Abstract
Transport metabolons have been discussed between carbonic anhydrase II (CAII) and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1), to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 µM), while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity.
Highlights
Maintenance of H+ homeostasis is important for all cell types, because protons are involved in many processes of metabolism and cellular function
The catalytically inactive mutant carbonic anhydrase II (CAII)-V143Y did not show an increase of rate of rise of proton concentration during application of CO2/ HCO32-buffered solution as compared to native oocytes, and there was no reduction of the rate of rise of proton concentration in the presence of EZA with this mutant (Fig. 2 B)
Effect of CAI, CAII and CAIII on NBCe1 transport activity We have recently shown that NBCe1 transport activity is enhanced by coexpression of CAII or after injection of CAII-protein into Xenopus oocytes [12], and that catalytic activity of CAII is important for this interaction
Summary
Maintenance of H+ homeostasis is important for all cell types, because protons are involved in many processes of metabolism and cellular function. Cells possess a number of ion transporters, which are involved in the active transport of H+ or HCO32, like sodium hydrogen exchangers (NHE) [1] or the solute carrier family 4 of bicarbonate transporters (SLC4) [2,3]. CAII regulates the concentration of the substrate of the sodium bicarbonate cotransporter 1 (NBCe1) by catalyzing the hydration of CO2 and the dehydration of HCO32. We have previously shown an increase in transport activity of NBCe1 by CAII in Xenopus oocytes as determined by an increased membrane current, rate of rise of intracellular sodium concentration and membrane conductance [12]. Other groups could not find evidence for an interaction of CAII and NBCe1 by determining the effect on membrane conductance of NBCe1-expressing oocytes after an injection of CAII-protein [13] or determining change of membrane current of NBCe1+CAIIcoexpressing oocytes [14]
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