A Decade of Serotonin ResearchRegulated phosphorylation and trafficking of antidepressant-sensitive serotonin transporter proteins
Introduction
Serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) are presynaptic proteins specialized for the clearance of 5-HT following vesicular release at central nervous system (CNS) and enteric nervous system synapses Rudnick and Clark 1993, Barker and Blakely 1995. SERTs are also expressed on a number of specialized, nonneuronal cells, including platelets Rudnick 1977, Hranilovic et al 1996, lymphoblasts Faraj et al 1997, Khan et al 1996, and placental syncitiotrophoblasts (Balkovetz et al 1989). SERTs are high-affinity targets in vivo for tricyclic antidepressants such as imipramine, the serotonin-selective reuptake inhibitors (SSRIs), and nonselective stimulants including cocaine and amphetamines Fuller and Wong 1990, Barker and Blakely 1995. These substances are believed to exert at least a portion of their physiologic actions by elevating extracellular 5-HT concentrations and extending the duration, magnitude, and spatial spread of 5-HT signals. Prolonged treatments of rats with antidepressants can down-regulate 5-HT clearance capacity (Piñeyro et al 1994) and modulate 5-HT release and 5-HT receptor sensitivity de Montigny et al 1990, Wade et al 1996, suggesting an interdependence among molecular events responsible for 5-HT signaling.
Recent studies with targeted gene disruptions of SERTs (personal communication, D. Murphy) as well as with homologous dopamine (DA) transporters (DATs) (Giros et al 1996), are consistent with the idea that transporter expression represents one element of a coordinated, homeostatic process controlling firing rates, transmitter biosynthesis, release, and response. Total loss of transporter gene expression achieved with homozygous transporter knockouts induces a significant loss of the respective amine and compensatory changes in receptor messenger RNAs (mRNAs) and, for DAT knockouts, a dramatic prolongation in DA transmitter clearance times. Interestingly, though transporter gene and protein expression drop by 50% in heterozygotes, one sees a marginal impact on transport capacity, suggesting the presence of powerful posttranscriptional modulatory events regulating transporter expression.
Currently, the degree to which endogenous regulation of transporter expression or activity contributes to in vivo modulation of amine signaling is unknown. SERTs are the products of a single gene, localized in the human to chromosome 17q11.2 Ramamoorthy et al 1993, Gelernter et al 1995. Alternative splicing of the human SERT gene (hSERT, HTT) generates multiple transcripts with different 5′ noncoding sequences (Bradley and Blakely 1997). Alternative polyadenylation of the 3′ end of the hSERT mRNA also contributes to mRNA heterogeneity (Heils et al 1995), though these noncoding variants are of unknown functional significance. In vitro, SERT gene expression is regulated by both cyclic adenosine monophosphate (cAMP)-dependent and cAMP-independent pathways, and in vivo can be influenced by antidepressants and steroid hormones (reviewed in Blakely et al 1997). Recently, polymorphisms have been described in the hSERT promoter that appear to impact SERT gene and protein expression (Lesch et al 1996). In some populations, this polymorphism has been reported to confer a genetic basis for susceptibility to anxiety (Lesch et al 1996) and mental illness including depression Collier et al 1996, Flattem et al 1997 and autism (Cook et al 1997).
Following transcription, SERT mRNAs must be translated and SERT proteins must be folded and inserted into the plasma membrane, providing additional steps for potential regulation and/or alterations in disease states. SERT proteins in mammals are 630 amino acid polypeptides that appear to span the lipid bilayer 12 times with cytoplasmic NH2 and COOH termini Blakely et al 1991, Hoffman et al 1991, Ramamoorthy et al 1993. SERTs, like other members of the GAT1/NET gene family of Na+- and Cl−-dependent transporters, are N-glycosylated Qian et al 1995, Melikian et al 1996 and then transported to sites of expression in the plasma membrane. A major question that will be discussed below is how SERTs come to be localized in plasma membrane domains to affect optimal 5-HT clearance. Presumably, SERTs should be targeted in neurons near vesicular release sites, though one cannot rule out a priori that transporters might also be localized to plasma membranes of cell bodies or dendrites. Here SERTs could participate in nonvesicular, carrier-mediated efflux of 5-HT (Levi and Raiteri 1993), though the evidence for this rests largely on paradigms using nonphysiologic stimuli (e.g., amphetamines). We and colleagues have found that SERTs are localized to plasma membrane subdomains of both neuroendocrine cells (Schroeter et al 1997) and polarized nonneuronal cells (MDCK, LLC-PK1) after transfection (Gu et al 1996). These findings suggest that elements of SERT protein sequence or structure confer specificity in targeting and/or retention at specialized plasma membrane zones perhaps through direct interactions with specialized plasma membrane and cytoskeletal proteins that segregate membrane compartments.
Once at the cell surface, activity of SERTs appears to be modulatable. Multiple reports have demonstrated a capacity of native and heterologously expressed SERTs to be regulated rapidly after acute elevation/depletion of intracellular Ca2+, treatment with calmodulin inhibitors, or via activation of protein kinase C (PKC) and (nitric oxide synthetic) NOS/cyclic guanosine monophosphate pathways (reviewed in Blakely et al 1997). The changes in 5-HT uptake capacity reported are too rapid to reflect genetic contributions and can be shown to be insensitive to inhibitors of transcription and translation. Furthermore, the significant distances between 5-HT release sites and the cell body in neurons imposes an expectation that rapid regulatory mechanisms would, of necessity, be posttranslational and imposed on carriers already expressed in the plasma membrane or in recruitable membrane vesicles beneath the plasma membrane. Since the second messenger pathways noted above can be activated by presynaptic receptors, it is reasonable to speculate that these pathways may subserve presynaptic modulation of 5-HT clearance in vivo. Recently, Daws and coworkers have applied amperometric techniques to gather support for this idea, demonstrating that 5-HT clearance can be regulated by 5-HT1b receptor activation in vivo (Daws et al 1997).
