A salt bridge linking the first intracellular loop with the C terminus facilitates the folding of the serotonin transporter.
Bottom Line: The folding trajectory of solute carrier 6 (SLC6) family members is of interest because point mutations result in misfolding and thus cause clinically relevant phenotypes in people.The bulk of the resulting mutants SERT-F604Q, SERT-I608Q, and SERT-I612Q were retained in the endoplasmic reticulum, but their residual delivery to the cell surface still depended on SEC24C.Finally, mutation of Glu(615) at the end of the C-terminal α-helix to Lys reduced surface expression of SERT-E615K.
Affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.Show MeSH
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Mentions: The most important insight of the simulations was the salt bridge between the end of the C-terminal α-helix and IL-1 of SERT. We verified its relevance by introducing point mutations to create SERT-E615K, SERT-K153E, and SERT-R152E. The rationale for this approach was to assume that the single mutations ought to reduce surface expression by disturbing the ionic interaction between the C terminus and IL-1. This was the case: uptake of substrate by SERT-E615K for instance was reduced by 50% (Fig. 8, A and B, open circles). Similarly, the maximum velocity of substrate translocation by SERT-K153E was also decreased (Fig. 8A, closed triangles). Surprisingly, the phenotypic consequence of the SERT-R152E mutation was modest (Fig. 8B, closed triangles). However, if the mutations were combined, i.e. the charges in the first intracellular loop and at the end of the C-terminal α-helix were reversed, the results were unequivocal: the K153E mutation failed to rescue the E615K mutation; in fact, SERT-K153E/E615K (Fig. 8A, open squares) was less active than either single point mutant. In contrast, the R152E mutation counteracted the effect of mutating Glu615 to Lys such that the transport rate of SERT-R152E/E615K (Fig. 8B, open squares, and Table 1) approached that of wild type SERT (Fig. 8B, closed squares, and Table 1). This was also recapitulated if the ratio of mature and core glycosylated SERT was visualized by immunoblotting: the mature glycosylated band was less abundant in SERT-E615K (Fig. 3, inset, lane labeled E-K); however, it was restored to wild type levels in the double mutant SERT-R152E/E615K (Fig. 8B, inset, lane labeled DM). Finally, we also verified the cellular distribution of CFP-tagged SERT-E615K and SERT-R152E/E615K by confocal microscopy. It is evident that a substantial amount of SERT-E615K was retained within the cells (Fig. 8E) because a large portion of the YFP fluorescence did not co-localize with the fluorescently labeled plasma membrane (Fig. 8E, left-hand image). In contrast, in cells expressing SERT-R152E/E615K, the bulk of the YFP fluorescence co-localized with the fluorescence of myristoylated and palmitoylated CFP (Fig. 8F, left-hand image). Consistent with the uptake data, the ratio of mature to core glycosylated band of the single mutant SERT-R152E (Fig. 8B, inset, lane labeled R-E) was comparable with that of wild type SERT (Fig. 8B, inset, lane labeled WT); the cellular distribution also showed that SERT-R152E reached the cell surface (Fig. 8D) to the same extent as wild type SERT (Fig. 8C).
Affiliation: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.