Cellular proton buffering: Spatio-temporal dynamics

胞液pH的变化影响多种细胞过程，包括基因表达、代谢活性、细胞信号、细胞增殖和肿瘤生长。与细胞钙缓冲相比，我们对质子缓冲在生理过程中的动态变化和细胞分布的作用几乎一无所知，例如，有证据表明，细胞质质子缓冲有几个成分，其中一些可能有助于缓冲能力的动态和局部变化，从而影响pH变化的幅度和动力学。酸碱偶联的膜转运体和碳酸氢酶活性可能会影响细胞内和胞外的pH瞬变；然而，关于快速的H+/HCO3-跨细胞膜转运和碳酸氢酶对质子缓冲的细胞分布的相关性，人们知之甚少。在这项研究中，我们想要分析细胞中主要的酸/碱调节因子-碳酸氢钠共转运体(NBCe1，SLC4A4)的作用，以及胞内碳酸氢酶II的活性和分布，以实现胞质质子的明显缓冲。通过用染料和/或微电极测量培养的星形胶质细胞(这些蛋白是天然表达的)和非洲爪哇卵母细胞(这些转运蛋白将在其中异源表达)的细胞内pH，酸碱通量和Na+的速率增加，碳酸氢酶II的活性将与细胞H+缓冲相关。我们的目标是评估质子缓冲能力的动态和局部变化，这决定了细胞和组织中pH瞬变的程度，并将尝试使用数学方法来模拟时空质子缓冲。这些结果对于深入了解pH变化的产生和调节，以及对依赖于pH的细胞过程的调控具有重要意义。

Changes in cytosolic pH affect multiple cellular processes, including gene expression, metabolic activity, cellular signalling, cell proliferation and tumor growth. We know virtually nothing about the role of dynamic changes and cellular distribution of proton buffering for physiological processes, in contrast to cellular buffering of calcium e.g. There is evidence, however, that cytoplasmic proton buffering has several components, some of which may contribute to dynamic and local changes of buffering capacity, and hence on the amplitude and kinetics of pH changes. Acid-/base-coupled membrane transporters and carbonic anhydrase activity may affect pH transients both intra- and extracellularly; very little is known, however, about the relevance of fast H+/HCO3 - transport across cell membranes and the cellular distribution of carbonic anhydrases for proton buffering. In the present study, we want to analyse the role of the sodium-bicarbonate cotransporter (NBCe1, SLC4A4), a prime acid/base regulator in cells, and the activity and distribution of cytosolic carbonic anhydrase II, for ‘apparent’ cytosolic proton buffering. By measuring intracellular pH with dyes and/or microelectrodes in cultured astrocytes, where these proteins are natively expressed, and in Xenopus oocytes, in which these transport proteins will be heterologously expressed, the rate of acid/base flux and Na+ rise, and the carbonic anhydrase II activity will be correlated with cellular H+ buffering. We aim to evaluate the dynamics and local changes of proton buffer capacity, which determine the extent of pH transients in cells and tissues, and will try to model spatio-temporal proton buffering using mathematical approaches. The results are important for gaining insight into the generation and modulation of pH changes, and into the regulation of pHdependent cellular processes.