The major kinetic alterations typically observed in acute modulation paradigms are changes in transport Vmax(Blakely et al 1997), suggesting silencing/activation of resident plasma membrane SERTs or endocytosis/recruitment of SERT proteins to regulate transport capacity rather than the ability to recognize 5-HT. 5-HT transport by mammalian SERTs is believed to be electroneutral such that membrane potential provides no net driving force for 5-HT transport (Rudnick and Clark 1993), and thus changes in membrane potential are not anticipated to regulate 5HT transport per se; however, such arguments neglect the presence of charged residues in predicted transmembrane domains Blakely et al 1991, DeFelice and Blakely 1996 and voltage-induced conformational effects that might enhance or suppress kinase-mediated regulation. Recently, SERTs have been shown to exhibit 5-HT-gated ion channel activity that is blocked by antidepressants Mager et al 1994, Corey et al 1994b, Galli et al 1997 and regulated by PKC (Qian et al 1997). SERT currents increase in magnitude at hyperpolarized potentials and, like 5-HT uptake, are dependent on extracellular Na+ and Cl−. Although the physiologic role of this channel activity remains unclear, we have found that 5-HT-activated currents provide a powerful and time-resolved assessment of 5-HT transport activity and can be used to monitor SERT regulation in single cells, studies we will describe below.
Given rapid changes in SERT activity or expression levels seen with stimuli leading to activated second-messenger dependent protein kinases, it is reasonable to speculate that phosphorylation might control SERT activity or surface density in a manner analogous to regulation of G-protein coupled receptors (GPCRs) or ion channels Levitan 1994, Ferguson et al 1996. Canonical sites for protein phosphorylation on SERT cytoplasmic domains have been described and, as we will detail, SERTs are now known to be phosphorylated in transfected cells via stimuli that trigger activation of PKC, PKA, and PKG (Ramamoorthy et al 1998). Increased SERT phosphorylation is also evident when cellular phosphatases [particularly phosphatase 2A (PP2A)] are inhibited, suggesting active surveillance of the SERT phosphorylation state as part of a regulatory kinase–phosphatase cycle. Over the same time course as phosphorylation proceeds, PKC activation leads to SERT proteins being removed from the plasma membrane (Qian et al 1997) with a consequent reduction in 5-HT uptake capacity (Vmax). In preliminary studies (Ramamoorthy et al 1997), we have observed that 5-HT can modulate SERT phosphorylation state and perhaps kinase-mediated trafficking of transporter protein. Following a review of studies that lead to these conclusions, we will present a hypothetical model that organizes findings of specialized sites of expression with data suggesting transporter surface number is actively regulated. Since changes in expression locale or density would impact 5-HT signaling in much the same way that SSRIs reduce clearance capacity, we speculate that interference with normal regulatory mechanisms might represent a strategy for the development of novel antidepressants. Furthermore, elucidation of SERT regulatory pathways may identify genes encoding SERT accessory proteins, which if defective, could predispose to unregulated SERT in disease states.
Section snippets
SERTs are organized in plasma membrane subdomains: studies on chromaffin cells
Neurons are highly polarized cells with highly complex dendritic branching patterns and secretory specializations often located centimeters to meters away from cell bodies. The molecular basis for this morphological polarity is a fascinating and still largely unwritten chapter in neurobiology. Over the past decade, it has become clear that ion channels and receptors that support the electrical and chemical signaling of neurons and the responses of their targets are organized into stable
SERT plasma membrane expression levels are regulated by cellular kinases and phosphatases
Although it is arguably more physiologically relevant to explore SERT regulation in a cellular context supporting 5-HT release, such as intact 5-HT neurons, cytoplasmic levels of 5-HT and release of unlabeled amine into the extracellular medium can compromise measures of transport rates and transport regulation. Furthermore, expression levels in vivo are fairly low, reducing the opportunity to assess trafficking of SERT proteins or measure SERT behavior in single cells. Motivated by such
SERTs are regulated phosphoproteins
As noted above, SERTs bear canonical sites for Ser/Thr phosphorylation, and activation of PKCs leads to a loss of SERT proteins from the cell surface, at least in transfected HEK-293 cells. This could occur because PKC enhances general endocytosis rates (Backer and King 1991), with the consequence that all membrane proteins exhibit increased steady-state internalization; however, in general, transporters display varying responses to such manipulations, and in 293-hSERT and RBL-293 cells, SERTs
An initial model for localized and regulated SERT plasma membrane expression
We now wish to propose a hypothetical model based on the available data from our lab and colleagues on SERT regulation as well as work on homologous transporters and other regulated ion channels and receptors (Figure 2). We offer this model to stimulate thought as to areas needed for future investigation, suspecting that several elements may be wrong or, at present, only superficially understood. We propose that SERTs (here labeled T to provide general reference to other transporters in the
Acknowledgements
We wish to thank the National Institutes of Health (NIDA, NINDS) and the National Alliance for Research on Schizophrenia and Depression (NARSAD) for their support to R.D.B. and L.J.D. in completion of the studies cited and generation of this review.
This work was presented at the Neuroscience Discussion Forum “A Decade of Serotonin Research” held at Amelia Island, Florida in November 1997. The conference was sponsored by the Society of Biological Psychiatry through an unrestricted educational
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