Renouncing electroneutrality is not free of charge: switching on electrogenicity in a Na + - coupled phosphate cotransporter.

A. Bacconi , L.V. Virkki, J. Biber, H. Murer and I.C. Forster

Institute of Physiology and Centre of Human Integrative Physiology (CHIP), University of Zurich , Zurich Switzerland.

Renal type IIa Na + -coupled inorganic phosphate (P i ) cotransporters (NaPi-IIa) mediate divalent P i transport in an electrogenic manner, whereas the renal type IIc isoform (NaPi-IIc) is electroneutral . They share high sequence identity but a differential tissue expression in adults and weaning kidney respectively. This study aimed to the identification of the amino acids involved in the determination of the different kinetic behavior of the two type II isoforms. Dual uptake ( 32 P i / 22 Na) assays confirmed that NaPi-IIc displayed Na + -coupled P i cotransport with a 2:1 (Na + :P i ) stoichiometry compared with 3:1 established for NaPi-IIa. This suggested that the electrogenicity of NaPi-IIa arises from the interaction of an additional Na + ion compared to NaPi-IIc. To identify the molecular elements responsible for the functional difference between isoforms, we used chimera and amino acid replacement approaches. Transport activity of chimeras constructed with NaPi-IIa and NaPi-IIc indicated that residues within the first 6 transmembrane domains (TMDs) were essential for the electrogenicity of NaPi-IIa. A sequence comparison between electrogenic and electroneutral isoforms revealed differences in charge and polarity of residues clustered in 3 areas, one of which included part of the predicted 3 rd TMD. Here, substitution of 3 residues with their NaPi-IIa equivalents in NaPi-IIc (S189A, S191A, G195D) resulted in a transporter that displayed a 1:1 charge:P i coupling, a 3:1 Na + :P i stoichiometry and transient currents that resembled presteady-state relaxations. Its weaker voltage dependency and 10-fold lower apparent P i affinity compared to NaPi-IIa indicated that other residues important for the NaPi-IIa kinetic fingerprint exist. Our findings demonstrate that through a minimal number of side chain substitutions we can affect a switch from electroneutral to electrogenic cotransporter function, concomitant with the appearance of a novel cosubstrate interaction site.