Effect of human carbonic anhydrase II on the activity of the human electrogenic Na/HCO3 cotransporter NBCe1-A in Xenopus oocytes

Abstract

Others report that carbonic anhydrase II (CA II) binds to the C termini of the anion exchanger AE1 and the electrogenic Na/HCO3 cotransporter NBCe1-A, enhancing transport. After injecting oocytes with NBCe1-A cRNA (Day 0), we measured NBC current (INBC) by two-electrode voltage clamp (Day 3), injected CA II protein + Tris or just Tris (Day 3), measured INBC or the initial rate at which the intracellular pH fell (dpHi/dt) upon applying 5% CO2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured INBC or dpH i/dt (Day 4). Because dpHi/dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. INBC slope conductance was unaffected by injecting CA II. Moreover, EZA had identical effects in CA II versus Tris oocytes. Thus, injected CA II does not enhance NBC activity. In a second protocol, we made a fusion protein with enhanced green fluorescent protein (EGFP) at the 5′ end of NBCe1-A and CA II at the 3′ end (EGFP-e1-CAII). We measured INBC or dpH i/dt (days 3-4), exposed oocytes to EZA, and measured INBC or dpHi/dt (Day 3-4). dpHi/dt was greater in oocytes expressing EGFP-e1-CA II versus EGFP-e1, and EZA eliminated the difference. Thus, fused CA II was functional. Slope conductances of EGFP-e1-CAII versus EGFP-e1 oocytes were indistinguishable, and EZA had no effect. Thus, even when fused to NBCe1-A, CA II does not enhance NBCe1-A activity. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.