Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase

Prasad et al. 10.1073/pnas.0501432102.

Supporting Information

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Fig. 6. Saturation kinetic analysis of 5-hydroxytryptamine (5-HT) transport by human serotonin transporter (hSERT), Pro339Leu (P339L), and Ile425Val (I425V). Plots are derived from mean activities obtained in three or more separate experiments. Kinetic values obtained were the following: hSERT, V_max, 416.2 ± 19 fmol per well per min: K_m, 1.00 ± 0.17 mM; Pro339Leu, V_max, 1.2 ± 0.11 fmol per well per min: K_m, ND; Ile425Val, V_max, 789.5 ± 28 fmol per well per min; K_m, 0.56 ± 0.13 mM.

Fig. 7. Impact of SERT-coding variants on total and cell surface [¹²⁵I]RTI-55 (3b-(4-iodophenyl)tropan-2b-carboxylic acid methyl ester) binding. HeLa cells transiently transfected with hSERT or one of the SERT-coding variants were subjected to intact cell-binding assays at 4°C with the cocaine analog [¹²⁵I]RTI-55(5 nM) as described in Materials and Methods. (A) Total binding values as defined with paroxetine (10 mM) as displacer. (B) Surface labeling by [¹²⁵I]RTI-55 as defined with 5-HT (100 mM) as displacer. In vehicle-treated cells, hSERT total binding (fmol per 10⁶ cells) was 618.2 ± 27.9, and the surface binding was 173.8 ± 6.6. Results for A and B reflect mean values ± SEM of three separate experiments normalized to hSERT (100%). Binding levels were analyzed by means of a one-way ANOVA followed by post hoc Dunnett’s tests comparing mutant means with hSERT (*, P < 0.05 taken as significant).

Fig. 8. Modulation of hSERT and coding variants by phorbol ester. Transfected cells treated with 10 or 100 m M b-phorbol 12-myristate 13-acetate (b-PMA) for 15 min in the presence or absence of the PKC antagonist bisindolylmaleimide (BIM) were assayed for 5-HT transport as described in Materials and Methods. P339L was not tested due to low basal transport activity. Results represent mean values ± SEM of three separate experiments normalized to each mutant’s vehicle-treated control (100%). Data were analyzed using a two-way ANOVA followed by a post hoc Bonferonni test to evaluate differences in PMA effects comparing hSERT with each coding variant (*, P < 0.05 taken as significant). Stauro, staurosporine; BIM-I, bisindolylmaleimide I.

Table 1. Human SERT nonsynonymous variants

NCBI, National Center for Biotechnology Information; ECL, extracellular loop; TM, transmembrane domain; Dep, depression; OCD, obsessive-compulsive disorder.

1. Di Bella, D., Catalano, M., Balling, U., Smeraldi, E. & Lesch, K. P. (1996) Am. J. Med. Genet. 67, 541-545.

2. Cargill, M., Altshuler, D., Ireland, J., Sklar, P., Ardlie, K., Patil, N., Lane, C. R., Lim, E. P., Kalayanaraman, N., Nemesh, J., et al. (1999) Nat. Genet. 22, 231-238.

3. Glatt, C. E., DeYoung, J. A., Delgado, S., Service, S. K., Giacomini, K. M., Edwards, R. H., Risch, N. & Freimer, N. B. (2001) Nat. Genet. 27, 435-438.

4. Ozaki, N., Goldman, D., Kaye, W. H., Plotnicov, K., Greenberg, B. D., Lappalainen, J., Rudnick, G. & Murphy, D. L. (2003) Mol. Psychiatry 8, 895, 933-936.

Table 2. Sequence conservation at sites of human SERT-coding variants

hSERT, human SERT; rSERT, rat ; mSERT, mouse; gpSERT, guinea pig; bovSERT, bovine; dSERT, Drosophila melanogaster; ceSERT, Caenorhabditis elegans; hNET, human norepinephrine transporter; hDAT, human dopamine transporter; hGAT1, human GABA transporter type 1.

Table 3. Pharmacological sensitivities of human SERT-coding variants

K_i values (nM ± SEM) were determined from three or more separate SERT uptake inhibition assays. *, P < 0.05, one-way ANOVA with a post hoc Dunnett’s test comparing variant with hSERT for the same compound.

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