Further Characterization of the Electrogenicity and pH Sensitivity of the Human Organic Anion-Transporting Polypeptides OATP1B1 and OATP1B3

Abstract

Organic anion-transporting polypeptides (OATPs) are involved in the liver uptake of many endogenous and xenobiotic compounds, such as bile acids and drugs, respectively. Using Xenopus laevis oocytes and Chinese hamster ovary (CHO) cells expressing rat Oatp1a1, human OATP1B1, or OATP1B3, the sensitivity of these transporters to extracellular/intracellular pH (pHo/pHi) and changes in plasma membrane potential (⌬⌿) was investigated. In X. laevis oocytes, nonspecific plasma membrane permeability increased only at pHo below 4.5. Above this value, both using oocytes and CHO cells, extracellular acidification affected differently the specific transport of taurocholic acid (TCA) and estradiol 17␤-D-glucuronide (E 2 17␤G) by Oatp1a1 (stimulation), OATP1B1 (inhibition), and OATP1B3 (stimulation). Changes in substrate uptake in the presence of valinomycin (K ϩ -ionophore), carbonyl cyanide 3-chlorophenylhydrazone and nigericin (protonophores), and amiloride (Na ϩ /H ϩ -inhibitor) and cation replacement in the medium were studied with fluorescent probes for measuring substrate uptake (cholylglycyl amidofluorescein) and changes in pHi (SNARF-4F) and ⌬⌿ [DilC 1 (5)]. The results suggest that activity of these three carriers is sodium/potassium-independent and affected differently by changes in pHo and ⌬⌿: Oatp1a1 was confirmed to be an electroneutral anion exchanger, whereas the function of both OATP1B1 and OATP1B3 was markedly affected by the magnitude of ⌬⌿. Moreover, electrophysiological measurements revealed the existence of a net anion influx associated to OATP1B1/OATP1B3-mediated transport of TCA, E 2 17␤G, and estrone-3-sulfate. Furthermore, a leakage of Na ϩ through OATP1B1 and OATP1B3, which is not coupled to substrate transport, was found. In conclusion, these results suggest that OATP1B1 and OATP1B3 are electrogenic transporters whose activity may be strongly affected under circumstances of displacement of local pH.

Molecular Characterization of Zebrafish Oatp1d1 (Slco1d1), a Novel Organic Anion-transporting Polypeptide

“…Oatp1d1 activity was affected by pH o and pH i changes, which in turn induced changes in proton fluxes across the plasma membrane (Figs. 7 (A and B) Ϫ ] antiport, which was previously proven for several mammalian OATPs/Oatps (26,45). In contrast, when [H ϩ ] gradient was reversed (pH i Ͻ pH o ) through intracellular acidification or extracellular alkalization, we observed significant reduction in Oatp1d1 transport activity (Fig.…”

“…The majority of OATPs/ Oatps, including OATP1A2, OATP1B1, and OATP1B3, function as [HCO 3 Ϫ ] antiporters (15,26,45,46). However, some rat transporters, namely Oatp1a1 and Oatp1a4, use glutathione for the antiport (47,48).…”

Mechanistic interpretation of conventional Michaelis–Menten parameters in a transporter system

“…two times the number of microrate constants), there are only 9 unknown variables, since k 4 is assumed to be the same value for each compound, as they were measured against the same transporter in the same expression system and k 4 is purely transporter-specific. While there has been evidence k 4 can be partially influenced by exchanged substrates in limited cases (12), it is assumed here that k 4 is substrate-independent. Compound pair analysis is of practical value, as Michaelis-Menten parameters are typically interpreted in the context of other compounds.…”

Targeted Drug Delivery to Treat Pain and Cerebral Hypoxia

“…For example, substrate affinity can be increased by protonation of a conserved histidine residue at the extracellular side of transmembrane domain 3 (Leuthold et al, 2009). Similarly, studies using Xenopus laevis oocytes, Caco-2 cells, and Chinese hamster ovary cells showed that OATP1A2, OATP1B1, and OATP1B3 transport function is dependent upon an electrogenic gradient and that these transporters are strongly affected by fluctuations in external pH (Kato et al, 2010;Martinez-Becerra et al, 2011). However, pH dependence of OATP1B1-and OATP1B3-mediated transport remains controversial since Mahagita et al (2007) showed that estrone-3-sulfate transport via either OATP1B1 or OATP1B3 was unaffected by changes in extracellular pH.…”