Functional consequences of homo- but not hetero-oligomerization between transporters for the biogenic amine neurotransmitters

Abstract

Before this study, the human norepinephrine transporter (hNET) was the only member of the biogenic amine neurotransmitter transporter family that had not been demonstrated to be a functional homo-oligomer. Here, using two forms of the transporter, I155C and hNET-myc, with distinct antigenicity and inhibitor sensitivity, we demonstrated that hNET exists as a homo-oligomer. hNET I155C is a functional mutant and is sensitive to inactivation by the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate, while hNET-myc is resistant to inactivation by this reagent. Coimmunoprecipitation of these two forms demonstrated that a physical interaction exists between norepinephrine transporter monomers. Further characterization of this physical interaction has revealed that the activity of norepinephrine transporters depends on interactions between monomers. Because norepinephrine transporters and serotonin transporters are the only two members of the neurotransmitter transporter family endogenously expressed in the cell membrane of the same cells, placental syncytiotrophoblasts, we tested the ability of norepinephrine transporters and serotonin transporters to associate and function in a hetero-oligomeric form. Similarly, coexpression of hNET-myc with serotonin transporter-FLAG showed a physical interaction in coimmunoprecipitation assays. However, coexpression of serotonin and norepinephrine transporters did not sensitize norepinephrine transporter activity to inhibition by citalopram, a selective serotonin transport inhibitor. Thus, the norepinephrine transporter-serotonin transporter physical association did not produce functional consequences. Based on this, we propose that the transporters for biogenic amine neurotransmitters interact functionally in homo- but not hetero-oligomeric forms.

Fig. 1

Modified COOH-terminal amino acid sequence of hNETmyc. hNET amino acid residues are underlined and the c-myc tag is in bold.

Fig. 2

Characterization of human norepinephrine transporter (hNET)-myc and I155C. (a) Antibody specificity. Each panel represents a western blot of HeLa cells expressing either hNET, I155C or hNETmyc. The upper blot was visualized with anti-NET antibody and the lower blot with antibody against c-myc. (b) Sensitivity to inactivation. HeLa cells expressing either hNET (hNET wild-type), hNET-myc or I155C were incubated with either 100 μM cocaine or 2.5 mM [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) for 10 min and then assayed for transport activity. All data plotted represent means ± SE, are based on triplicate determinations and are representative of three separate experiments. DA, Dopamine.

Fig. 3

Physical association between human norepinephrine transporter (hNET) monomers, serotonin transporter (SERT) and dopamine transporter (DAT). (a) Coprecipitation of hNET-myc with I155C. HeLa cells transfected with I155C, hNET-myc or both constructs were solubilized in the presence of 10 mM N-ethylmaleimide (NEM), treated with rat anti-mouse protein A sepharose beads and, where indicated, antibody against c-myc. The immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and blotted with anti-NET antibody as described in Experimental procedures. (b) Physical association between three neurotransmitter transporters. HeLa cells transiently cotransfected with equal amounts of hNET-myc/SERT-FLAG (lane 1), myc-SERT/SERT-FLAG (lanes 2 and 5), FLAG-DAT/hNET-myc (lane 3), DAT-myc/SERT-FLAG (lane 4), DAT-myc/FLAG-DAT (lane 6) or only with SERT-FLAG (lane 7) were treated with 10 mM NEM and harvested, solubilized and treated with protein A beads and, where indicated, antibody against c-myc. The immunoprecipitates were separated by SDS-PAGE and were blotted with biotinylated anti-FLAG antibody as described in Experimental procedures.

Fig. 4

Quantitation of expressed human norepinephrine transporter (hNET) constructs. HeLa cells were transfected with mixtures of hNET-myc and I155C cDNA such that the total DNA per well was constant; however, the percent of hNET-myc in the mixture varied from 0 to 100% as indicated. Extracts of the cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were visualized by western blot analysis using (a) anti-myc and (b) anti-NET antibodies. The relative amount of each hNET construct was estimated by comparison with standard curves using cells expressing either hNET-myc or I155C. The values are expressed in terms of the amount of cell protein required in the standard curve to give an equivalent signal.

Fig. 5

Inactivation of human norepinephrine transporter (hNET)-myc and I155C hNET mixtures by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). HeLa cells were transfected with mixtures of hNET-myc and I155C, as described in the legend to Fig. 3, and were assayed for dopamine (DA) transport. The expression of I155C relative to total hNET was determined by quantitative immunoblotting and is indicated on the x-axis. The normalized mean of three separate experiments is shown, with error bars indicating SD. Transport rates before (●) and after (◯) treatment with 2.5 mM MTSET for 10 min. The solid line is the best fit to the control transport rates. The shortdashed line is the activity expected if no interaction occurred between resistant and sensitive forms and the amount of inactivation was equal to the amount of activity contributed by I155C. The long-dashed line represents the calculated activity if hNET was a dimer and modification of both subunits was required for inactivation of activity in that dimer. The dot-dashed line represents the calculated activity if modification of one subunit in a dimer inactivated all the activity of that dimer. All calculations assumed random dimer formation. Mean nonspecific uptake was determined in HeLa cells transfected with the parent vector pBluescript SK II(–) alone and was subtracted from total uptake to yield specific DA uptake.

Fig. 6

The association between serotonin transporter (SERT) and human norepinephrine transporter (hNET) does not lead to functional coupling. HeLa cells were transiently transfected with hNET or a mixture of hNET and SERT cDNA as described in Experimental procedures. Cells were incubated with the indicated concentrations of citalopram for 10 min before the addition of (a) 20.5 nM [³H]serotonin (5-HT) or (b) 28.7 nM dopamine (DA). Influx was determined after an additional 10 min incubation as described in Experimental procedures.