Basolateral carbonic anhydrase IV in the proximal tubule is a glycosylphosphatidylinositol-anchored protein

Abstract

Carbonic anhydrase (CA) IV facilitates HCO(3) reabsorption in the renal proximal tubule by catalyzing the reversible hydration of CO(2). CAIV is tethered to cell membranes via a glycosylphosphatidylinositol (GPI) lipid anchor. As there is basolateral as well as apical CAIV staining in proximal tubule, the molecular identity of basolateral CAIV was examined. Biotinylation of confluent monolayers of rat inner medullary collecting duct cells stably transfected with rabbit CAIV showed apical and basolateral CAIV, and in the cell transfectants expressing high levels of CAIV, a transmembrane form was targeted to the basolateral membrane. Basolateral expression of CAIV ( approximately 46 kDa) was confirmed in normal kidney tissue by Western blotting of vesicle fractions enriched for basolateral membranes by Percoll density fractionation. We examined the mode of membrane linkage of basolaterally expressed CAIV in the kidney cortex. CAIV detected in basolateral or apical membrane vesicles exhibited similar molecular size by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis following deglycosylation, and was equally sensitive to phosphatidylinositol-specific phospholipase C digestion, indicating that CAIV is expressed on the basolateral membrane as a GPI-anchored protein. Half of the hydratase activity of basolateral vesicles was resistant to SDS denaturation, compatible with being CAIV. Thus, GPI-anchored CAIV resides in the basolateral membrane of proximal tubule epithelia where it may facilitate HCO(3) reabsorption via association with kNBC1.

The role of carbonic anhydrases in renal physiology

“…83 Furthermore, we found no evidence for expression of a B3 kDa larger molecular weight species corresponding to a putative transmembrane form of CAIV in kidney cortex. Expression of CAIV as a transmembrane protein has been reported in normal pancreatic tissues, where CAIV expressed on the luminal or apical surface of ductal cells was insensitive to PI-PLC cleavage and was detected by a polyclonal antisera raised against the COOH terminal tail that is cleaved upon GPI-anchor attachment.…”

Proximal Nephron

“…The α-CA enzymes, expressed primarily in mammals, are divided broadly into membrane CAs (CAIV, CAIX, CAXII, CAXIV and CAXV), cytosolic CAs (CAI, CAII, CAIII, CAVII and CAXIII), mitochondrial CAs (CAVA and CAVB), and secreted CAs (CAVI) (for details, please refer to http://www.genenames.org) (417). In the kidney, CAII, a 29 kDa protein, and CAIV, a ~35 kDa protein, are predominantly expressed in humans and rabbits, while CAXII and CAXIV are also expressed in rodents (67; 356; 416; 417; 455; 504). CAII is localized in the cytoplasm of most nephron segments including proximal convoluted tubules, proximal straight tubules, the thick ascending limbs, distal tubules and collecting ducts (417).…”

“…In proximal tubules, CAIV is localized in both apical and basolateral membranes of the S1 and S2 segments (67; 417), where CAXII is expressed primarily in basolateral membranes (417). Approximately 95% of total CA activity in the kidney is mediated by the cytosolic CAII, while the remaining 5% of the CA activity in the kidney is mediated by the membrane-associated CAIV and CAXII (416; 417). …”

Substrate-dependent Interference of Carbonic Anhydrases with the Glutamine Transporter SNAT3-Induced Conductance

“…E.g., SNAT3 and CAIV are both expressed the at the basolateral membrane of the proximal tubulus [44][45], where glutamine uptake is mainly used to fuel urea production. It might be hypothesized that CAIV support the supply H + , and CAII dissipates the H + accumulation ("pH microdomain") around SNAT3 transport pores by a 'push and pull' mechanism, already discussed by Purkerson & Schwartz [46] for other acidbase transporters in the kidney.…”