Cytosolic H Plus Microdomain Developed Around Ae1 During Ae1-Mediated Cl-/Hco3- Exchange
JOURNAL OF PHYSIOLOGY-LONDON(2011)
Abstract
Non-technical summaryMost cellular processes are exquisitely sensitive to pH. Consequently our cells have a range of processes directed to control cellular pH. Plasma membrane transport proteins move acid or base across the plasma membrane to regulate pH precisely. We studied AE1 (also called Band 3) of erythrocytes and kidney cells, which rapidly transports the base, bicarbonate. AE1's high transport rate, combined with the surprisingly slow rates of H+ diffusion in cytosol, led us to wonder whether AE1 changes the pH of its local environment. The key findings were that H+ diffusion through the cytosol occurs at 0.6 mu m s-1, and along the inner surface of the plasma membrane at only 0.01 mu m s-1. We estimated that the size of the region of altered pH (H+ microdomain) around AE1 is 0.3 mu m in diameter. pH-regulatory transporters, like AE1, have differential effects on their immediate environment, with implications for the regulation of nearby pH-sensitive proteins.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 similar to 1 mu m 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 mu m 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.
MoreTranslated text
AI Read Science
Must-Reading Tree
Example
![](https://originalfileserver.aminer.cn/sys/aminer/pubs/mrt_preview.jpeg)
Generate MRT to find the research sequence of this paper
Chat Paper
Summary is being generated by the instructions you defined