McAlear SD et al. (2006), Electrogenic Na/HCO3 cotransporter (NBCe1) vari...

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J Gen Physiol 2006 Jun 01;1276:639-58. doi: 10.1085/jgp.200609520.

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Electrogenic Na/HCO3 cotransporter (NBCe1) variants expressed in Xenopus oocytes: functional comparison and roles of the amino and carboxy termini.

McAlear SD , Liu X , Williams JB , McNicholas-Bevensee CM , Bevensee MO .

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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 CO(2)/HCO(3)(-) solution displayed all the hallmarks of an electrogenic Na(+)/HCO(3)(-) cotransporter: (a) a DIDS-sensitive pH(i) recovery following the initial CO(2)-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 HCO(3)(-) dependencies (apparent K(M) of 4-6 mM) and external Na(+) dependencies (apparent K(M) of 21-36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (-60 mV) expressing NBCe1-A exhibited peak HCO(3)(-)-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 (V(m) = -60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO(3)(-)-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.

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Species referenced: Xenopus laevis
Genes referenced: lum slc4a1 slc4a4 tbx2

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	Figure 1. Wild-type and truncated rat NBCe1 variants. NBCe1-A, -B, and -C differ at the amino and/or carboxy termini. The putative membrane topology of NBCe1 is based on mapping of AE1 by Taylor et al. (2001). Truncations examined in this study include hemagglutinin (HA)-tagged NBCe1s with stretches of the cytoplasmic amino or carboxy terminus removed. (Inset, top left) At the amino terminus, NBCe1-A has 41 unique amino acids that differ from 85 amino acids in the B and C variants. At the carboxy terminus, NBCe1-C has 61 unique amino acids that differ from 46 amino acids in the A and B variants.
	Figure 2. Activity of rat brain NBCe1-B expressed in an oocyte. (A) pHi (top trace) and voltage (bottom trace) were measured simultaneously in an NBCe1-B–injected oocyte that was initially bathed in a nominally HCO3−-free, HEPES-buffered solution. The oocyte was exposed to a solution containing 1.5% CO2/10 mM HCO3− during segment ae. Electrogenic NBC activity was evident from the instantaneous hyperpolarization (b'), and the pHi recovery (bc) following the initial CO2-induced acidification (ab). DIDS blocked the pHi recovery (cd), and partially reversed the hyperpolarization (c'). (B) The same experimental protocol in panel A was performed on an H2O-injected oocyte. Exposing the oocyte to CO2/HCO3− had no effect on Vm (a'b'), and elicited no pHi recovery (bc) following the initial decrease in pHi (ab). 200 μM DIDS generated only a small hyperpolarization (c').
	Figure 3. Current–voltage (I-V) relationship of NBCe1-C expressed in oocytes. (A) I-V plots were obtained from an NBCe1-C–expressing oocyte initially bathed in ND96 (diamonds), followed by 10 min in 5% CO2/33 mM HCO3− (CB) (squares), and then after 2 min in the CO2/HCO3− solution containing 200 μM DIDS (triangles). (B) The same experimental protocol in A was performed on an H2O-injected oocyte.
	Figure 4. I-V relationships of the three NBCe1 variants. (A) HCO3−-dependent I-V plots were obtained from oocytes expressing NBCe1-A (closed diamonds), NBCe1-B (open squares), and NBCe1-C (closed triangles). For each data point, n ≥ 11. (Inset) The I-V plots are magnified near the reversal potentials (IM = 0) for NBCe1-B and C. (B) HCO3−-dependent I-V plots from oocytes injected with 25 ng of NBCe1-A cRNA (closed diamonds, redrawn from A), 2 ng of NBCe1-A cRNA (open diamonds, n = 3), or 25 ng of HA-tagged NBCe1-AHA cRNA (closed squares, n = 6). (C) HCO3−-dependent I-V plots from oocytes injected with 0.5 ng of NBCe1-AHA cRNA (closed diamonds, n = 3), 25 ng of NBCe1-BHA cRNA (open squares, n = 4), or 25 ng of NBCe1-CHA cRNA (closed triangles, n = 4). Error bars smaller than symbols are not shown.
