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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.

Abstract The gammaproteobacterium Thiomicrospira crunogena XCL–2 is a hydrothermal vent chemolithoautotroph that has a carbon concentrating mechanism (CCM), which is functionally similar to that of cyanobacteria. At hydrothermal vents, dissolved inorganic carbon (DIC) concentrations and pH values fluctuate over time, with CO2 concentrations ranging from 20 μM to greater than 1 mM, therefore having a CCM would provide an advantage when CO2 availability is very low as CCMs generate intracellular DIC concentrations much higher than extracellular, thereby providing sufficient substrate for carbon fixation. The CCM in T. crunogena includes α–carboxysomes (intracellular inclusions containing form IA RubisCO and carbonic anhydrase), and also presumably requires at least one active HCO3 µ transporter to generate the elevated intracellular concentrations of DIC. To determine whether RubisCO itself might be adapted to low CO2 concentrations, the KCO2 for purified carboxysomal RubisCO was measured (250 μM SD ±; 40) and was much greater than that of whole cells (1.03 μM). This finding suggests that the primary adaptation by T. crunogena to low–DIC conditions has been to enhance DIC uptake, presumably by energy–dependent membrane transport systems that are either ATP–dependent and/or dependent on membrane potential (δ ψ). To determine the mechanism for active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis andδ ψ. After separate incubations with the ATP synthase inhibitor DCCD and the protonophore CCCP, intracellular ATP was diminished, as was the concentration of intracellular DIC and fixed carbon, despite the absence of an inhibitory effect on δ ψ in the DCCD–incubated cells. In some organisms, DCCD inhibits the NDH–1 and bc1 complexes so it was necessary to verify that ATP synthase was the primary target of DCCD in T. crunogena. Both electron transport complex activities were assayed in the presence and absence of DCCD and there was no significant difference between inhibited (309.0 μmol/s for NDH–1 and 3.4 μmol/s for bc1) and uninhibited treatments (271.7 μmol/s for NDH–1 and 3.6 μmol/s for bc1). These data support the hypothesis that an ATP–dependent transporter is primarily responsible for HCO3 µ transport in T. crunogena. The ATP–dependent transporter solute–binding protein gene (cmpA) from Synechococcus elongatus PCC 7942, was used to perform a BLAST query. Tcr_1153 was the closest match in the T. crunogena genome. However, the gene neighborhood and the result of a maximum likelihood tree suggest that Tcr_1153 is a nitrate transporter protein. Work is underway to find the genes responsible for this ATP–dependent transporter.

No description available.