Arsenic is a biologically toxic metalloid which dissolves in water to form arsenic acid. Its chemical similarity with phosphoric acid suggests that the inorganic arsenate (As_i^V) could enter the organism via epithelial phosphate transporters. The type IIb Na⁺/P_i cotransporter performs a Na⁺-coupled transport of inorganic phosphate (P_i) in the kidney and intestine.

We studied the As_i^V interactions with NaP_i-IIb and identified which partial reactions of the transport cycle are targeted by using biophysical techniques.

As_i^V inhibited the electrogenic response to P_i and decreased the apparent affinity for P_i. The steady-state activity of As_i^V alone was 30% of the maximum response to P_i. As_i^V inhibited mobile charge movement in a dose-dependent manner. To investigate the As_i^V interaction at the molecular level, we performed Voltage Clamp Fluorometry assays using the mutant S155C. The addition of As_i^V quenched the emitted fluorescence at the hyperpolarizing extreme in a dose-dependent manner. This indicates that the microenvironment of the fluorophore at Cys-155 changes in accordance with the probability of the substrate occupying the anionic binding site.

We propose that As_i^V interacts with NaP_i-IIb at the P_i 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.