Publications

• Isobutyrylcarnitine as a Biomarker of OCT1 Activity and Interspecies Differences in its Membrane Transport.

  Jensen O., Matthaei J., Klemp H.G., Meyer M.J., Brockmoeller J., Tzvetkov M.V.

  Genome-wide association studies have identified an association between isobutyrylcarnitine (IBC) and organic cation transporter 1 (OCT1) genotypes. Higher IBC blood concentrations in humans with active OCT1 genotypes and experimental studies with mouse OCT1 suggested an OCT1-mediated efflux of IBC ... >> More

  Genome-wide association studies have identified an association between isobutyrylcarnitine (IBC) and organic cation transporter 1 (OCT1) genotypes. Higher IBC blood concentrations in humans with active OCT1 genotypes and experimental studies with mouse OCT1 suggested an OCT1-mediated efflux of IBC. In this study, we wanted to confirm the suggested use of IBC as an endogenous biomarker of OCT1 activity and contribute to a better understanding of the mechanisms behind the association between blood concentrations of carnitine derivatives and OCT1 genotype. Blood and urine IBC concentrations were quantified in healthy volunteers regarding intra- and interindividual variation and correlation with OCT1 genotype and with pharmacokinetics of known OCT1 substrates. Furthermore, IBC formation and transport were studied in cell lines overexpressing OCT1 and its naturally occurring variants. Carriers of high-activity OCT1 genotypes had about 3-fold higher IBC blood concentrations and 2-fold higher amounts of IBC excreted in urine compared to deficient OCT1. This was likely due to OCT1 function, as indicated by the fact that IBC correlated with the pharmacokinetics of known OCT1 substrates, like fenoterol, and blood IBC concentrations declined with a 1 h time delay following peak concentrations of the OCT1 substrate sumatriptan. Thus, IBC is a suitable endogenous biomarker reflecting both, human OCT1 (hOCT1) genotype and activity. While murine OCT1 (mOCT1) was an efflux transporter of IBC, hOCT1 exhibited no IBC efflux activity. Inhibition experiments confirmed this data showing that IBC and other acylcarnitines, like butyrylcarnitine, 2-methylbutyrylcarnitine, and hexanoylcarnitine, showed reduced efflux upon inhibition of mOCT1 but not of hOCT1. IBC and other carnitine derivatives are endogenous biomarkers of hOCT1 genotype and phenotype. However, in contrast to mice, the mechanisms underlying the IBC-OCT1 correlation in humans is apparently not directly the OCT1-mediated efflux of IBC. A plausible explanation could be that hOCT1 mediates cellular concentrations of specific regulators or co-substrates in lipid and energy metabolism, which is supported by our <i>in vitro</i> finding that at baseline intracellular IBC concentration is about 6-fold lower alone by OCT1 overexpression. << Less

  Front. Pharmacol. 12:674559-674559(2021) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

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

  Yee S.W., Buitrago D., Stecula A., Ngo H.X., Chien H.C., Zou L., Koleske M.L., Giacomini K.M.

  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 classif ... >> More

  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. << Less

  FASEB J. 34:15734-15752(2020) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• Crystal structure of the carnitine transporter and insights into the antiport mechanism.

  Tang L., Bai L., Wang W.H., Jiang T.

  CaiT is a membrane antiporter that catalyzes the exchange of L-carnitine with gamma-butyrobetaine across the Escherichia coli membrane. To obtain structural insights into the antiport mechanism, we solved the crystal structure of CaiT at a resolution of 3.15 A. We crystallized CaiT as a homotrimer ... >> More

  CaiT is a membrane antiporter that catalyzes the exchange of L-carnitine with gamma-butyrobetaine across the Escherichia coli membrane. To obtain structural insights into the antiport mechanism, we solved the crystal structure of CaiT at a resolution of 3.15 A. We crystallized CaiT as a homotrimer complex, in which each protomer contained 12 transmembrane helices and 4 l-carnitine molecules outlining the transport pathway across the membrane. Mutagenesis studies revealed a primary binding site at the center of the protein and a secondary substrate-binding site at the bottom of the intracellular vestibule. These results, together with the insights obtained from structural comparison with structurally homologous transporters, provide mechanistic insights into the association between substrate translocation and the conformational changes of CaiT. << Less

  Nat. Struct. Mol. Biol. 17:492-496(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Functional characterization of residues within the carnitine/acylcarnitine translocase RX2PANAAXF distinct motif.

  De Lucas J.R., Indiveri C., Tonazzi A., Perez P., Giangregorio N., Iacobazzi V., Palmieri F.

  The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane alpha-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by usi ... >> More

  The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane alpha-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using molecular modelling of the carrier suggest that R275, N280 and F284 are involved in substrate binding during acylcarnitine/carnitine translocation. Furthermore, functional analysis of mutations of residues Pro278 and Ala279 in A. nidulans, together with kinetic data in reconstituted liposomes, suggest a predominant structural role for these amino acids. << Less

  Mol. Membr. Biol. 25:152-163(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The reconstituted carnitine carrier from rat liver mitochondria: evidence for a transport mechanism different from that of the other mitochondrial translocators.

  Indiveri C., Tonazzi A., Palmieri F.

  The transport mechanism of the reconstituted carnitine carrier purified from rat liver mitochondria was investigated kinetically. The half-saturation constant (Km) for carnitine on the internal side of the liposomal membrane (8.7 mM) was found to be much higher than that determined for the externa ... >> More

  The transport mechanism of the reconstituted carnitine carrier purified from rat liver mitochondria was investigated kinetically. The half-saturation constant (Km) for carnitine on the internal side of the liposomal membrane (8.7 mM) was found to be much higher than that determined for the external surface (0.45 mM). The exclusive presence of a single transport affinity for carnitine on each side of the membrane indicated a unidirectional insertion of the carnitine carrier into the proteoliposomes, most probably right-side-out with respect to mitochondria. Under these defined conditions bisubstrate initial velocity studies of homologous (carnitine/carnitine) and heterologous (carnitine/acylcarnitine) antiport were performed by varying both the internal and external substrate concentrations. The kinetic patterns obtained showed that the ratio Km/Vmax is not influenced by the second (non-varied) substrate, which indicates a ping-pong mechanism. The carnitine carrier thus differs from all other mitochondrial carriers analyzed so far in the reconstituted state, for which a common sequential type of reaction mechanism has been found. << Less

  Biochim. Biophys. Acta 1189:65-73(1994) [PubMed] [EuropePMC]