GPI-anchored carbonic anhydrase IV displays both intra- and extracellular activity in cRNA-injected oocytes and in mouse neurons

Abstract

Soluble cytosolic carbonic anhydrases (CAs) are well known to participate in pH regulation of the cytoplasm of mammalian cells. Membrane-bound CA isoforms—such as isoforms IV, IX, XII, XIV, and XV—also catalyze the reversible conversion of carbon dioxide to protons and bicarbonate, but at the extracellular face of the cell membrane. When human CA isoform IV was heterologously expressed in Xenopus oocytes, we observed, by measuring H + at the outer face of the cell membrane and in the cytosol with ion-selective microelectrodes, not only extracellular catalytic CA activity but also robust intracellular activity. CA IV expression in oocytes was confirmed by immunocytochemistry, and CA IV activity measured by mass spectrometry. Extra- and intracellular catalytic activity of CA IV could be pharmacologically dissected using benzolamide, the CA inhibitor, which is relatively slowly membrane-permeable. In acute cerebellar slices of mutant mice lacking CA IV, cytosolic H + shifts of granule cells following CO 2 removal/addition were significantly slower than in wild-type mice. Our results suggest that membrane-associated CA IV contributes robust catalytic activity intracellularly, and that this activity participates in regulating H + dynamics in the cytosol, both in injected oocytes and in mouse neurons.

Evidence from mathematical modeling that carbonic anhydrase II and IV enhance CO2 fluxes across Xenopus oocyte plasma membranes

“…If ϳ76% of total oocyte CA IV is located in intracellular vesicles, then, to be consistent with our simulations, only about one-third could be available to catalyze cytosolic reactions. • On the other hand, if extracellular CA IV is confined to a shell only 0.1 m thick (A S ϭ 10,000), then we calculate that ϳ78% of total CA activity would be in the cytosol, a figure virtually identical to the estimate of Schneider et al (21). In this scenario, to be consistent with our simulations, virtually all vesicular CA IV would have to be available to catalyze cytosolic reactions with ϳ100% efficiency.…”

“…The preliminary immunohistochemistry work of Nakhoul et al (16) on oocytes expressing CA IV has shown that CA IV is distributed both near the plasma membrane and near the intracellular vesicles. Recently, Schneider et al (21) used a mass spectrometry approach to measure CA activity in oocytes expressing CA IV, with measurements on intact oocytes representing the extracellular activity of the CA IV, and measurements on lysed oocytes representing total (i.e., extracellular ϩ intracellular) CA activity. These authors concluded that only ϳ24% of the total CA activity is on the extracellular surface of the oocyte, and that ϳ76% is in intracellular vesicles.…”

How carbonic anhydrases and pH buffers facilitate the movement of carbon dioxide through biological membranes. Focus on “Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase II enhances CO2 fluxes across Xenopus oocyte plasma membranes”; “Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes”; and “Evidence from mathematical mo

“…This effect was blocked by extracellular acetazolamide and substantially slowed by acetazolamide injected into the cytosol, implying that CA IV, as heterologously expressed in oocytes, is present on both the extracellular surface and intracellularly. This result confirms earlier work (7,9). Because the surface electrode is "cis" to the surface CA IV, and the intracellular electrode is "cis" to the intracellular CA IV, how can one reach intuitive conclusions about the effect of the enzyme?…”

Intracellular and Extracellular Carbonic Anhydrases Cooperate Non-enzymatically to Enhance Activity of Monocarboxylate Transporters

“…A possible explanation for this effect is that the increase in intracellular buffering power makes up for the decrease in the proton gradient due to the CO 2 -induced decrease in intracellular pH from 7.3 in HEPES-buffered to 6.8 in CO 2 /HCO 3 Ϫ -buffered solution. Determination of CAIV Expression and Activity-We have shown previously that CAIV can display not only extracellular, but also robust intracellular catalytic activity in CAIV-express- ing Xenopus oocytes and in mouse neurons in situ (38). Therefore we determined the expression level of intra-and extracellular CAIV in Xenopus oocytes at different amounts of injected cRNA.…”