Electrogenic Na/HCO3 Cotransporter (NBCe1) Variants Expressed in Xenopus Oocytes: Functional Comparison and Roles of the Amino and Carboxy Termini

Abstract

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.

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) stimulates the electrogenic Na/HCO 3 cotransporter NBCe1-A expressed in Xenopus oocytes

“…The percentage of the NBC-mediated inward current that is Na ϩ independent (10-20%) is similar to that seen in whole-cell voltage-clamp experiments (18%) where bath HCO 3 Ϫ drives NBC-mediated transport into oocytes (8). Further studies are required to determine if the Na ϩ -independent NBCe1-A current is due to transporter slippage (24) or a Na ϩ -independent (e.g., HCO 3 Ϫ ) conductance.…”

“…We previously reported that applying bath HCO 3 Ϫ to create symmetrical Na ϩ and HCO 3 Ϫ gradients across inside-out macropatches (ϪV p ϭ Ϫ60 mV) from NBC-expressing oocytes elicited small DIDS-reversible inward currents (8). Here, we stimulated these currents by increasing the Na ϩ and HCO 3 Ϫ gradients using a nominally HCO 3 Ϫ -free pipette solution containing low (10 mM) Na ϩ .…”

“…Similar to other BTs, NBCe1 is inhibited by stilbene derivatives such as DIDS (6,8,(15)(16)(17), which can inhibit NBCe1 from the intracellular side of the plasma membrane (6). As shown in Rundown of NBCe1-A Activity in Excised Macropatches.…”

“…The Frömter laboratory has used both inside-out and outside-out configurations of the technique to examine sensitivity of rat NBCe1-A to cytosolic ATP (6) and Ca 2ϩ levels (5). More recently, our group has used the inside-out configuration to characterize NBCe1 variants truncated at their amino termini (8). The technique provided 2 -induced current; those data points were also included in the 100-M group.…”

“…For NBCe1, the unique amino terminus of the A variant stimulates activity, whereas the different amino terminus of the B and C variants inhibits activity (8). Such automodulation may involve additional binding proteins and signaling molecules.…”