Role of AQP9 in transport of monomethyselenic acid and selenite

Abstract

AQP9 is an aquaglyceroporin with a very broad substrate spectrum. In addition to its orthodox nutrient substrates, AQP9 also transports multiple neutral and ionic arsenic species including arsenic trioxide, monomethylarsenous acid (MAs^III) and dimethylarsenic acid (DMA^V). Here we discovered a new group of AQP9 substrates which includes two clinical relevant selenium species. We showed that AQP9 efficiently transports monomethylselenic acid (MSeA) with a preference for acidic pH, which has been demonstrated in Xenopus laevis oocyte following the overexpression of human AQP9. Specific inhibitors that dissipate transmembrane proton potential or change the transmembrane pH gradient, such as FCCP, valinomycin and nigericin did not significantly inhibit MSeA uptake, suggesting MSeA transport is not proton coupled. AQP9 was also found to transport ionic selenite and lactate, with much less efficiency compared with MSeA uptake. Selenite and lactate uptake via AQP9 is pH dependent and inhibited by FCCP and nigericin, but not valinomycin. The selenite and lactate uptake via AQP9 can be inhibited by different lactate analogs, indicating that their translocation share similar mechanisms. AQP9 transport of MSeA, selenite and lactate is all inhibited by a previously identified AQP9 inhibitor, phloretin, and the AQP9 substrate arsenite (As^III). These newly identified AQP9 selenium substrates imply that AQP9 play a significant role in MSeA uptake and possibly selenite uptake involved in cancer therapy under specific microenvironments.

Keywords: AQP9; Arsenic trioxide (AsIII); Glycerol; Lactate; Liver; Monomethyselenic acid (MSeA); Selenite; Water.

Fig. 1. Monomethyselenic acid (MSeA) transport by AQP9

Transport of MSeA by human AQP9 was examined under different pH conditions. A: Time course of MSeA uptake via AQP9. MSeA was added in transport buffer at a final concentration of 100μM. B. Transport of MSeA under different pH. MSeA was added in transport buffer at a final concentration of 100μM for 60 minutes. C: Inhibition of MSA transport by different organic acids and AQP9 substrate As^III Transport assay was performed under pH 5.5 for 60 minutes. D. Effect of FCCP, valinomycin and nigericin on MSeA transport by hAQP9. Oocytes were pretreated with FCCP (20 μM), valinomycin (100 μM), or nigericin (10 μM) for 60 min at pH 5.5. The open bars represent arsenic uptake via AQP9, and the solid bars are the water injected controls.

Fig. 2. Lactate transport by AQP9

Transport of Lactate species by AQP9 was examined under different pH conditions. A: Time course of lactate uptake via AQP9. Lacate was added at a final concentration of 1mM. B. Transport of lactate under different pH. Lactate was added in transport buffer at a final concentration of 1mM for 60 minutes. C: Inhibition of lactate transport by different AQP9 substrates. Transport assay was performed under pH 5.5 for 60 minutes. D. Effect of FCCP, valinomycin and nigericin on lactate transport by AQP9. Oocytes were pretreated with FCCP (20 μM), valinomycin (100 μM), or nigericin (10 μM) for 60 min at pH 5.5. The open bars represent arsenic uptake via AQP9, and the solid bars are the water injected controls.

Fig. 3. Selenite transport by AQP9

Transport of selenite by human AQP9 was examined under different pH conditions. A: Time course of lactate uptake via human AQP9. Selenite was added in transport buffer at a final concentration of 1mM for 60 minutes. B. Transport of lactate under different pH. Selenite was added in transport buffer at a final concentration of 1mM for 60 minutes. C: Inhibition of lactate transport by different AQP9 substrates. Transport assay was performed under pH 5.5 for 60 minutes. D. Effect of FCCP, valinomycin and nigericin on selenite transport by AQP9. Oocytes were pretreated with FCCP (20 μM), valinomycin (100 μM), or nigericin (10 μM) for 60 min at pH 5.5. The open bars represent arsenic uptake via AQP9, and the solid bars are the water injected controls.

Fig. 4. Hypothetical pathway of MSeA and selenite uptake and cellular metabolism in cells

Inorganic selenite has two uptake pathways: under physiological pH, it is facilitated via ZIP8 with Zn and bicarbonate, which is predicted a major pathway under these conditions; when under acidic microenvironment while ZIP8 is not expressed, uptake of Se could be by AQP9. MSeA cellular uptake is mainly dependent on AQP9 expression, such as in liver tissue.