Intracellular pH was monitored at the cytosolic surface of plasma membrane solute transporters (Na+/H+/nucleoside co-transporters, or Cl-/HCO3- exchangers), using pH-sensitive fluorescent proteins (FPs), dual emission green FP (deGFP4) and a monomeric red FP Nectarine (mNect), whose development and characterization are also reported here.
Human concentrative nucleoside transporter, hCNT3, mediates Na+/H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in HEK293 cells. pH changes at the intracellular surface of hCNT3 were monitored by fusing mNect to the cytoplasmic N-terminus of hCNT3 (mNect.hCNT3) or an inactive hCNT3 mutant (mNect.hCNT3-F563C). Cells were incubated at the permissive pH for H+-coupled nucleoside transport, pH 5.5, under both Na+-free and Na+-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mM uridine, providing the first direct evidence for H+-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H+ flux) in the presence or absence of Na+. This suggests that in acidic Na+-containing conditions, 1 Na+ and 1 H+ are transported/uridine molecule, while in acidic Na+-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule and intestine, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3.
Microdomains, discrete regions of altered cytosolic solute concentration, are enhanced by rapid solute transport and slow diffusion rates. pH-regulatory membrane transporters, like the Cl-/HCO3- exchanger AE1, could nucleate H+ microdomains, since AE1 has a rapid transport rate and cytosolic H+ diffusion is slow. As AE1 drives Cl-/HCO3- exchange, differences in pH, near and remote from AE1, were monitored simultaneously by deGFP4 fused to AE1 (deGFP4.AE1) and mNect.hCNT3-F563C. deGFP4.AE1-mNect.hCNT3-F563C distance was varied by co-expression of different amounts of the two proteins in HEK293 cells. As the deGFP4.AE1-mNect.hCNT3-F563C distance increased, mNect.hCNT3-F563C detected the cytosolic pH change with a time delay and reduced rate of pH change, compared to deGFP4.AE1. Carbonic anhydrase activity was essential for H+ microdomain formation. H+ diffusion along the plasma membrane was 60-fold slower than to the cytosolic ER-surface. During physiological HCO3- transport, a H+ microdomain 0.3 µm in diameter develops around AE1, which will affect nearby pH-sensitive processes.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:AEU.10048/1697 |
| Date | 06 1900 |
| Creators | Johnson, Danielle Elaine |
| Contributors | Dr. Joseph Casey, Physiology and Biochemistry, Dr. James Young, Physiology, Dr. Larry Fliegel, Biochemistry, Dr. Todd Alexander, Physiology and Pediatrics, Dr. Gergely Lukacs, McGill University, Department of Physiology |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 97384510 bytes, application/pdf |
| Relation | Johnson, D. E., and Casey, J. R. 2009. Bicarbonate Transport Metabolons. in Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural and Disease Applications, Supuran, C.T. and Winnum, J. Eds, Wiley Chapter 20, pp 415-437., Johnson, D.E., H.W. Ai, P. Wong, J.D. Young, R.E. Campbell, and J.R. Casey. 2009. Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport. J Biol Chem. 284:20499-20511. |
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