Carbonic anhydrases enhance activity of endogenous Na-H exchangers and not the electrogenic Na/HCO3 cotransporter NBCe1-A, expressed in Xenopus oocytes

Abstract

Key points: According to the ${\mathrm{HCO}}_3^{-}$ metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO₃ cotransporter NBCe1-A speeds transport by regenerating/consuming ${\mathrm{HCO}}_3^{-}$ . The present work addresses published discrepancies as to whether cytosolic CAs stimulate NBCe1-A, heterologously expressed in Xenopus oocytes. We confirm the essential elements of the previous experimental observations, taken as support for the ${\mathrm{HCO}}_3^{-}$ metabolon hypothesis. However, using our own experimental protocols or those of others, we find that NBCe1-A function is unaffected by cytosolic CAs. Previous conclusions that cytosolic CAs do stimulate NBCe1-A can be explained by an unanticipated stimulatory effect of the CAs on an endogenous Na-H exchanger. Theoretical analyses show that, although CAs could stimulate non- ${\mathrm{HCO}}_3^{-}$ transporters (e.g. Na-H exchangers) by accelerating CO₂ / ${\mathrm{HCO}}_3^{-}$ -mediated buffering of acid-base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carrying ${\mathrm{HCO}}_3^{-}$ , ${\mathrm{CO}}_3^{=}$ , or ${\mathrm{NaCO}}_3^{-}$ ion pairs.

Abstract: The ${\mathrm{HCO}}_3^{-}$ metabolon hypothesis predicts that cytosolic carbonic anhydrase (CA) binds to NBCe1-A, promotes ${\mathrm{HCO}}_3^{-}$ replenishment/consumption, and enhances transport. Using a short step-duration current-voltage (I-V) protocol with Xenopus oocytes expressing eGFP-tagged NBCe1-A, our group reported that neither injecting human CA II (hCA II) nor fusing hCA II to the NBCe1-A carboxy terminus affects background-subtracted NBCe1 slope conductance (G_NBC ), which is a direct measure of NBCe1-A activity. Others - using bovine CA (bCA), untagged NBCe1-A, and protocols keeping holding potential (V_h ) far from NBCe1-A's reversal potential (E_rev ) for prolonged periods - found that bCA increases total membrane current (ΔI_m ), which apparently supports the metabolon hypothesis. We systematically investigated differences in the two protocols. In oocytes expressing untagged NBCe1-A, injected with bCA and clamped to -40 mV, CO₂ / ${\mathrm{HCO}}_3^{-}$ exposures markedly decrease E_rev , producing large transient outward currents persisting for >10 min and rapid increases in [Na⁺ ]_i . Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔI_m and d[Na⁺ ]_i /dt, it does not reduce G_NBC . In oocytes not expressing NBCe1-A, CO₂ / ${\mathrm{HCO}}_3^{-}$ triggers rapid increases in [Na⁺ ]_i that both hCA II and bCA enhance in concentration-dependent manners. These d[Na⁺ ]_i /dt increases are inhibited by EZA and blocked by EIPA, a Na-H exchanger (NHE) inhibitor. In oocytes expressing untagged NBCe1-A and injected with bCA, EIPA abolishes the EZA-dependent decreases in ΔI_m and d[Na⁺ ]_i /dt. Thus, CAs/EZA produce their ΔI_m and d[Na⁺ ]_i /dt effects not through NBCe1-A, but endogenous NHEs. Theoretical considerations argue against a CA stimulation of ${\mathrm{HCO}}_3^{-}$ transport, supporting the conclusion that an NBCe1-A- ${\mathrm{HCO}}_3^{-}$ metabolon does not exist in oocytes.

Keywords: NBCe1-A; NHE; SLC4A4; bicarbonate; carbonic anhydrase; metabolon.

Figure 1:. Functional expression of wild-type and tagged NBCe1-A constructs does not differ