	Figure 5. Larger HCO3−-induced currents in oocytes expressing NBCe1-A than NBCe1-C. (A) A 5% CO2/33 mM HCO3− solution elicited an outward current in the NBCe1-A–expressing oocyte (AWT) that was 4.5-fold higher than the current in the NBCe1-C-expressing oocyte (CWT). An H2O-injected oocyte displayed no HCO3−-induced outward current. (B) Summary of H2O-subtracted, HCO3−-induced currents from experiments similar to those shown in A. n ≥ 7 from two batches of oocytes. (C) The mean normalized luminescence (Norm. Lum.) was similar for the A and C variants. n ≥ 12 from two batches of oocytes.
	Figure 6. External HCO3−-dependencies of NBCe1-B and -C. (A) A voltage-clamped oocyte (Vh = −60 mV) expressing NBCe1-C was exposed to different HCO3−-containing solutions at a constant [Cl−] of 7.6 mM. (B) The experimental protocol used in A was performed on an H2O-injected oocyte. (C) Normalized HCO3−-induced currents as a function of external [HCO3−] for oocytes expressing NBCe1-B (r2 = 0.99). HCO3−-induced outward currents in experiments similar to that in A were normalized to the mean currents elicited by the flanking exposures to the standard 5% CO2/33 mM HCO3− solution. (D) Normalized HCO3−-induced currents as a function of external [HCO3−] for oocytes expressing NBCe1-C (r2 = 0.99).
	Figure 7. External Na+ dependencies of NBCe1-A and -C. (A) A voltage-clamped oocyte (Vh = −60 mV) expressing NBCe1-C was exposed to 5% CO2/33 mM HCO3− solutions containing different [Na+]s. (B) The experimental protocol used in A was performed on an H2O-injected oocyte. (C) Normalized HCO3−-induced currents as a function of external [Na+] for oocytes expressing NBCe1-A (r2 = 0.98). HCO3−-induced outward currents at different Na+ concentrations were normalized to the mean currents elicited by the flanking exposures to the 33 mM HCO3− solution containing “full” 98.5 mM Na+. (D) Normalized HCO3−-induced currents as a function of external [Na+] for oocytes expressing NBCe1-C (r2 = 0.97).
	Figure 8. Inhibited NBCe1 activity elicited by removing regions of the cytosolic amino terminus. (A) Exposing oocytes to a solution containing 5% CO2/33 mM HCO3− elicited an outward current in the oocyte expressing wild-type NBCe1-C (CWT), but little/no current in the oocyte expressing either CΔN426 or CΔN213. (B) Summary of HCO3−-induced outward currents from experiments similar to those shown in A. For each bar, n ≥ 3 from two batches of oocytes. SEM values for the CΔN426 or CΔN213 data are small. (C) Compared to the mean normalized luminescence (Norm. Lum.) for CWT, mean Norm. Lum. was 1.5-fold greater for CΔN426, and similar for CΔN213 in oocytes from the same two batches in B. n ≥ 10 for each bar.
	Figure 9. Changes in NBCe1 activity elicited by removing the different amino termini. (A, left) An oocyte expressing mutant AΔN43 displayed a peak HCO3−-induced outward current that was ∼50% smaller than seen in the oocyte expressing AWT. (A, right) An oocyte expressing mutant CΔN87 displayed a peak HCO3−-induced outward current that was approximately threefold larger than seen in the oocyte expressing CWT. (B) Summary of HCO3−-induced outward currents from experiments similar to those shown in A. n ≥ 15 for each bar. (C, left) The mean normalized luminescence (Norm. Lum.) was 10% lower for AΔN43 compared with AWT. n ≥ 19 for each bar. (C, right) The mean Norm. Lum. was 33% higher for CΔN87 compared with CWT. n ≥ 23 for each bar.
	Figure 10. Current–voltage (I-V) relationships from oocytes expressing wild-type and amino-terminal truncations of NBCe1 variants. (A) Representative experiment on an oocyte expressing CΔN87 in which I-V plots were obtained with the oocyte initially bathed in ND96 (diamonds), followed by 1 min in 5% CO2/33 mM HCO3− (circles) and then 10 min in the physiological buffer (squares), and finally in the HCO3− solution containing 200 μM DIDS (triangles). (B) HCO3−-dependent I-V plots for NBCe1-A (closed diamonds), AΔN43 (open diamonds), NBCe1-C (closed triangles), and CΔN87 (open triangles). Data were obtained from experiments similar to that shown in A. n = 3 for each data point from a single batch of oocytes. SEM bars smaller than symbol sizes are not shown. Similar results were obtained from a second batch of oocytes.
