Deorphaning a solute carrier 22 family member, SLC22A15, through functional genomic studies

Abstract

The human solute carrier 22A (SLC22A) family consists of 23 members, representing one of the largest families in the human SLC superfamily. Despite their pharmacological and physiological importance in the absorption and disposition of a range of solutes, eight SLC22A family members remain classified as orphans. In this study, we used a multifaceted approach to identify ligands of orphan SLC22A15. Ligands of SLC22A15 were proposed based on phylogenetic analysis and comparative modeling. The putative ligands were then confirmed by metabolomic screening and uptake assays in SLC22A15 transfected HEK293 cells. Metabolomic studies and transporter assays revealed that SLC22A15 prefers zwitterionic compounds over cations and anions. We identified eight zwitterions, including ergothioneine, carnitine, carnosine, gabapentin, as well as four cations, including MPP⁺ , thiamine, and cimetidine, as substrates of SLC22A15. Carnosine was a specific substrate of SLC22A15 among the transporters in the SLC22A family. SLC22A15 transport of several substrates was sodium-dependent and exhibited a higher Km for ergothioneine, carnitine, and carnosine compared to previously identified transporters for these ligands. This is the first study to characterize the function of SLC22A15. Our studies demonstrate that SLC22A15 may play an important role in determining the systemic and tissue levels of ergothioneine, carnosine, and other zwitterions.

Keywords: carnitine; carnosine; ergothioneine; metabolomic; transporter.

Fig. 1.

Phylogeny tree analysis, structural analysis and plasma expression of SLC22A15. (A) Circular tree of the human SLC22A and SLC22B transporter family using 28 protein sequences. Six new members of the SLC22 family are included here. Blue font represents cation transporters. Red font represents anion transporters, and green font represents zwitterion transporters. Pink font represents sugar transporters. SLC22 family members with unknown substrates are in black font, including SLC22A15, which is the focus of this study. ^†Six new members in the SLC22 family. (B). Surface representations of the calculated electrostatic potential (−5 to 5 kT e-1) of the human SLC22A1, SLC22A6 and SLC22A4, which are characterized cation, anion and zwitterion transporters, respectively, and the human SLC22A15. The cross-section allows for visual inspection of the predicted binding pockets with blue color estimated as having positive charges and red being estimated as having negative charges. (C). Western blot of cellular components expressing SLC22A15 with C-terminal Myc-DDK Tag. SLC22A15 is expressed on the plasma membrane and the size is slightly above 52 kDa. When the plasma membrane fraction is deglycosylated (degly), the size is reduced to ~52 kDa.

Fig. 2.

Metabolomic study showing the three major groups of metabolites that are significantly different between SLC22A15 and EV cells. (A) zwitterions, (B) acylcarnitines, (C) monoacylglycerols. These metabolites reached the significant threshold of p-adjusted value < 0.01 (Supplemental Table 1). (D) Chemical structures of the zwitterions, four acylcarnitine and four monoacylglycerols.

Fig. 3.

