by an excess of the other inhibitor. Inhibition of phosphorylation by Na + and stimulation by ibogaine occurred at concentrations that induced outward opening and inward opening, respectively, as measured by the accessibility of cysteine residues in the extracellular and cytoplasmic permeation pathways, respectively. The results are consistent with a mechanism of SERT regulation that is activated by the transport of 5-HT, which increases the level of inward-open SERT and may lead to unwinding of the TM5 helix to allow phosphorylation.(1) reported a rare missense mutation in the coding region of serotonin transporter (SERT) in two unrelated families. Within these families, individuals with the mutation (Ile425Val, or I425V) were more strongly affected with several psychiatric disorders, including obsessive-compulsive disorder and autism spectrum disorders. Subsequently, additional families with this mutation and others in SERT have been identified (2-8).In transfected cells, the I425V mutation led to enhanced 5-HT transport relative to WT SERT (5, 9). The enhanced transport was similar to the up-regulation of 5-HT transport that had been observed in rat basophilic leukemia (RBL-2H3) cells on stimulation of adenosine A3 receptors with subsequent NO formation and cGMP production (10). Results with heterologously expressed SERT suggest that the I425V mutation interferes with this pathway either by directly increasing basal SERT phosphorylation or by inhibiting SERT dephosphorylation (11).Mutation of serine and threonine residues on the cytoplasmic surface of SERT led to the identification of Thr276 as a potential cGMP-dependent phosphorylation site (12). From homology models based on prokaryotic and eukaryotic homologs, this residue is located near the cytoplasmic end of transmembrane helix 5 (TM5), a location that agrees with cysteine scanning accessibility studies (13) that also have shown this position to be poorly accessible to aqueous reagents. The unlikely location of a phosphorylation site in a transmembrane helix, and the inaccessibility of this position, cast some doubt on the proposal that Thr276 is the site of cGMP-stimulated phosphorylat...

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“…Accordingly, they accumulate within the synaptic vesicles, where they dissipate the proton gradient; therefore, 5-HT increases within the cytosol. In addition, amphetamines effectively trigger a sustained Na ϩ inward current (Schicker et al, 2012). The cytosolic accumulation of both Na ϩ and substrate allows for reverse transport.…”

Section: Introductionmentioning

confidence: 99%

Amphetamine Action at the Cocaine- and Antidepressant-Sensitive Serotonin Transporter Is Modulated by αCaMKII

et al. 2015

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Serotonergic neurotransmission is terminated by reuptake of extracellular serotonin (5-HT) by the high-affinity serotonin transporter (SERT).Selective 5-HT reuptake inhibitors (SSRIs) such as fluoxetine or escitalopram inhibit SERT and are currently the principal treatment for depression and anxiety disorders. In addition, SERT is a major molecular target for psychostimulants such as cocaine and amphetamines. Amphetamine-induced transport reversal at the closely related dopamine transporter (DAT) has been shown previously to be contingent upon modulation by calmodulin kinase II␣ (␣CaMKII). Here, we show that not only DAT, but also SERT, is regulated by ␣CaMKII. Inhibition of ␣CaMKII activity markedly decreased amphetamine-triggered SERT-mediated substrate efflux in both cells coexpressing SERT and ␣CaMKII and brain tissue preparations. The interaction between SERT and ␣CaMKII was verified using biochemical assays and FRET analysis and colocalization of the two molecules was confirmed in primary serotonergic neurons in culture. Moreover, we found that genetic deletion of ␣CaMKII impaired the locomotor response of mice to 3,4-methylenedioxymethamphetamine (also known as "ecstasy") and blunted D-fenfluramine-induced prolactin release, substantiating the importance of ␣CaMKII modulation for amphetamine action at SERT in vivo as well. SERT-mediated substrate uptake was neither affected by inhibition of nor genetic deficiency in ␣CaMKII. This finding supports the concept that uptake and efflux at monoamine transporters are asymmetric processes that can be targeted separately. Ultimately, this may provide a molecular mechanism for putative drug developments to treat amphetamine addiction.

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Unifying Concept of Serotonin Transporter-associated Currents

Cited by 103 publications

References 46 publications

Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate

Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate

Control of serotonin transporter phosphorylation by conformational state

Amphetamine Action at the Cocaine- and Antidepressant-Sensitive Serotonin Transporter Is Modulated by αCaMKII

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