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 (I(NBC)) by two-electrode voltage clamp (Day 3), injected CA II protein + Tris or just Tris (Day 3), measured I(NBC) or the initial rate at which the intracellular pH fell (dpH(i)/dt) upon applying 5% CO2 (Day 4), exposed oocytes to the permeant CA inhibitor ethoxzolamide (EZA), and measured I(NBC) or dpH(i)/dt (Day 4). Because dpH(i)/dt was greater in CA II than Tris oocytes, and EZA eliminated the difference, injected CA II was functional. I(NBC) 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 I(NBC) or dpH(i)/dt (days 3-4), exposed oocytes to EZA, and measured I(NBC) or dpH(i)/dt (Day 3-4). dpH(i)/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.
Highlights
Because the reports of Vince and Reithmeier (4 – 6) that cytosolic carbonic anhydrase II (CA II) binds to the LDADD motif on the cytoplasmic C terminus of the Cl-HCO3 exchanger AE1, Sterling et al [7, 8] have measured rates of intracellular pH change in HEK293 cells transiently transfected with AE1 and concluded that CA II enhances AE1-mediated HCO3Ϫ transport
Effect of Injected CA II on CO2-induced pHi Changes—In Fig. 1, we outline our experimental design for studies in which we examined the effect of injected, recombinant, human CA II protein or the effect of CA II fused to the C terminus of NBCe1-A
The following narrative applies to the pHi experiments done for studies in which we examined the effect of injected CA II protein
Summary
Expression of NBCe1-A and Co-injection with CA II in Xenopus Oocytes cDNA encoding human NBCe1-A had been cloned into the pGH19 expression vector [17] by Dr Inyeong Choi [10]. (iii) We used AgeI to cut the silently mutated EGFP from the aforementioned vector and ligated it nondirectionally into the newly created AgeI site in the 5Ј untranslated region of NBCe1-A. When translated, this construct would consist of EGFP that has its NcoI site silently mutated, followed by a 20-amino acid linker (QLWQINSPSAEFGLGGLAGK), which in turn would be followed by the start methionine of NBCe1-A. PHi Experiments on EGFP-e1-CAII and EGFP-e1—The nominally CO2/HCO3Ϫ-free solution contained (in mM) 42.4 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 32.5 HEPES, and 33 sodium gluconate at a pH of 7.50. Parallel experiments (not shown) proved that 1:1000 Me2SO does not affect the NBCe1-A current
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