Extracellular Loop 3 of NBCe1‐A Are Closely Associated at the Dimer Interface

The basolateral electrogenic sodium bicarbonate cotransporter NBCe1‐A plays a critical role in absorption of the filtered bicarbonate load in the kidney. We previously determined that NBCe1‐A consists of 14 transmembrane helices and exists as a dimer in the lipid bilayer. The molecular structure of the dimer interface between the two NBCe1‐A monomers remains unknown. In the present study, we discovered that transmembrane segment (TM) 5 and 6 from each of the NBCe1‐A monomer is closely self associated at the interface of the NBCe1‐A dimer. Our conclusion is based on the observations: 1) individual cysteine substituted amino acids at the beginning (Tyr567‐Ser582) and the end (Gln643‐Asp647) of extracellular loops 3 is self cross‐linked to form a NBCe1‐A dimer in the plasma membrane; 2) the cross‐linked dimers are sensitive to DTT treatment indicating disulfide bond formation; 3) the NBCe1‐A dimer is resistant to NEM pre‐treatment suggesting the cross‐linking is formed during protein synthesis. Further biotin maleimide labeling analysis revealed that the extracellular aqueous/lipid interface of TM 5 is at amino acid Tyr566 and TM 6 is at Ile648, indicating that the extracellular loop 3 consists of 82 amino acids. Our findings suggest that the extracellular loop 3 from each of the NBCe1‐A monomer may interact to form a domain‐like structure on the surface of the transporter that carries an uncharacterized physiological function.