Abteilung für Allgemeine Zoologie, Fachbereich Biologie, University of Kaiserslautern, D-67653 Kaiserslautern, Germany.
Proc Natl Acad Sci U S A. 2013 Jan 22;110(4):1494-9. doi: 10.1073/pnas.1221213110. Epub 2013 Jan 7.
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.
可溶性细胞溶质碳酸酐酶（CA）是众所周知的参与哺乳动物细胞质的 pH 调节。膜结合的 CA 同工型 - 如同工型 IV、IX、XII、XIV 和 XV - 也催化二氧化碳到质子和碳酸氢盐的可逆转换，但在细胞膜的细胞外表面。当人 CA 同工型 IV 在非洲爪蟾卵母细胞中异源表达时，我们通过用离子选择性微电极测量细胞膜外表面和细胞质中的 H+，观察到不仅有细胞外催化 CA 活性，还有强大的细胞内活性。通过免疫细胞化学和质谱法测量 CA IV 活性来证实卵母细胞中的 CA IV 表达。使用相对较慢的膜渗透性 CA 抑制剂苯甲酰胺可以对 CA IV 的细胞外和细胞内催化活性进行药理学剖析。在缺乏 CA IV 的突变小鼠的急性小脑切片中，颗粒细胞在 CO2 去除/添加后细胞质 H+的转移明显比野生型小鼠慢。我们的结果表明，膜相关的 CA IV 具有强大的细胞内催化活性，这种活性参与调节细胞质中的 H+动力学，无论是在注射的卵母细胞还是在小鼠神经元中。
