The SLC4 family of bicarbonate transporters plays a vital role in acid-base homeostasis by regulating pH within organelles, the cytosol, and extracellular fluids. NBCe1, a key member of this family, is crucial for maintaining acid-base and electrolyte balance across various human tissues. Dysfunction of NBCe1 can result in severe proximal tubular acidosis and is implicated in conditions like type 2 diabetes and prostate cancer. Although NBCe1 cotransports bicarbonate with sodium ions, its exact stoichiometry remained uncertain. Using cryo-electron microscopy with a novel composition mask method to improve 3D reconstruction resolution, we resolved NBCe1 structures both with and without sodium bicarbonate at 1.9 to 2.1 Å, confirming a 1:1 sodium-to-substrate cotransport ratio.
Given the critical functions of SLC4 transporters, their activities require tight regulation. Borate transporters in plants, homologous to the SLC4 family, serve as an excellent model for examining these regulatory processes. We used Arabidopsis thaliana Bor1 (AtBor1), which is vital for boron homeostasis, as a model to investigate structure and regulation. Applying the composition mask method in cryo-EM data processing, we improved resolution and identified three distinct active states from a heterogeneous Bor1 sample: inward-facing, occluded, and a hybrid dimer of inward-facing/occluded conformations. High-resolution structures of AtBor1 in inactive and active states revealed two regulatory mechanisms: an auto-inhibitory motif at the carboxyl terminus obstructing the substrate pathway and Thr410 phosphorylation, which is essential for borate transport. These insights into the fast regulation of SLC4 transporters have significant implications for both plant and human health.