A, I_NBC–V_m relationships acquired from oocytes expressing e1, eGFP-e1, or eGFP-e1-CAII (injected with cRNA 4 days before recording). With the oocyte first exposed to ND96, we voltage-clamp to V_h = spontaneous V_m, and then step to V_m = −160 mV for 60 ms, return to V_h for 60 ms, step to Vm = –140 mV, return to V_h, and so on, increasing V_m during the step by 20 mV with each cycle, peaking at step to +20 mV (see inset to panel A for a depiction of the protocol waveform). After turning off the clamp, we switch to 5% CO₂/33 mM ${\text{HCO}}_3^{-}$ and, within 6 s, V_m reaches a nadir between −120 mV and −140 mV. At this point, we voltage clamp to V_h ≅ E_rev and then repeat the I–V protocol. For each oocyte, I_NBC vs. V_m is the difference I vs. V_m in CO₂/${\text{HCO}}_3^{-}$ and I vs. V_m in ND96. The main plot shows mean I_NBC (± SD) vs. V_m for 36 oocytes expressing each of the three NBCe1 constructs. The fitted slopes represent their mean NBCe1-A-dependent slope conductance, G_NBC. B, Representative western blot of biotinylated (surface) protein (equivalent to 0.7 oocytes per lane) from the same oocytes from which we previously acquired the I_NBC–V_m relationships, with no-biotin control samples on the same blot (left panel). The right panel displays a representative western blot of the total protein expression (equivalent to 0.24 oocytes per lane). C, Surface expression, quantified by densitometry and normalized to the values for oocytes injected in parallel with e1. Legend for column color applies to panels C–D. D, Total protein expression, quantified by densitometry and normalized to the values for e1 expressing oocytes. E, Surface expression for each group of 12 oocytes analyzed in panel C, normalized to the mean slope conductance. We assess statistical significance for panels C–E by unpaired-samples t-tests with Welch’s correction and the Holm-Bonferroni (Holm, 1979) method (α = 0.05). Number of comparisons for each test group is m = 3. Statistics Table 1 reports the unadjusted p-values and corrected α values for all analyses in Fig. 1. The numbers in parentheses at the bottom of each bar in panels C–E indicate the number of groups of 12 oocytes analyzed for each type of cRNA injected. The bars in panels C–D report mean ± SD, with each data point used to calculate the mean represented by open square symbols. V_h, holding potential.

Figure 2:. Tethering hCA II to the Ct of NBCe1-A does not increase G_NBC

A, I_m–V_m relationships acquired in ND96 and 5% CO₂/33 mM ${\text{HCO}}_3^{-}$ from oocytes expressing eGFP-e1-CAII (injected with cRNA 4 days before recording). We first obtain I–V relationships in ND96 and 5% CO₂/33 mM ${\text{HCO}}_3^{-}$. We use the I–V protocol described in the Fig. 1 legend, except that the duration of V_m steps and return to V_h was 1 s in the middle panel, and 30 s in the right panel. Regardless of step duration, we incubate oocytes for 3 h in 400 μM EZA to inhibit CA II activity, and then repeat the I–V protocols. Data for each of the three step-duration protocols come from separate groups of oocytes. I_m values are mean ± SD. B, Bars represent the mean G_NBC (± SD) values derived from I–V relationships like those in panel A with each data point used to calculate the mean represented by open symbols. Unpaired-samples t-tests with Welch’s correction generate unadjusted p-values for comparisons of I–V step duration for Pre-EZA data (black bars). Paired-samples t-tests generate unadjusted p-values for comparisons of Pre-EZA (black bars) vs. post-EZA (gray bars). For both the unpaired and paired analyses, we used the Holm-Bonferroni method (α = 0.05, m = 3) to judge significance. For analysis of pre-EZA data, † denotes significance vs. 60-ms protocol, and ‡ denotes significance vs. 1-s protocol. Analyses of pre- vs. post-EZA data reveal no differences. C, CO₂-induced acidification in a representative oocyte expressing eGFP-e1-CAII, pre- (black trace) and post-inhibition (gray trace) with 400 μM EZA. D, Comparison of maximal rates of CO₂-induced acidification from cells expressing eGFP-e1 (white bar) or expressing eGFP-e1-CAII, pre- (black bar) and post-400 μM EZA (gray bar). An unpaired-samples t-test with Welch’s correction generates unadjusted p-values to assess differences between eGFP-e1 versus eGFP-e1-CAII († denotes significance). A paired-samples t-test generates unadjusted p-values to assess pre- vs. post EZA (* denotes significance). α = 0.05 is not adjusted because we perform a single unpaired or paired test. Statistics Table 2 reports unadjusted p-values and α values for panels B and D. The numbers in parentheses in panels B–D indicate the number of oocytes for each group. Bars represent the mean ± SD in panels B and D with each data point used to calculate the mean represented by open symbols.

Figure 3:. hCA II injected into the oocyte does not increase G_NBC

A, I_m–V_m relationships from oocytes injected 4 days before recording with 1.5 ng cRNA encoding eGFP-e1 and then injected again 1 day before recording with 50 mM Tris (Top) or 300 ng recombinant hCA II (Bottom). I–V relationships are acquired as described in Fig. 2A, using a separate group of oocytes for each of the three step duration protocols (60 ms, left; 1 s, center; or 30 s, right). I_m values are mean ± SD. B, Mean G_NBC values (± SD) derived from I_m–V_m relationships like those in panel A, before and after the oocytes were incubated for 3 h in 400 μM EZA to inhibit the activity of the injected hCA II. Unpaired-samples t-tests with Welch’s correction generate unadjusted p-values for comparisons of I–V step duration for Pre-EZA data (black bars). Because not all oocytes survived the 3-h EZA incubations, we also used unpaired-samples (rather than paired-samples) t-tests for comparisons of Pre-EZA (black bars) vs. post-EZA (gray bars). The Holm-Bonferroni method (α = 0.05, m = 3) was used to judge significance. For analysis of pre-EZA data, † denotes significance vs. 60-ms protocol, and ‡ denotes significance vs. 1-s protocol. Analyses of pre- vs. post-EZA data reveal no differences. C, CO₂-induced acidification of a representative oocyte expressing eGFP-e1 and injected 24 h earlier with 300 ng hCA II. The black pre-EZA and the gray post-3h EZA record are obtained from the same oocyte. D, Comparison of the mean maximal rates of CO₂-induced acidification (± SD) from cells expressing eGFP-e1 and injected with 50 mM Tris (white bar), or expressing eGFP-e1 but injected with 300 ng recombinant hCA II, pre- (black bar) and post-400 μM EZA (gray bar). An unpaired-samples t-test with Welch’s correction generates unadjusted p-values to assess differences between eGFP-e1+Tris vs. eGFP-e1+hCAII injected oocytes († denotes significance). A paired-samples t-test generates unadjusted p-values to assesses pre- vs. post EZA incubation (* denotes significance). α = 0.05 is not adjusted because we perform a single unpaired or paired test. For panels B and D, unadjusted p-values and α values are presented in Statistics Table 3. The numbers in at the bottom of each bar in panels B & D indicate the number of oocytes for each group with each data point used to calculate the mean represented by open symbols.

