Remarkable commonalities of electrogenic and electroneutral Na+–phosphate cotransporters
Year: 2009
Type: article
Abstract: Transporter gene families show that related electrogenic and electroneutral transporters have evolved for a common substrate (e.g. Murer et al. 2004; Romero et al. 2004), indicating that while there may be a common motif for transporting a given substrate, subtle differences in this motif may render a transporter electroneutral or electrogenic (e.g. Bacconi et al. 2005; Choi et al. 2007). A particular case in point is the solute carrier family 34 (SLC34), members of which serve to maintain body inorganic phosphate (Pi) homeostasis by secondary active Na+-coupled transport of Pi in renal, intestinal, and other tissues. Of the three gene products of the SLC34 family, SLC34A1 (NaPi-IIa) and SLC34A3 (NaPi-IIc) are localized to the apical membrane of proximal tubular epithelial cells, where they reabsorb filtered inorganic phosphate. SLC34A2 (NaPi-IIb) is found in the apical membrane of intestinal epithelia, where it absorbs dietary phosphate (Virkki et al. 2007).
NaPi-IIa and NaPi-IIb are electrogenic: they transport 3 Na+ and 1 divalent Pi (HPO42−) per transport cycle leading to the translocation of one net charge per Pi (Forster et al. 1999). NaPi-IIc exhibits a 2 : 1 Na+ : HPO42− transport stoichiometry and is electroneutral (Segawa et al. 2002; Bacconi et al. 2005). Surprisingly, substitution of three key residues in NaPi-IIc with their NaPi-IIa equivalents (S189A, S191A and G195D) resulted in a switch from electroneutral 2 : 1 Na+ : Pi cotransport to electrogenic 3 : 1 Na+ : Pi cotransport (Bacconi et al. 2005).
In this issue of The Journal of Physiology, Ghezzi et al. (2009) present findings that significantly advance our understanding of the mechanistic features that distinguish electroneutral and electrogenic Na+–Pi cotransporters. They expressed NaPi-IIc in Xenopus laevis oocytes, and used a combination of radiotracer (32P) flux assays, chemical modification of transporter at a single engineered cysteine residue and voltage-clamp fluorometry to determine the order of ligand binding to the transporter in its outward-facing conformation.
Using steady-state influx and efflux assays, the authors demonstrate that Pi cannot interact with the transporter in the absence of Na+, implying that Na+ is the first ligand to bind to the transporter. In uptake experiments, the rate of Na+–Pi cotransport was measured by varying the concentration of one substrate while maintaining the concentration of the cosubstrate constant, and the experiment was repeated at different concentrations of the invariant cosubstrate. The results of these studies suggest that the concentration of Na+ in the extracellular fluid is the main determinant of the maximum rate of Na+–Pi cotransport. Importantly, at saturating concentrations of extracellular Na+, the maximum rate of transport appeared to be independent of the concentration of Pi. These results imply that Na+ is also the last ligand to bind to the outwardly-facing transporter before 2 Na+ : 1 Pi cotranslocation.
To further examine the binding order, the authors introduced a novel cysteine residue in the third extracellular loop of NaPi-IIc (S437C), and examined this mutant by using sulfhydryl chemical modification with a fluorescent probe and voltage-clamp fluorometry. Fluorescence intensity emitted by the fluorophore is a function of its microenvironment and, therefore, experimental manoeuvres that alter the fluorescence can be deduced to cause local transporter conformational changes at the labelled cysteine residue. When S437C was labelled with a reporter fluorophore, Na+–Pi cotransport was blocked but, remarkably, neither Na+ nor Pi binding was affected. This allowed the authors to study steady-state changes in fluorescence under a variety of experimental conditions to assess ligand-induced conformational changes of the transporter. Both Na+ and Pi binding to the transporter induced conformational changes. Sodium-induced conformational changes were observed in the absence of Pi. Conformational changes caused by binding of Pi as well as the competitive transport inhibitor, phosphonoformic acid, strictly required the presence of Na+, confirming that Na+ is the first ligand to bind to the transporter – a scenario that excludes a random order of ligand binding.
Given the 2 : 1 Na+ : Pi stoichiometry of NaPi-IIc, it would now seem plausible to assume a Na+→Pi→Na+ binding order. Amazingly, in the absence of Pi, Na+-induced conformational changes of the transporter exhibited a sigmoidal dependence on external Na+, suggesting that at least two Na+ ions interact cooperatively with NaPi-IIc before Pi binding. Not surprisingly for this electroneutral transporter, the conformational changes induced by Na+ were not voltage dependent, probably because Na+ binding occurs outside of the membrane electric field. Moreover, similar to the electrogenic NaPi-IIb, NaPi-IIc binds Li+ even though this ion cannot drive cotransport. Reconciling the radiotracer flux and fluorometric data, the authors propose a Na+→Na+→Pi→Na+ ligand binding order to NaPi-IIc, which is akin to the binding order of the electrogenic NaPi-IIb (Virkki et al. 2006).
Thus, three Na+ ions interact with electrogenic and electroneutral type II Na+–Pi cotransporters. Whereas all three bound Na+ ions are cotransported with Pi in NaPi-IIa/b, only two are cotranslocated with Pi in NaPi-IIc. A key conclusion is that ion/substrate binding stoichiometry and cotransport stoichiometry are not equivalent in NaPi-IIc. Clearly, the bound yet untranslocated Na+ ion cannot thermodynamically contribute to the concentrative capacity of NaPi-IIc. What is the function of the ‘extra’ Na+ ion bound to the transporter? The authors propose that it acts as a catalytic activator of cotransport. It will be illuminating to test for the existence of this feature in other Na+-coupled cotransporters (Rudnick, 2006).
Source: The Journal of Physiology
Author: Sepehr Eskandari
Institution: California State Polytechnic University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2069962919