The conductance properties of Asp555 in the electrogenic Na/HCO3 cotransporter SLC4A4/NBCe1

Abstract

The electrogenic Na/HCO3 cotransporter SLC4A4 (NBCe1) is important for HCO−3 reabsorption in the proximal tubules of the kidney. By sequence comparison between electrogenic transporters and electroneutral transporters, we previously identified Asp555 in NBCe1 as a residue that affects electrogenic HCO−3 current. In this study, we further characterized the HCO−3 conductance of the mutant transporter D555E (substitution of Asp555 with a Glu) using two‐electrode voltage clamp. In oocytes expressing D555E, the application of CO2/HCO−3 caused a negative shift of the reversal potential for HCO−3 current. However, the current progressively decreased at positive voltages that otherwise should be favorable for HCO−3 conductance. The current was larger at negative voltages. No inward current was produced at low [Na+]o that should reverse the direction of Na+/HCO−3 movement as in wild type NBCe1. In the steady state condition, which was achieved by superfusing oocytes with Na+‐free CO2/HCO−3 for 15 min, the application of a series of [Na+]o produced HCO−3 currents. The half maximum effective concentration of external Na+ was 2 mM for D555E, significantly smaller than 12 mM for wild type NBCe1. The substitution of Asp555 with an Asn (D555N) produced a progressive decrease in HCO−3 current at positive voltages and a negative shift of the reversal potential for the current. Reciprocal substitutions of Glu742 in the electroneutral Na/HCO3 cotransporter SLC4A7 (NBCn1) with Asp or Gln failed to produce electrogenic HCO−3 current. We conclude that the steric properties near Asp555 in NBCe1 likely contribute to ion translocation of the transporter.