Figure 4:. hCA II injected into the oocyte does not increase the HCO3−-dependent rate of pH_i increase, even when NBCe1-A operates at high rates for long periods

A, Representative recordings of pH_i, V_m, and I_m from oocytes injected 4 days before recording with 1.5 ng cRNA encoding eGFP-e1 and then injected again 1 day before recording with 50 mM Tris or B, 300 ng recombinant hCA II. Switching from ND96 to 5% CO₂/33 mM ${\text{HCO}}_3^{-}$ solution (gray shading and “CO₂/${\text{HCO}}_3^{-}$” label) initiates a CO₂-induced acidification. Once pH_i reaches its acidic nadir, we voltage-clamp oocyte V_m at −120 mV then switch to 0 mV to promote high NBCe1-A activity. The symbol key above panels C–H is applicable to each of these panels. C, Mean (±SD) values derived from maximal rates of CO₂-induced acidification. D, Using the I–V protocol depicted in the inset, we record I_NBC–V_m relationship at the V_m nadir (IV_#1 in panels A & B). E, I_NBC–V_m relationship at the pH_i nadir (IV_#2 in panels A & B). F, changes in I_m at the instant of the switch of V_h to 0 mV. G, I_NBC–V_m relationship the switch of V_h to 0 mV. (IV_#3 in panels A & B). H, maximal rates of alkalinization, from experiments like those in panels A & B. The numbers in parentheses at the bottom of each bar in panels C, F and H indicate the number of oocytes tested for each group with each data point used to calculate the mean represented by symbols (see key at top of the figure). For panels C, F and H, unpaired-samples t-tests with Welch’s correction generate unadjusted p-values for comparisons of Tris vs. hCA II. The Holm-Bonferroni method determines significance (α = 0.05, m = 2, † denotes significance). For panel D, E and G, unadjusted p-values from comparisons of mean G_NBC from eGFP-e1+Tris vs. eGFP-e1+hCA II oocytes recorded by IV_#1, IV_#2 or IV_#3 are all > 0.05 and therefore not significant whether α is naive, or adjusted for three tests of the null hypothesis (one for each I–V; m=3). Statistics Table 4 reports the unadjusted p-values and corrected α values. Values are mean ± SD in panels F–H.

Figure 5:. Tethering hCA II to the Ct of NBCe1-A does not increase G_NBC in a B&D-like protocol.

A, Representative recordings of pH_i, V_m, and I_m from oocytes injected 4 days before recording with 1.5 ng cRNA encoding eGFP-e1 or B, 1.5 ng cRNA encoding the eGFP-e1-CAII fusion protein (described in Fig. 2A; Lu et al., 2006). V_h is set at −40 mV in ND96 and the oocyte remains in voltage-clamp during all bath solution switches for the entire protocol. Periods during which the oocyte is perfused with ND96 are not shaded or labeled. Periods during which the oocyte is perfused with 5% CO₂/33 mM ${\text{HCO}}_3^{-}$ buffer are shaded gray and labeled “CO₂/${\text{HCO}}_3^{-}$ ”. 10 µM EZA in ND96 is perfused for 10 min before a second exposure to 5% CO₂/33 mM ${\text{HCO}}_3^{-}$ (plus 10 µM EZA). This period is labeled “10 μM EZA” and shaded cyan. I–V relationships are acquired by stepping V_h in 20 mV increments from −120 mV to +20 mV for 10 s each. The double-staircase V_h step sequence deduced from B&D is −40, −60, −80, −100, −120, −20, 0, and then 20 mV without an inter-step period when V_h is returned to −40 mV and can be seen in the V_m traces in panels A & B. A magnified example of the V_h command during the I–V acquisitions is also displayed as an inset to panel G. C, Mean initial pH_i values at points a₁ (Pre-EZA) and a₂ (+10 μM EZA) are reported for either eGFP-e1 or eGFP-e1-CAII oocytes. D, 10 µM EZA inhibits the CA II-catalyzed rate of CO₂-induced acidification for the eGFP-e1-CAII fusion protein but not eGFP-e1 expressing oocytes. E, We observe no significant changes in ΔI_m or G_NBC following inhibition of the CA II fused to NBCe1-A (E–H). Paired t-tests are performed to compare the difference between a₁ vs. a₂, or Pre EZA vs. +10 µM EZA on the same groups of oocytes in panel C–E and H (significance denoted by *). Unpaired t-tests with Welch’s correction compare eGFP-e1 vs. eGFP-e1-CAII at points a₁ or a₂ (for Panel C), or during Pre EZA or +10 µM EZA periods in panels D, E and H (significance denoted by †). The Holm-Bonferroni method determines significance for panels C–E (α = 0.05, m=2). For panel H, unadjusted p-values are all > 0.05, so differences in the mean G_NBC are not significant even if α is naïve or adjusted for four tests of the null hypothesis (m = 4). Statistics Table 5C-E & 5H presents the unadjusted p-values and adjusted α values. The numbers in parentheses in panels C–E and H indicate the number of oocytes for each group with the individual data points used to calculate each mean overlaid as symbols on the bars. Values are mean ± SD in panels C–H.

