Functional expression of the 11 human Organic Anion Transporting Polypeptides in insect cells reveals that sodium fluorescein is a general OATP substrate

Abstract

Organic Anion Transporting Polypeptides (OATPs), encoded by genes of the Solute Carrier Organic Anion (SLCO) family, are transmembrane proteins involved in the uptake of various compounds of endogenous or exogenous origin. In addition to their physiological roles, OATPs influence the pharmacokinetics and drug-drug interactions of several clinically relevant compounds. To examine the function and molecular interactions of human OATPs, including several poorly characterized family members, we expressed all 11 human OATPs at high levels in the baculovirus-Sf9 cell system. We measured the temperature- and inhibitor-sensitive cellular accumulation of sodium fluorescein and fluorescein-methotrexate, two fluorescent substrates of the OATPs, OATP1B1 and 1B3. OATP1B1 and 1B3 were functional in Sf9 cells, showing rapid uptake (t1/2(fluorescein-methotrexate) 2.64 and 4.16 min, and t1/2(fluorescein) 6.71 and 5.58 min for OATP1B1 and 1B3, respectively) and high-affinity transport (Km(fluorescein-methotrexate) 0.23 and 0.53 μM, and Km(fluorescein) 25.73 and 38.55 μM for OATP1B1 and 1B3, respectively) of both substrates. We found that sodium fluorescein is a general substrate of all human OATPs: 1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, while fluorescein-methotrexate is only transported by 1B1, 1B3, 1A2 and 2B1. Acidic extracellular pH greatly facilitated fluorescein uptake by all OATPs, and new molecular interactions were detected (between OATP2B1 and Imatinib, OATP3A1, 5A1 and 6A1 and estradiol 17-β–D-glucuronide, and OATP1C1 and 4C1 and prostaglandin E2). These studies demonstrate for the first time the applicability of the insect cell system for the functional expression of the entire human OATP family, and for drug-OATP interaction screening.

Structural similarities between some common fluorophores used in biology and marketed drugs, endogenous metabolites, and natural products

“…Thus ( Fig 4A) w e illustr ate the dr ugs closest to fluor escein in t-SNE space (as per Figur e 1B), si nce fluor escein is a ver y com m on fluor ophor e, i s also w idely used in ophthalm ology (e.g. [54; 55]), and can enter cells via a var iety of tr anspor ter s [56] such as m onocar boxylate tr anspor ter s (SLC16A1, SLC16A4) [57], SLCO1B1/3B1 [58; 59] and SLC 22A20 [60] (see also Table 1). Fluor escein is sim ilar in t-SNE space ( Fig 4A) to a var iety of dr ugs.…”

Membrane Transporters Contributing to PGE₂ Distribution in Central Nervous System

“…PGT, MATR1, SLC21A2, PHOAR2 100 b) 55) , 331 a) 56) Br, 30) Eye, 107) Ov, 108) St 109) SLCO2B1 OATP2B1 OATP-B, SLC21A9 N.D. 44,49) Br, 46) Eye, 46,99,107) Li, 40) Si 110) SLCO3A1 OATP3A1 OATP-D, OATP3A1_v1, SLC21A11 56 a) 75) , 218 a) 81) Eye, 99) Br and Te 81) SLCO3A1 OATP3A1_v2 OATP-D, SLC21A11 372 a) 81) Br and Te 81)…”

“…OATP-H, SLC21A20 N.D. 49) N.D. SLC22A1 OCT1 HOCT1 657 a) 57) Br, 64) Li, 111) Lu 112) SLC22A2 OCT2 -28.9 a) 57) Br, 64,113) Ki, 103,111) Lu, 112) Si 103) SLC22A6 OAT1 HOAT1, PAHT, ROAT1 970 a) 57) Br, 114) Ki, 115,116)…”

“…Oatp1c1 is specifically expressed along the plasma membranes of brain capillary endothelial cells, and has been shown to contribute to brain uptake of triiodothyronine and thyroxine. Since hOATP1C1 accepts PGE 2 as a substrate, 49) and OATP1C1mediated transport is inhibited by PGE 2 , 50) this suggests that OATP1C1 functions as a PGE 2 importer at the BBB. Current knowledge on transporters for PGE 2 at the BBB is described according to information on transporters' recognition to PGE 2 and localization of transporters by histological examinations.…”

“…37,78) However, Oatp1a1 may not be involved in PG absorption from the CP epithelium because Oatp1a1 accepts PGE 2 only under acidic conditions. 41,49) In contrast, Oatp1a4 and Oatp2b1 (a.k.a. Moat1) are likely expressed at the basolateral side (facing the capillaries) of the CP epithelium.…”