Inactivation mechanisms of Na+/HCO₃⁻ cotransporter NBCe1 by phosphorylation

The solute carriers (SLC) superfamily comprises 66 families with more than 450 members. The Na+/ HCO₃⁻ cotransporter NBCe1 (SLC4A4) of SLC4 family plays critical roles in intracellular pH regulation and transepithelial transport of fluid and electrolytes. Here, we explored the structural mechanisms of NBCe1-A regulation by two phosphorylation modules: P-loop in the amino-terminal domain and H-loop in the transmembrane domain. Mimic-phosphorylation of P-loop or H-loop substantially decreases NBCe1-A activity. Inhibition of NBCe1 by P-loop is abolished by mutations to specific basic residues in the fourth intracellular loop (IL4) in the carrier domain and IL3/IL6 in the scaffold. Inhibition by H-loop is abolished by specific mutations to IL3. We conclude that: (1) P-loop inactivates NBCe1-A by binding to the carrier and the scaffold; (2) H-loop blocks NBCe1-A by interacting with IL3 in the scaffold. Our findings have implications for studying the structural mechanisms for the regulation of other SLCs by phosphorylation. Wu et al. show that phosphorylation-mimicking of P-loop and H-loop regions inhibits NBCe1-A activity via distinct interactions with intracellular loops. These findings reveal dual regulatory mechanisms with broader relevance to SLC modulation.