A topological, 12-transmembrane (TM) domain model, organizing the five hSERT-coding variants identified in autism subjects, is shown in a
. The three novel hSERT variants uncovered in autism probands, Ile425Leu, Phe465Leu and Leu550Val, are located within predicted TM domains, whereas the previously reported Gly56Ala and Lys605Asn substitutions occur in the cytoplasmic amino and carboxy termini of hSERT, respectively. Previously, we demonstrated that the lymphoblasts homozygous for the Ala56 allele demonstrate elevated SERT activity, relative to Gly56 homozygous lines (Prasad et al. 2005
; Sutcliffe et al. 2005
). Here, we extended these studies to the lines derived from the autism subjects that carry single alleles of Ile425Leu, Phe465Leu or Leu550Val. We measured SERT activity in these lines and, in parallel, measured l
-glutamate transport activity as a measure of specificity. As shown in b
, SERT activity was elevated in all three variant lines relative to the activity found in the cells bearing wild-type alleles at each of the mutated sites. All 5-HT transport in the variant-expressing cells' activity was citalopram sensitive, diminishing the possibility of induction of other transport systems capable of transporting 5-HT such as PMAT (Zhou et al. 2007
) or OCT3 (Kekuda et al. 1998
). More importantly, no change was observed in l
-glutamate transport, indicating a measure of specificity for the transport alterations to hSERT, as well as stability of cellular bioenergetics, such as ion gradients or membrane potential that could indirectly impact SERT. Rather, these initial findings suggest an intrinsic, gain-of-function property produced by each of the hSERT variants.
Figure 1 Location and 5-HT transport activity of autism-associated SERT-coding variants. (a) Autism-associated variants are overlaid on a 12-TM model of a single SERT subunit, with NH2 and COOH termini oriented inside the cell. Variants in extramembrane domains (more ...)
In order to establish the impact of Ile425Leu, Phe465Leu and Leu550Val on SERT activity in a model system devoid of other influences arising from the cells of the affected subjects, we transiently expressed the five cDNAs individually into HeLa cells for comparison with wild-type hSERT. Regulation of hSERT activity by intracellular signalling pathways can be lost or altered if the transporter expression level greatly exceeds that found in natively expressing cells. Thus, we titrated hSERT plasmids used for transfection to achieve a level of expression comparable to that found in cultured lymphoblast lines and mast cells. With saturation kinetic studies (), we observe significant reductions in 5-HT KM for all variants; Ile425Leu, Phe465Leu and Leu550Val also exhibit significantly elevated transport capacity (Vmax). The 5-HT transport activity of all variants retains sensitivity to the highly specific hSERT antagonist paroxetine, and thus we attribute the gain-of-function properties to the introduced constructs, rather than ectopic stimulation of non-specific uptake systems.
Kinetic impact of autism-associated SERT-coding variants. (Values are expressed as the mean of at least three experiments±s.e.m. *p<0.05, **p<0.01 versus hSERT control, Student's t-test.)
Analysis of whole-cell radioligand-binding experiments with hSERT-transfected cells reveals a striking difference for the three novel variants in relative surface expression (a,b). Thus, whereas Ile425Leu, Phe465Leu and Leu550Val display equivalent levels of total SERT protein, as inferred from paroxetine-sensitive [125I]RTI-55 binding to transfected cells (a), these variants exhibit significantly enhanced binding of the radioligand under conditions (5-HT displacement, 4°C) adopted to reveal the surface pool of transporters (b). These data suggest that a significant component of the elevation of SERT activity seen in transport studies for these variants arises from altered surface trafficking.
Figure 2 Analysis of protein expression of autism-associated SERT-coding variants. Impact of SERT-coding variants on total and cell surface [125I]RTI-55 binding. HeLa cells transiently transfected with hSERT or one of the SERT-coding variants were subjected to (more ...)
To gain further support for surface trafficking alterations, we biotinylated transfected cells using the membrane-impermeant reagent NHS-SS biotin and compared the level of SERT proteins in total and biotinylated (surface) fractions by immunoblotting with a human SERT-specific antibody. Our studies demonstrate that as with total SERT binding, total protein levels for all hSERT variants were equivalent to wild-type hSERT (c,e), whereas surface proteins were significantly elevated for Ile425Leu, Phe465Leu and Leu550Val (d,f). In these experiments, we actually observed diminished Gly56Ala SERT protein in surface membranes when compared with wild-type SERT. These findings reinforce our contention that the gain-of-function transport property of the Gly56Ala variant arises from catalytic activation and that gain of catalytic activity can, unexpectedly, accompany a reduction in surface expression. Additionally, they ascribe to Ile452Leu, Phe465Leu and Leu550Val a mechanistic perturbation distinct from that impacting Gly56Ala, which leads to elevated transporter surface expression.
Previously, we demonstrated that the hSERT variants Gly56Ala and Lys605Asn not only display a gain of transport activity but also exhibit a striking loss of regulation through PKG and p38 MAPK pathways (Prasad et al. 2005
). To determine whether this is a common property of other autism-associated hSERT variants, we returned to the HeLa model to explore sensitivity of variants to 8Br-cGMP or anisomycin, which activate PKG or p38 MAPK, respectively. As described previously, wild-type SERT displays an elevation of activity in response to these agents that can be attenuated by PKG or p38 MAPK inhibitors, whereas hSERT Gly56Ala is insensitive to these agents (a
). In contrast to Gly56Ala, the cells expressing Ile425Leu, Phe465Leu and Leu550Val exhibit significant PKG- and p38 MAPK-stimulated SERT activity, though percentage stimulation was not as great as that seen with wild-type SERT, possibly a consequence of their elevated basal function. Regardless, these findings are consistent with a distinct mechanism supporting the functional perturbations leading to the gain-of-function alterations exhibited by Ile425Leu, Phe465Leu and Leu550Val.
