Arsenate interactions with thetype IIbNa+/Picotransporter

Maria Daniela Renna1, Anne-Kristine Meinild2 and Ian C Forster2

1Laboratory of Cellular and Molecular Physiology, Dept. of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy.

2Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Switzerland.

Background: Arsenic is a biologically toxic metalloid which dissolves in water to form arsenic(V) acid(H3AsO4).Its chemical similarity withphosphoric acid (H3PO4) suggests that one candidate pathway through which the arsenic salt, inorganic arsenate (AsiV), could enter mammalian organisms to exert its toxic effects is via epithelial phosphate transporters. For example, type II sodium-phosphate cotransporters (SLC34 family) perform an essential physiological role bymediating Na+-coupled transport of inorganic phosphate (Pi) in the kidney and intestine.Although Pi is the physiologically relevant substrate, in vitrouptake studies have established thatAsiV competitively inhibits Pi transport and, importantly,isalso transported in the absence of Pi. Moreover, theapparent affinity for AsiVwas reported to be similarto Pi for the intestinalNaPi-IIb(Villa-Belosta and Sorribas, (2010) Toxicol. Appl.Pharmacol. 247, 36-40).

Goal: To elucidate the interactions AsiVwith NaPi-IIb and identify which partial reactions of the transport cycle are targettedby using biophysical techniques.

Methods: We expressed flounder NaPi-IIb in Xenopusoocytes and used conventional two-electrode voltage clamp (TEVC), and TEVC combined with time-resolved fluorometry (voltage clamp fluorometry,VCF) to monitor the conformational changes associated with substrate interaction.For VCF experiments, we used a mutant (S155C), which displays WT-like transport kinetics. When labeled with thefluorophore MTS-TAMRA,S155C yieldsrobust substrate and voltage-dependent changes in emitted fluorescence intensity (F) (Virkki et al., (2006) JBC281: 28837-28849).

Results: In 100 mM Na+ (pH 7.4) and a holding potential =-50mV, AsiV (1 mM)inhibited theelectrogenic response to Pi(1 mM) by 40% and increased the apparent affinity for Pi from 50 to 200 M.The steady-state activity ofAsiV alone was 30% of the maximum response to Pi.AsiV inhibited mobile charge movement in a dose dependent manner (KiAs150 uM) and had marginal effect on voltage dependent partial reactions. To investigate the AsiVinteraction at the molecular level, we performed VCF assays. In 100 mM Na+ alone, F showed a sigmoidal dependence on membrane potential. Theaddition of either Pi(1 mM) orAsiV(1 mM) quenched the emitted fluorescence at the hyperpolarizing extremeby 50% and70%, respectively. Interestingly, when both Pi (1 mM) and AsiV (1mM) were present, F was quenched by the same amount as for 1 mM Pi alone. Moreover, the dose-dependence of AsiVobtained from VCFconfirmed the findings from presteady-state analysis. This established that the microenvironment of the fluorophore at Cys-155 changes in accordance with the probability of the substrate (Pi or AsiV) occupying the anionic binding site.

Conclusions:

We propose that AsiV interacts with NaPi-IIb at the Pi binding site, but with significantly lower apparent affinity. Our findings can be incorporated into a revised kinetic scheme for the cotransport cycle in which the rates associated with partial reactions for substrate interaction are dependent on the specific anionic substrate.