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.

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This thesis characterizes adenosine signalization in Drosophila and describes response to adenosine in various cell types. Extracellular adenosine mediates most of its physiological effects through its receptors but recent data also indicate that adenosine transport also has important physiological functions. It was shown in this thesis that adenosine stimulates only cAMP second messenger system in Drosophila cells endogenously expressing AdoR. The pharmacological profile of the DmAdoR was established using the cAMP functional assay. The utility of the agonist 2-chloroadenosine and antagonist SCH58261 were examined in flies in vivo and compared with phenotypes of DmAdoR mutants. The responses of Drosophila cells to adenosine mediated by adenosine transport were also examined. Different cell types exhibited striking differences in adenosine uptake and adenosine recycling that were closely connected with the regulation of carbohydrate and lipid metabolism. This thesis provides an important foundation for the study of interactions between adenosine receptor and adenosine transport.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 128-140).