Presenting Author Emory University School of Medicine
Background: The sodium bicarbonate transporters move HCO3– across cell membranes and regulate intracellular pH or transepithelial HCO3– transport. Some members of these transporters, such as NBCe1 and NBCe2, mediate electrogenic transport (1 Na+ and 1 CO32– or 2 HCO3–), while others, such as NBCn1 and NBCn2, mediate electroneutral transport (1 Na and 1 HCO3–). A structure/function study has shown that the loop between transmembrane (TM) 7 and 8 is responsible for electrogenicity. On the other hand, a recent report on the molecular dynamics simulations of NBCe1 cryoEM structure has proposed an ion binding site, which is located in transmembrane domain (TM) 5 and serves as a transient binding site to attract anions from the surrounding solution. In the present study, we tested whether this binding site is responsible for electrogenic Na/HCO3 transport.
Methods: Amino acid residues constituting the ion binding site of NBCe1 and NBCn1 were mutated and their effects on electrogenicity were evaluated by expressing the corresponding mutant transporters in Xenopus oocytes and assessing an Na/HCO3 transport current (INBC) in two-electrode voltage clamp. In addition, the loop between TM7 and TM8 was replaced and its effect on INBC was examined.
Results: 1) Replacing TM5 of NBCe1 with the corresponding TM5 of NBCn1 retained INBC, whereas a reciprocal replacement did not. 2) Replacing charged residues constituting the ion binding site in NBCn1 with the residues at the corresponding site in NBCe1 did not induce INBC. 3) Replacing the loop between TM7 and TM8 in NBCe1 with the corresponding loop in NBCn1 abolished INBC. 4) Replacing the loop in the NBCe1/TM5 chimeric transporter with the loop in NBCn1 abolished INBC.
Conclusion: We conclude that the NBCe1 electrogenicity is not associated with the ion binding site but determined by the loop between TM7 and TM8.