Figure 3 Impact of 8Br-cGMP and p38 MAPK on SERT activity of autism-associated hSERT-coding variants. (a) Activity modulation. HeLa cells transfected with hSERT or autism-associated hSERT-coding variants were examined for 5-HT transport activities as described (more ...)
Activation of protein kinase C (PKC) with phorbol esters such as β-PMA leads to a rapid reduction in 5-HT transport capacity and surface expression in hSERT-transfected cells (Qian et al. 1997
), a sensitivity significantly enhanced with the Gly56Ala and Lys605Asn mutants (Prasad et al. 2005
). To examine whether the novel autism coding variants display similar or distinct patterns of regulation to PKC activators as seen with Gly56Ala, wild-type- and mutant-transfected cells were treated with β-PMA (0.1–1
min). As shown in a
, wild-type (WT) hSERT displays under these conditions a dose-dependent sensitivity to β-PMA that can be blocked by either staurosporine or bisindolylmaleimide (BIM). Under these conditions, 1
μM β-PMA induces an approximately 30 per cent loss of SERT activity. As described previously (Prasad et al. 2005
), the Gly56Ala variant exhibits increased sensitivity to β-PMA, with an approximately 50 per cent loss of function evident under the same conditions. By contrast, the Ile425Leu, Phe465Leu and Leu550Val variants displayed comparable or even slightly reduced sensitivity to β-PMA when compared with wild-type hSERT.
Figure 4 Impact of PKC and PP2A on SERT activity of autism-associated hSERT-coding variants. (a) Activity modulation. HeLa cells transfected with hSERT or autism-associated hSERT-coding variants were examined for 5-HT transport activities as described in §2 (more ...)
The catalytic subunit of the serine/threonine phosphatase PP2A (PP2Ac) has been found to physically associate with SERTs in brain and transfected cells (Bauman et al. 2000
). PP2A inhibitors that bind to and inhibit PP2Ac, including okadaic acid, calyculin A and fostriecin, also trigger hSERT phosphorylation, reduced surface expression and loss of SERT activity (Blakely & Bauman 2000
), presumably due to loss of SERT-associated phosphatase activity that can attenuate SERT phosphorylation and internalization triggered by kinases such as PKC. Stimulatory phosphorylation, such as that triggered by PKG and p38 MAPK, may also depend upon SERT-associated PP2A activity to suppress phosphorylation at inhibitory sites (Samuvel et al. 2005
; Ramamoorthy et al. 2007
; Zhang et al. 2007
). To determine whether PP2A dysregulation might contribute commonly or uniquely to one or more of the autism-associated SERT variants, we treated transfected cells with increasing concentrations of the two most selective PP2Ac inhibitors available: calyculin A and fostriecin. As shown in b
, both drugs demonstrate dose-dependent inhibition of 5-HT transport activity for wild-type hSERT. This effect is significantly enhanced over the more modest sensitivity displayed by either Gly56Ala or Ile425Val.
hSERTs that are transiently transfected into HeLa cells express transporters in a reduced, heterologous system, where the changes in function, pharmacology and regulation can be rapidly assessed. Although this is a powerful paradigm, it is not without its limitations, chiefly the lack of sustained expression that can allow for the impact of chronically expressed hSERT variants to be evaluated. Typical stable cell models have the disadvantage of integration of transfected DNAs at different genomic loci for each cell line produced, introducing clone-to-clone variability in the levels of expression dictated by cis-acting transcriptional mechanisms unique to each site of integration. We therefore created stable cell lines that express the wild-type, Gly56Ala and Ile425Leu hSERT alleles at a common genomic locus (CHO-Flp-In cells). As observed with transient expression studies, stably expressing Gly56Ala cells demonstrate elevated function versus the 5-HT transport achieved by wild-type hSERT (a). Surprisingly, we found Ile425Leu cells to show a small reduction in 5-HT transport function. Exploration of the basis for these changes via western blots of total and surface proteins yielded even more remarkable results. In total extracts, we found that both Gly56Ala and Ile425Leu expression to be significantly reduced relative to wild-type hSERT (b). Despite elevated transport activity, Gly56Ala total protein levels were actually only approximately 50 per cent of wild-type, whereas Ileu425Leu expression was only approximately 25 per cent of wild-type. Surface expression of hSERT proteins was similarly reduced for both variants relative to wild-type SERT (c), and the losses here were even more profound. Thus, Gly56Ala and Ile425Leu reached only approximately 45 per cent and approximately 15 per cent of wild-type SERT surface expression, respectively. When we normalized 5-HT transport for the levels of surface expression in the same cells, we demonstrated as expected that Gly56Ala proteins exhibit enhanced catalytic function, with a turnover rate of approximately 250 per cent that of wild-type SERT (g). Ile425Leu was also strikingly elevated in its catalytic rate by this measure, achieving turnover rates equal to or slightly above those of Gly56Ala.
Figure 5 Altered activity of SERT variants is differently exhibited in CHO-Flp-In stable cells. (a) 5-HT transport activity of autism-associated SERT-coding variants in CHO-Flp-In stable cells. All variants were assayed for 5-HT transport activity as described (more ...)