Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: insights into the transport mechanism.

SLC4 transporters mediate the transmembrane transport of HCO3‾, CO32‾, and Cl‾ necessary for pH regulation, transepithelial H+/base transport and ion homeostasis. Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward facing (OF) structures of two SLC4 members (hAE1 and hNBCe1) with different transport modes (Cl‾/HCO3‾ exchange vs Na+-CO32‾ symport) revealed highly-conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein-ion coordination motifs are still unclear. In the present work we combined Site Identification by Ligand Competitive Saturation (SILCS) mapping and extensive Molecular Dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites (entry and central) in the OF states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, while in hNBCe1, a Na+ acts as an anchor for CO32‾ binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1, and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.