Carbonic anhydrases (CAs) are zinc metalloenzymes, which catalyze the reversible hydration of CO₂ to yield H⁺ and HCO₃^-. Defects and alterations in CA expression are related to a number of diseases, such as glaucoma, osteoporosis, epilepsy and cancer, which has promoted the search for CA inhibitors. Various isoforms are expressed nearly ubiquitously in living cells, some of which are located in the cytosol (e.g. CAI, CAII, CAIII), while others expose their catalytic activity at the extracellular membrane face (CAIV, CAIX, CAXII, CAXIV, CAXV). When human CA isoform IV (hCAIV) was heterologously expressed in Xenopus oocytes, we observed, by measuring H⁺ at the outer face of the cell membrane and in the cytosol with H⁺-selective microelectrodes, not only extracellular, but also intracellular catalytic CA activity. CA expression and CA activity in oocytes were confirmed by immunocytochemistry and mass spectrometry. Extra- and intracellular catalytic activity of CAIV could be pharmacologically dissected using the slowly membrane-permeable CA inhibitor benzolamide, and determined by mass spectrometry of intact and lysed oocytes. In acute cerebellar slices of mice deficient of CAIV, cytosolic [H⁺] shifts in granule cells following CO₂ removal/addition were significantly slower than in wild-type mice. Our results suggest that membrane-associated CAIV contributes robust catalytic activity intracellularly that participates in H⁺ dynamics in the cytosol, both in injected oocytes and in mouse neurons. These findings infer that also other extracellular CAs may contribute to intracellular enzymatic activity, which might be important for interpreting functions of membrane-bound CA isozymes in physiological and pathophysiological processes.

Supported by the Deutsche Forschungsgemeinschaft (DE 231/24)