Deborah Sterling, Reinhart AF Reithmeier, Joseph R Casey
Carbonic anhydrases are a widely expressed family of enzymes that catalyze the reversible reaction: CO2 + H2O HCO3 - + H+ . These enzymes therefore both produce HCO3 - for transport across membranes and consume HCO3 - that has been transported across membranes. Thus these enzymes could be expected to have a key role in driving the transport of HCO3 - across cells and epithelial layers. Plasma membrane anion exchange proteins (AE) transport chloride and bicarbonate across most mammalian membranes in a one-for-one exchange reaction and act as a model for our understanding of HCO3 - transport processes. Recently it was shown that AE1, found in erythrocytes and kidney, binds carbonic anhydrase II (CAII) via the cytosolic C-terminal tail of AE1. To examine the physiological consequences of the interaction between CAII and AE1, we characterized Cl- /HCO3 - exchange activity in transfected HEK293 cells. Treatment of AE1- transfected cells with acetazolamide, a CAII inhibitor, almost fully inhibited anion exchange activity, indicating that endogenous CAII activity is essential for transport. Further experiments to examine the role of the AE1/CAII interaction will include measurements of the transport activity of AE1 following mutation of the CAII binding site. In a second approach a functionally inactive CA mutant, V143Y, will be co-expressed with AE1 in HEK293 cells. Since over expression of V143Y CAII would displace endogenous wildtype CAII from AE1, a loss of transport activity would be observed if binding to the AE1 Cterminus is required for transport.
