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Correspondence to Mark O. Bevensee: bevensee{at}physiology.uab.edu
Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO2/HCO3– solution displayed all the hallmarks of an electrogenic Na+/HCO3– cotransporter: (a) a DIDS-sensitive pHi recovery following the initial CO2-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na+-dependent outward current under voltage-clamp conditions (–60 mV). All three variants had similar external HCO3– dependencies (apparent KM of 4–6 mM) and external Na+ dependencies (apparent KM of 21–36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (–60 mV) expressing NBCe1-A exhibited peak HCO3–-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current–voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (Vm = –60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO3–-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.
Abbreviations used in this paper: AE, anion exchanger; DIDS, 4,4'-diisothiocyanatostilbene-2,2' disulfonate; HA, hemagglutinin; HRP, horseradish peroxidase; NBC, Na/bicarbonate cotransporter; pHi, intracellular pH; PIP2, phosphatidylinositol 4,5-bisphosphate; SOC, single-oocyte chemiluminescence.
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