Figure 6:. hCA II injected into NBCe1-A expressing oocytes does not increase G_NBC when data is acquired using a reference B&D-like protocol

Representative recordings from oocytes injected 4 days prior to recording with 1.5 ng/oocyte cRNA for eGFP-e1. 1 day before recording, half of the eGFP-e1 expressing oocytes are injected with A, Tris buffer and the other half injected with B, 50 ng hCA II dissolved in Tris. Experiments were performed as in Fig. 5. C, Mean pH_i values at points a₁ (pre-EZA) and a₂ (10 μM EZA) are not significantly different but D, 10 µM EZA inhibited the hCA II-catalyzed rate of CO₂-induced acidification. However, as is observed for CA II tethered to the NBCe1-A Ct in Fig. 5, we do not observe concomitant changes in E, ΔI_m and H, G_NBC. Panels F and G present mean I_NBC–V_m relationships used to calculate G_NBC data in panel H. Statistical significance is determined as described for Fig. 5 and Statistics Table 6C–H reports unadjusted p-values and adjusted α values. The numbers in parentheses in panels C–E and H indicate the number of oocytes for each group with the individual data points used to calculate each mean overlaid as symbols on the bars. Values are mean ± SD in panels C–H.

Figure 7:. Are CA II-dependent changes seen when NBCe1-A is expressed at low levels?

0.15 ng/oocyte of injected eGFP-e1 cRNA yields NBCe1-A currents of equivalent magnitude to B&D, although this ~100 fold less cRNA mass injected per oocyte than in that study. 24 h prior to recording, we inject eGFP-e1 expressing oocytes with either A, 50 nl Tris buffer or B, 50 ng hCA II dissolved in 50 nl Tris buffer. We clamp V_h at –40 mV and expose oocytes to three periods in 5% CO₂/33 mM ${\text{HCO}}_3^{-}$ buffer (gray shading and “CO₂/${\text{HCO}}_3^{-}$” label). Prior to the 3^rd CO₂/${\text{HCO}}_3^{-}$ delivery, the oocyte is perfused for 10 min with 10 μM EZA to inhibit hCA II activity (EZA perfusion periods denoted with “10 μM EZA” label and shaded cyan). We record I–V relationships when the pH_i reaches its acidified nadir during each CO₂/${\text{HCO}}_3^{-}$ delivery (IV_#1, IV_#2, and IV_EZA), each voltage step being 20 mV and 10 s duration according to the B&D protocol. The difference between the maximal pH_i after CO₂ removal and the initial pH_i (b₁ – a₁) is minimized by a CO₂/${\text{HCO}}_3^{-}$ pre-pulse so that the comparable difference for the second CO₂/${\text{HCO}}_3^{-}$ pulse (b₂ – a₂) is substantially smaller than the first. Note that mean initial pH_i values corresponding to a₁ and a₂ for eGFP-e1+ Tris oocytes and eGFP-e1+ hCAII oocytes are substantially different (> 0.1 pH units) and in the case of eGFP-e1+ hCAII the difference is significant. C, Mean initial pH_i values for a₂ and a₃ (in 10 μM EZA) are not substantially or significantly different for either eGFP-e1+Tris oocytes or eGFP-e1+ hCAII oocytes. D, (dpH_i/dt)_max is significantly faster in hCA II injected vs. Tris injected oocytes and the activity of the injected hCA II significantly inhibited by EZA. E, The mean initial [Na⁺]_i before each CO₂/${\text{HCO}}_3^{-}$ exposure. For both eGFP-e1+ Tris oocytes and eGFP-e1+hCAII oocytes, the differences between initial [Na⁺]_i at a₁ vs. a₂ are significant, but at a₂ vs. a₃ are smaller and not significant. There are no significant differences in mean initial [Na⁺]_i when comparing points a₁, a₂ or a₃ between eGFP-e1+ Tris oocytes vs. eGFP-e1+hCAII oocytes. F, We record no significant hCA II-mediated differences in d[Na⁺]_i/dt, or G, ΔI_m. In panels C–G, * denotes the measured mean is significantly different from a₂ or period #2, determined by a paired t-test with the Holm-Bonferroni method applied (α = 0.05, m = 2). † indicates that the measured a₂ or period #2 means are significantly different between eGFP-e1+Tris oocytes and eGFP-e1+ hCAII oocytes, as determined by an unpaired t-test with Welch’s correction. α = 0.05 is not adjusted because we perform one unpaired t-test to evaluate the null hypothesis in each case. Statistics Table 7C–I presents the unadjusted p-values and adjusted or unadjusted α values. The numbers in parentheses in panels C–G indicate the number of oocytes for each group with the individual data points used to calculate each mean overlaid as symbols on the bars. Values are mean ± SD in panels C–G.