Uptake of zwitterions and cations in HEK293 Flp-In cells expressing SLC22A15 or other zwitterion transporters. (A) Uptake of six zwitterions and two cations that are canonical substrates of SLC22 family members or significant in the in vitro metabolomic study (see Fig. 2). Uptake of these compounds was significantly different between EV and SLC22A15 transiently transfected cells. †Compounds that were found to be significantly different between EV and SLC22A15 transfected cells in the metabolomic study (Fig. 2). The bars represent the mean uptake values from one experiment (± S.D. fold over EV) from three replicate wells. (B) Uptake of zwitterions or cations in cells expressing EV (white bars), SLC22A4 (grey bars), SLC22A5 (dark grey bars) and SLC22A15 (black bars). Uptake, expressed as fold uptake over uptake in EV, was performed in transiently transfected cells (HEK293 Flp-In). The bars represent the mean uptake values from one experiment (± S.D. fold over EV) from three to four replicate wells. All uptake values are significantly higher than EV cells, except for those label n.s. (not significant). Significance were determined by one-way analysis of variance followed by Dunnett’s multiple comparisons test (by comparisons to EV as control). (C) Kinetics of uptake of four zwitterions for SLC22A15. A representative curve for each substrate is shown in this figure. Kinetic parameters of uptake of ergothioneine, carnosine, carnitine and creatine for SLC22A15 are listed in Supplemental Table 4. The mean and S.D. of the kinetic parameters from two to three experiments are shown in Supplemental Table 4. (D) Sodium dependence studies of the uptake of four zwitterions in SLC22A15 overexpressing cells. Cells were pre-incubated with sodium buffer, lithium buffer or NMDG-chloride buffer and uptake was performed using the respective buffer. The figure shows a representative plot from one experiment (mean ± S.D. fold over EV) in triplicate wells. (E) Uptake of zwitterions and cations in HEK293 Flp-In cells stably expressing SLC22A15-GFP in the presence (grey bars) and absence of quinidine (black bars represent uptake in SLC22A15 expressing cells and white bars represent uptake in EV cells). Phenylalanine (1 mM) was used to inhibit endogenous amino acid transporter, SLC7A5 (LAT1). The data represent the uptake of metabolites or prescription drugs that are zwitterions or cations in SLC22A15 transfected cells compared to EV (mean ± SD). (F) [¹⁴C]-Carnitine efflux from SLC22A15 stably expressing cells and empty vector (EV) cells. SLC22A15 cells and EV cells were preloaded with trace amount of [¹⁴C]-carnitine and 20 μM carnitine for 30 min. Subsequently, cells were washed twice with cold HBSS. The efflux of [¹⁴C]-carnitine was measured following addition of HBSS buffer to the SLC22A15 and EV cells. Quinidine (0.5 mM) was included in the HBSS buffer (black squares) to inhibit SLC22A15.

Fig. 4.

Inhibition of SLC22A15-mediated uptake of [³H]-ergothioneine by different classes of compounds in HEK293 Flp-In cells stably expressing SLC22A15. Compounds used in these inhibition studies include (A) metabolites identified in the metabolomic study (Fig. 2) and other compounds in their metabolic pathways; (B) substrates and/or inhibitors of OCTs, OCTNs and OATs; (C) other zwitterion and cation drugs. Figure shows a representative plot from one experiment (mean ± S.D. from three replicate wells). All compounds were screened at 500 μM (black bar), except a few compounds at 100 μM (blue bar); (D) SLC22A15-mediated uptake of substrates in the presence of varying concentrations of HEPES, a commonly used buffering agent in uptake media. Uptake in control (EV) cells is also included in the graph; (i) SLC22A15-mediated uptake of four substrates in the presence and absence of HEPES. Two experiments were performed in transiently transfected cells. Data shown are representative of one experiment from triplicate wells (mean ± SD); (E) Percent uptake of [³H]-ergothioneine at various concentration of HEPES. IC₅₀ of HEPES is 4.8 mM. ^†Compounds that were found to be significantly different between EV and SLC22A15 transfected cells in the metabolomic study.

Fig. 5.

A volcano plot showing the 7,696 genes which are differentially expressed (measured with RNAseq) between EV cells and SLC22A15 cells at p-value adjusted < 0.05. The fold-changes (log₂) of transcript expression were calculated between HEK293 Flp-In EV/SLC22A15-GFP cells, and the –log₁₀(p-value adjusted) were plotted against the log₂(fold change) for the 7,696 transcripts. Each dot represents mean values (n = 4). The red dots represent p-adjusted (FDR) < 0.05, while the green dots represent p-adjusted (FDR) < 0.05 and log₂fold > 2- or < −2-fold changes in expression. Twelve genes that are labeled have p-adjusted < 1×10⁻¹⁰⁰ and log₂fold > 2.0- or < −2.0-fold changes in expression. The top-most significant genes, SLC22A15, GSTP1 and HSPA1A have p-value adjusted < 1×10⁻³⁰⁰. These three genes are plotted as p=1×10⁻³⁰⁰ in this figure.