Membrane Protein Research Group, Department of Physiology, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
J Physiol. 2011 Apr 1;589(Pt 7):1551-69. doi: 10.1113/jphysiol.2010.201483. Epub 2011 Feb 7.
Microdomains, regions of discontinuous cytosolic solute concentration enhanced by rapid solute transport and slow diffusion rates, have many cellular roles. pH-regulatory membrane transporters, like the Cl−/HCO3− exchanger AE1, could develop H+ microdomains since AE1 has a rapid transport rate and cytosolic H+ diffusion is slow. We examined whether the pH environment surrounding AE1 differs from other cellular locations. As AE1 drives Cl−/HCO3− exchange, differences in pH, near and remote from AE1, were monitored by confocal microscopy using two pH-sensitive fluorescent proteins: deGFP4 (GFP) and mNectarine (mNect). Plasma membrane (PM) pH (defined as ∼1 μm region around the cell periphery) was monitored by GFP fused to AE1 (GFP.AE1), and mNect fused to an inactive mutant of the Na+-coupled nucleoside co-transporter, hCNT3 (mNect.hCNT3). GFP.AE1 to mNect.hCNT3 distance was varied by co-expression of different amounts of the two proteins in HEK293 cells. As the GFP.AE1–mNect.hCNT3 distance increased, mNect.hCNT3 detected the Cl−/HCO3− exchange-associated cytosolic pH change with a time delay and reduced rate of pH change compared to GFP.AE1. We found that a H+ microdomain 0.3 μm in diameter forms around GFP.AE1 during physiological HCO3− transport. Carbonic anhydrase isoform II inhibition prevented H+ microdomain formation. We also measured the rate of H+ movement from PM GFP.AE1 to endoplasmic reticulum (ER), using mNect fused to the cytosolic face of ER-resident calnexin (CNX.mNect). The rate of H+ diffusion through cytosol was 60-fold faster than along the cytosolic surface of the plasma membrane. The pH environment surrounding pH regulatory transport proteins may differ as a result of H+ microdomain formation, which will affect nearby pH-sensitive processes.
微区是溶质浓度不连续的细胞质区域，由于溶质快速转运和扩散缓慢而增强，具有许多细胞功能。pH 调节膜转运蛋白，如 Cl−/HCO3− 交换体 AE1，可以形成 H+微区，因为 AE1 具有快速转运速率，而细胞质 H+扩散缓慢。我们研究了 AE1 周围的 pH 环境是否与其他细胞位置不同。由于 AE1 驱动 Cl−/HCO3− 交换，因此通过使用两种 pH 敏感荧光蛋白：deGFP4（GFP）和 mNectarine（mNect）的共聚焦显微镜监测 AE1 附近和远处的 pH 差异：GFP 融合到 AE1（GFP.AE1）监测质膜（PM）pH（定义为细胞周围的 1 μm 区域），mNect 融合到 Na+-偶联核苷协同转运体的无活性突变体 hCNT3（mNect.hCNT3）监测细胞质 pH 变化。通过共表达两种蛋白的不同量来改变 GFP.AE1 到 mNect.hCNT3 的距离。随着 GFP.AE1–mNect.hCNT3 距离的增加，mNect.hCNT3 检测到与 Cl−/HCO3− 交换相关的细胞质 pH 变化的时间延迟和降低的 pH 变化速率，与 GFP.AE1 相比。我们发现，在生理 HCO3− 转运过程中，直径为 0.3 μm 的 H+微区围绕 GFP.AE1 形成。碳酸酐酶同工酶 II 抑制阻止了 H+微区的形成。我们还使用融合到内质网（ER）胞质面的 ER 驻留钙连蛋白（CNX.mNect）的 mNect 测量了从 PM GFP.AE1 到 ER 的 H+运动速度。H+穿过细胞质的扩散速度比沿着质膜胞质面的扩散速度快 60 倍。pH 调节转运蛋白周围的 pH 环境可能会因 H+微区的形成而不同，这将影响附近的 pH 敏感过程。