Figure 8:. Are CA II-dependent changes in G_m or G_NBC seen when NBCe1-A is expressed at low levels?

Mean (±SD) I_m–V_m relationships from eGFP-e1 expressing oocytes injected with either A, 50 nl Tris buffer or B, 50 ng hCA II dissolved in 50 nl Tris buffer, acquired in 10 s steps from the IV_#1 (at the pH_i nadir during the CO₂/${\text{HCO}}_3^{-}$ pre-pulse), IV_#2 (at the pH_i nadir during the 2^nd CO₂/${\text{HCO}}_3^{-}$ exposure) and IV_EZA (at the pH_i nadir during the CO₂/${\text{HCO}}_3^{-}$ + 10 μM EZA exposure) time-points in Fig. 7. Legend keys in panels A and B indicate the symbols used to plot the data from IV_#1, IV_#2 and IV_EZA (Fig. 7). We calculate C, G_m and D, G_NBC from the I_m–V_m relationships in panels A & B. We record no significant differences in G_m or G_NBC pre- and post-hCA II inhibition by the EZA (C & D). Statistics Table 8C & D presents the unadjusted p-values and adjusted or unadjusted α values for all statistical comparisons. The numbers in parentheses in panels C & D indicate the number of oocytes for each group with the individual data points used to calculate each mean overlaid as symbols on the bars. Bar values represent the mean ± SD.

Figure 9:. Does CA purified from bovine erythrocytes and injected into oocytes expressing eGFP-e1 influence function differently from the recombinant hCA II?

eGFP-e1 expressing oocytes are injected with 50 ng bCA and data acquired according to the protocol described in the legend for Fig. 7. A, Representative pH_i, [Na⁺]_i, V_m and I_m traces from a single oocyte. B, Steady state pH_i recovers to a significantly more alkaline pH after a CO₂/${\text{HCO}}_3^{-}$ pre-pulse (a₁ vs. a₂) prior to the initiation of the 2nd CO₂/${\text{HCO}}_3^{-}$ exposure and falls to a significantly more acidic pH_i immediately prior to the initiation of the 3rd CO₂/${\text{HCO}}_3^{-}$ exposure (a₃), following 10 min in 10 μM EZA. C, Perfusion with 10 μM EZA also significantly inhibits the activity of the injected bCA as reported by the mean CO₂-induced (dpH_i/dt)_max, but no significant bCA-mediated differences in D, initial [Na⁺]_i, (d[Na⁺]_i/dt)_max, E, or F, ΔI_m, before each CO₂/${\text{HCO}}_3^{-}$ exposure are recorded. G, The mean (±SD) I_m–V_m relationship from eGFP-e1 expressing oocytes injected with 50 ng bCA. We record no significant differences in H, G_m or I, G_NBC pre- and post-bCA inhibition by the EZA. Paired-samples t-tests compare the differences between each successive CO₂/${\text{HCO}}_3^{-}$ exposure in panels B–F and H and I and the Holm-Bonferroni analyses determine significance (α = 0.05, m = 2). Statistics Table 9B–F and 9H and 9I present the unadjusted p-values and adjusted α’s. The numbers in parentheses in panels B–F and H and I indicate the number of oocytes for each group with the individual data points used to calculate each mean overlaid as symbols on the bars. Values are mean ± SD in panels B–F and H and I.

Figure 10:. Are bCA-dependent changes seen when injected in oocytes expressing non eGFP-tagged NBCe1-A?

