Carbonic anhydrase II fused to the human electrogenic Na/HCO3 cotransporter (hNBCe1‐A) does not enhance the activity of the transporter in Xenopus oocytes

Abstract

It has been reported that carbonic anhydrase II (CAII) binds to the C termini of AE1 and hNBCe1-A (e1), thereby enhancing transport. Here we have fused e1 at its 5′ end to EGFP and at its 3′ end to human CAII to create 5′ EGFP-e1-3′ CAII. We expressed the fusion construct in Xenopus oocytes and confirmed its delivery to the plasma membrane via laser scanning microscopy of EGFP and immunocytochemistry of EGFP and CAII. Exposing oocytes to a pH-7.50 out-of-equilibrium solution containing 5% CO2 but virtually no HCO3 (“pure CO2”) caused pHi to decrease faster in cells expressing 5′ EGFP-e1-3′ CAII than in control cells expressing 5′ EGFP-e1 (71 vs 32×10−4pH units/s, n=28 vs 22). For cells expressing 5′ EGFP-e1-3′ CAII, a 3-hour incubation with ethoxzolamide (EZA; 400 μM) reduced the acidification rate nearly to 5′ EGFP-e1 levels. Thus, the CAII is functional. Before EZA incubation, cells expressing 5′EGFP-e1-3′CAII and cells expressing 5′EGFP-e1 had identical 5%CO2/33mM HCO3−induced NBC currents. The e1-dependent slope conductances were of 16.5 and 16.8 μS, respectively, measured by two-electrode voltage clamp. EZA had no effect on the e1-dependent slope conductance of either 5′EGFP-e1-3′CAII or 5′EGFP-e1 oocytes after 3 hours incubation. We conclude that, at least in oocytes, CAII does not enhance activity of NBCe1-A. (Supported by NIH DK30344)