Voltage clamp fluorometric measurements on a Na+‐coupled Pi cotransporter reveal new insights into substrate binding order.

Voltage clamp fluorometry (VCF) combines conventional voltage clamp and fluorescence measurements to detect protein conformational changes, which are sensed by a covalently attached fluorophore. We have applied VCF to a type IIb Na + ‐coupled P i cotransporter (NaPi‐IIb) expressed in Xenopus oocytes. We introduced a novel cysteine in the putative 3 rd extracellular loop (S448C) and labelled the protein with tetramethylrhodamine methanethiosulfonate. Labelling blocked cotransport, but we detected voltage‐ and substrate‐ dependent changes in fluorescence (Δ F ), which suggested that i) substrate interaction still occurred in the labelled protein and ii) S448C lies in an environment where conformational changes occur. Hyperpolarizing membrane potentials ( V m ) and increasing Na + or Li + concentration quenched Δ F . Presteady‐state charge movements saturated at hyper and depolarising V m , whereas F saturated only at depolarising V m , indicating that the two signals provide insight into different underlying physical processes. Interpreting the Na + , Li + and P i ‐dependencies of Δ F suggested that the substrate binding order differs from our previous model, whereby two (rather than one) Na + ions precede P i binding, and that only the second Na + binding transition is voltage‐dependent. Moreover, we show that Li + , which does not drive cotransport, interacts with the first Na + binding step. The results were incorporated in a new model of the transport cycle of type II Na + /P i cotransporters, the validity of which is supported by simulations that predict the voltage and substrate dependency of the experimental data. Supported by SNF and GRS (Switzerland)