A, e1 expressing oocytes injected with 23 nl H₂O or B, 50 ng bCA dissolved in H₂O as described in by B&D are voltage-clamped to V_h = –40 mV and exposed to three periods of 5% CO₂/33 mM ${\text{HCO}}_3^{-}$ using the same protocol as in Fig. 7. C, Mean steady-state initial pH_i before each of the three periods in CO₂/${\text{HCO}}_3^{-}$ buffer. In e1+H₂O oocytes, initial pH_i before the second period in CO₂/${\text{HCO}}_3^{-}$ buffer (a₂) is elevated by ~0.1 pH unit, when compared to the pH_i before the first CO₂/${\text{HCO}}_3^{-}$ exposure (a₁), but not significantly so, and the pH_i before the final CO₂/${\text{HCO}}_3^{-}$ exposure in 10 μM EZA (a₃) is not significantly different from the pH_i before the second CO₂/${\text{HCO}}_3^{-}$ exposure (a₂). In e1+bCA injected oocytes, initial pH_i before the second period in CO₂/${\text{HCO}}_3^{-}$ buffer (a₂) is elevated by ~0.3 pH units and the difference is statistically significant (Statistics Table 10C). After the second CO₂/${\text{HCO}}_3^{-}$ exposure and a 10 min incubation in 10 μM EZA (a₃), the pH_i in e1+bCA oocytes return to a value 0.16 pH units lower than measured at a₂ and 0.1 pH units higher measured at a₁. D, 10 μM EZA significantly inhibits the activity of the injected bCA as reported by the mean rate of CO₂-induced acidification, which prior to EZA incubation is significantly faster in bCA injected oocytes than H₂O injected controls (Statistics Table 10D). E, The initial [Na⁺]_i before each CO₂/${\text{HCO}}_3^{-}$ exposure reported increases incrementally in e1+H₂O oocytes, but none of the increases are significant, nor is the difference in mean initial [Na⁺]_i at time point a₂ when comparing e1+H₂O with e1+bCA injected oocytes (Statistics Table 10E). The incremental increase in initial steady-state [Na⁺]_i before each CO₂/${\text{HCO}}_3^{-}$ exposure in e1+bCA oocytes was significant (Statistics Table 10E). F, d[Na⁺]_i/dt in e1+H₂O oocytes is significantly slower during the first CO₂/${\text{HCO}}_3^{-}$ buffer exposure (#1) than the second (#2) or third (EZA) which are almost identical (Statistics Table 10F). bCA injected e1 expressing oocytes exhibit d[Na⁺]_i/dt that is incrementally slower during each CO₂/${\text{HCO}}_3^{-}$ period, the difference between period #2 and during the EZA incubated CO₂/${\text{HCO}}_3^{-}$ exposure being significant (Statistics Table 10F). G, Peak ΔI_m magnitude does not significantly change for all three periods in CO₂/${\text{HCO}}_3^{-}$ buffer in e1+H₂O oocytes, but a delta ΔI_m peak magnitude following 10 min perfusion with 10 μM EZA is significantly less than that the previous #2 CO₂/${\text{HCO}}_3^{-}$ exposure (Statistics Table 10G). In panels C–G, paired-samples t-tests compare the differences between the means for each parameter measured during each successive CO₂/${\text{HCO}}_3^{-}$ exposure on either e1+ H₂O injected or e1+bCA injected oocytes (significance determined by the Holm-Bonferroni method and indicated by *, α = 0.05, m = 2). Unpaired t-tests with Welch’s correction compare the means for each measured parameter from e1+ H₂O vs. e1+bCA oocytes (significance indicated by † symbol, α = 0.05 is not adjusted because we perform one unpaired t-test to evaluate the null hypothesis in each case). The numbers in parentheses in panels C–G indicate the number of oocytes for each group with the individual data points used to calculate each mean overlaid as symbols on the bars. Bar values represent the mean ± SD.

Figure 11:. Are bCA-dependent changes in G_m or G_NBC seen when injected in oocytes expressing non eGFP-tagged NBCe1-A?

Mean (±SD) I_m–V_m relationships from e1 expressing oocytes injected with either A, 23 nl H₂O buffer or B, 50 ng bCA dissolved in 23 nl H₂O. As in Fig. 8, I_m–V_m relationships for e1+H₂O and e1+bCA injected oocytes are acquired at 3 time-points; IV_#1, IV_#2, and IV_EZA (see panels Fig. 10A & B). The differences in C, G_m or D, G_NBC calculated from IV_#1 or IV_#2 from e1+H₂O vs. e1+bCA oocytes are not significant. The differences pre- and post-EZA incubation from e1+H₂O or e1+bCA oocytes (#2 vs. EZA) are also not significant. This indicates that neither the presence of bCA nor inhibition of its activity by EZA directly influences G_NBC. Statistics Table 11C & D presents the unadjusted p-values and adjusted or unadjusted α values for all statistical comparisons. The numbers in parentheses in panels C & D indicate the number of oocytes for each group with the individual data points used to calculate each mean overlaid as symbols on the bars. Bar values, represent the mean ± SD.