	Figure 11. Lost activity of CΔC97 expressed in oocytes. (A) pHi (top trace), voltage (middle trace), and current (bottom trace) were measured simultaneously in an NBCe1-C–injected oocyte that was initially bathed in ND96. The oocyte was then voltage clamped and exposed to a solution containing 5% CO2/33 mM HCO3− during segment ae. Electrogenic NBC activity was evident from the instantaneous outward current (a'), and the pHi recovery (bc) following the initial CO2-induced acidification (ab). Removing Na+ blocked the pHi recovery (cd), and reversed the hyperpolarization (c'). (B) The same experimental protocol in A was performed on a CΔC97-injected oocyte. Exposing the oocyte to CO2/HCO3− elicited only a small outward current (a'), and minimal pHi recovery (bc) following the initial decrease in pHi (ab).
	Figure 12. Lost activity of CΔC97 expressed in oocytes due to poor expression at the plasma membrane. (A) Summary data of the segment-bc pHi recovery rates from experiments similar to those shown in Fig. 11, top traces. n ≥ 4 for each bar. (B) Summary of H2O-subtraced, HCO3−-induced currents from experiments similar to those shown in Fig. 11, bottom traces. For each bar, n ≥ 6 from two batches. (C) Immunoblot analysis of total microsomal protein from single oocytes injected with H2O, CΔC97 cRNA, and CWT cRNA. An NBCe1 antibody labeled bands of the expected sizes: ∼130 kD for CWT and ∼118 kD for CΔC97. No labeling was observed in protein from an H2O-injected oocyte. (D) The mean normalized luminescence (Norm. Lum.) for oocytes injected with CΔC97 and H2O are similar. n ≥ 10 from two batches of oocyte.
	Figure 13. Reduced activity and low surface expression of AΔC46 and CΔC61 expressed in oocytes. (A) 5% CO2/33 mM HCO3− elicited an outward current in an oocyte expressing NBCe1-A (AWT) that was markedly larger than the current in the oocyte expressing AΔC46. (B) Summary of H2O-subtracted, HCO3−-induced currents from experiments similar to those shown in A. n ≥ 6 for each bar from two batches of oocytes. (C) Immunoblot analysis of total microsomal protein from single oocytes injected with H2O, AWT cRNA, or AΔC46 cRNA. An NBCe1 antibody labeled bands of the expected sizes: ∼130 kD for AWT and ∼120 kD for AΔC46. (D) The mean normalized luminescence (Norm. Lum.) for oocytes expressing the truncated NBCs were markedly less than the Norm. Lum. for oocytes expressing the corresponding wild-type NBCs. n ≥ 11 for each bar from two batches of oocytes.
	Figure 14. NBCe1 activity from inside-out macropatches excised from oocytes. (A) NBC-mediated inward currents were elicited by exposing patches to a low-Cl−, 5% CO2/33 mM HCO3− solution ± 200 μM DIDS with the patch pipette (−Vp = −60 mV) containing the same low-Cl−, 33 mM HCO3− solution. (B) H2O-injected oocyte. The HCO3− solution without or with DIDS did not elicit any change in current. (C) CWT-injected oocyte. The HCO3− solution elicited a small inward current that was reversible when the patch was returned to ND96. (D) CΔN87-injected oocyte. The inward current elicited by the HCO3− solution was larger than the current seen in C, and completely inhibited by DIDS. (E) AWT-injected oocyte. The HCO3− solution elicited a small, reversible inward current. (F) AΔN43-injected oocyte. No change in current was observed when the patch was exposed to HCO3−.
	Figure 15. Summary macropatch data from experiments similar to those shown in Fig. 14 on oocytes injected with H2O, CWT, CΔN87, AWT, and AΔN43. The mean HCO3−-induced macropatch current for CΔN87 is 3.2-fold larger than the mean current for CWT (†, P = 0.03). The mean HCO3−-induced currents for AWT and CWT are similar (§, P = 0.32), and both are larger than the mean current seen in H2O-injected oocytes (P < 0.05). The mean current for AΔN43 is 71% smaller than that of AWT (*, P < 0.01). n values are shown under bars.

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