Figure 12:. bCA injected in to the cytoplasm does not enhance NBCe1-A slope conductance (G_NBC)

A, I_m–V_m relationships from oocytes injected 4 days before recording with 1.5 ng cRNA for e1 and then injected again 1 day before recording with 23 nl H₂O (Top) or 50 ng bCA in 23 nl H₂O (Bottom). I–V relationships are acquired as described in Fig. 2A, using a separate group of oocytes for each of the three step duration protocols (60 ms, left; 1 s, center; or 30 s, right). B, G_NBC calculated from the I_m–V_m relationships in panel A, before and after the oocytes are incubated for 3 h in 400 μM EZA to inhibit the activity of the injected bCA. The number of replicates for each group is indicated at the base of each bar. Unpaired-samples t-tests with Welch’s correction are performed to compare the effect of increasing the I–V step duration from 60 ms to 1 s, and 1 s to 30 s († denotes significance vs. 60 ms protocol, ‡ denotes significance vs. 1 s protocol). We perform paired-samples t-tests to compare the differences in G_NBC pre- and post-EZA incubation. Although we observe significant differences in G_NBC as a factor of I–V protocol step duration, the 3 h EZA incubation did not result in significantly different G_NBC in H₂O or bCA injected e1 expressing oocytes for any of the I–V step durations employed. The Holm-Bonferroni method is applied to determine significance in all tests (α = 0.05, m=3). Statistics Table 12 reports the unadjusted p-values and corrected α values. C, CO₂-induced acidification of a representative oocyte expressing e1 and injected 24 h earlier with 50 ng bCA. We obtain the black pre-EZA and the gray post-3h EZA record from the same oocyte. D, Mean (dpH_i/dt)_max for oocytes expressing e1 and injected with 23 nl H₂O (white bar), or oocytes expressing e1 and injected with 50 ng bCA pre-EZA incubation (black bar) then post-EZA incubation (gray bar). Unpaired-samples t-test with Welch’s correction assess the significance of the differences between e1+H₂O versus e1+bCA injected oocytes († denotes significance). A paired-samples t-test assesses the significance of the reduction in (dpH_i/dt)_max for e1+bCA injected oocytes pre- and post EZA incubation (* denotes significance). α = 0.05 is not adjusted because we perform one unpaired or one paired t-test to evaluate the null hypothesis in each case. Statistics Table 12 reports the unadjusted p-values. The numbers in parentheses in panels B and D indicate the number of oocytes for each group. Values are mean ± SD in panels A, B and D, with the individual data points used to calculate each mean represented by open symbols in panels B and D.

Figure 13:. Does the N-terminal eGFP tag inhibit functional upregulation of NBCe1-A by hCA II?

We inject e1 expressing oocytes with 50 ng hCA II and data is acquired according to the protocol described in the legend for Fig. 7 to assess whether in the hCA II preparation can upregulate NBCe1-A function if the transporter lacks the eGFP tag on the N-terminus. A, Representative pH_i, V_m, I_m, and [Na⁺]_i traces from a single oocyte. B, Steady-state initial pH_i at a₂ is significantly more alkaline than at a₁ and at point a₃, following 10 min in 10 μM EZA. C, Perfusion with 10 μM EZA also significantly inhibits the activity of the injected hCA II as reported by the mean rate of CO₂-induced acidification (#2 vs. 10 μM EZA), but we record no significant CA-mediated differences in initial D, [Na⁺]_i, E, d[Na⁺]_i/dt, or F, ΔI_m. G, I–V relationships (legend key indicates symbols used to plot data from IV_#1, IV_#2 and IV_EZA recorded in each bath solution) determine that H, G_m or I, G_NBC are not significantly different pre- and post-hCA II inhibition by the EZA. Paired-samples t-tests compare the differences between each successive CO₂/${\text{HCO}}_3^{-}$ exposure in panels B–F, H and I. Holm-Bonferroni analyses determine significance (α = 0.05, m = 2). Unadjusted p-values and adjusted α are presented in Statistics Table 13B–F, H and I. * denotes significance compared to point a₂ in panels B and D or compared to period #2 in panels C, E–F and H–I. The numbers in parentheses in panels B–F, H and I indicate the number of oocytes for each group. Values are mean ± SD in panels B–I, with the individual points used to calculate each mean shown by each bar in panels B–F and panels H and I represented by open symbols.

Figure 14:. Injected hCA II significantly enhances the CO₂-induced increase in d[Na⁺]_i/dt in H₂O injected oocytes

Recordings from control oocytes injected 4 days prior to recording with 9.2 nl H₂O (no NBCe1-A) and 1 day before recording with either A, 50 nl Tris buffer or B, 50 ng hCA II in 50 nl Tris observing the same protocol as in Fig. 7. V_h is clamped at –40 mV and oocytes are exposed to three periods of 5% CO₂/33 mM ${\text{HCO}}_3^{-}$ (gray shading and “CO₂/${\text{HCO}}_3^{-}$” label). Prior to the 3^rd CO₂/${\text{HCO}}_3^{-}$ delivery, the oocyte is perfused for 10 min with 10 μM EZA to inhibit CA II activity (cyan shading and “10 μM EZA” label). C, Although not expressing NBCe1-A, pH_i always recovers to a more alkaline pH after each CO₂/${\text{HCO}}_3^{-}$ exposure in hCA II injected oocytes (points a₁ vs. a₂ vs. a₃). D, hCA II injected oocytes acidify significantly faster than Tris injected oocytes, and this activity is inhibited by 10 μM EZA to a rate similar to that from the Tris injected oocytes. E, The initial [Na⁺]_i is larger in hCA II injected oocytes than Tris injected oocytes, but the differences are not significant. However, the increase in initial [Na⁺]_i before each successive CO₂/${\text{HCO}}_3^{-}$ exposure in both Tris and hCA II injected oocytes is significant. F, EZA inhibition of hCA II activity results in a significant decrease in the d[Na⁺]_i/dt for cells injected with hCA II, not observed in the Tris injected cells. I–V relationships are acquired during the recording of the data exampled in panels A and B for H₂O injected oocytes plus 50 nl Tris and H₂O injected oocytes plus 50 ng hCA II. G, The mean G_m for each condition is plotted in panel. We perform paired-samples t-tests to compare the differences between each successive CO₂/${\text{HCO}}_3^{-}$ exposure in panels C–G (* denotes a significant difference from point a₂ or period #2). Holm-Bonferroni analyses determine significance (α = 0.05, m = 2). † indicates that the measured a₂ or period #2 means are significantly different between H₂O + Tris vs. H₂O + 50 ng hCA II oocytes, as determined by an unpaired t-test with Welch’s correction. α = 0.05. Statistics Table 14C–H reports the unadjusted p-values and adjusted or unadjusted α values in for all analyses. The numbers in parentheses in panels C-G indicate the number of oocytes for each group. Bar values are mean ± SD in panels C–G with the individual points used to calculate each mean represented by solid symbols for H₂O + Tris oocytes and open symbols for H₂O + hCAII oocytes.

Figure 15:. Injected bCA significantly enhances the CO₂-induced increase in d[Na⁺]_i/dt in H₂O injected oocytes

Recordings from control oocytes injected 4 days prior to recording with H₂O (no NBCe1-A) and 1 day before recording with either A, 23 nl H₂O or B, 50 ng bCA in 23 nl H₂O observing the same protocol as in Fig. 7 and Fig. 14. C, The initial pH_i immediately before successive CO₂/${\text{HCO}}_3^{-}$ periods is not significantly different in the H₂O injected oocytes. In bCA injected oocytes the initial pH_i is significantly increased at point a₂ vs. a₁ but is not significantly different between points a₂ and a₃. D, bCA injected oocytes acidify significantly faster than H₂O injected oocytes, and this activity is significantly inhibited by 10 μM EZA. E, [Na⁺]_i significantly increases in bCA injected oocytes but not in H₂O injected oocytes after each period in CO₂/${\text{HCO}}_3^{-}$ solution and we observe during EZA perfusion a significant decrease in the F, d[Na⁺]_i/dt for cells injected with bCA, but not those injected with H₂O. We acquire I–V relationships during the recording of the data exampled in panels A and B for H₂O injected oocytes plus 50 nl H₂O and H₂O injected oocytes plus 50 ng bCA. G, We measure no significant differences in G_m between successive periods in CO₂/${\text{HCO}}_3^{-}$ or between H₂O + H₂O vs. H₂O + bCA injected oocytes. For panels C–G, * denotes a significant difference from point a₂ or period #2 by paired-samples t-test. Holm-Bonferroni analyses determine significance (α = 0.05, m = 2). † indicates that the measured a₂ or period #2 means are significantly different between H₂O + H₂O vs. H₂O + 50 ng bCA oocytes, as determined by an unpaired t-test with Welch’s correction (α = 0.05). Statistics Table 15C-G presents the unadjusted p-values and adjusted or unadjusted α values or all analyses. The numbers in parentheses in panels C-G indicate the number of oocytes for each group. Bar values are mean ± SD in panels C-G, with the individual points used to calculate each mean represented by solid symbols for H₂O + H₂O oocytes and open symbols for H₂O + bCA oocytes.

Figure 16:. The activity of the endogenous Xenopus oocyte Na-H exchanger (XL-NHE) is modulated in a dose dependent manner by CA

Recordings from control oocytes injected 4 days prior to recording with 9.2 nl H₂O (no NBCe1-A) and 1 day before recording with either A, 50 nl 50 mM Tris buffer B, 50 ng hCA II or C, 300 ng hCA II dissolved in 50 nl Tris buffer or D, 23 nl H₂O, E, 50 ng bCA, or F, 300 ng bCA dissolved in 23 nl H₂O. Cells are acidified by switching from ND96 to 5% CO₂/33 mM ${\text{HCO}}_3^{-}$. After 5 min, 50 µM EIPA is introduced to the 5% CO₂/33 mM ${\text{HCO}}_3^{-}$ solution in the bath. Panel B displays a 10 min ND96 washout step, followed by a second CO₂-induced acidification to show that the EIPA block is reversible. In panel C, EIPA block reverses when we switch the bath solution back to 5% CO₂/33 mM ${\text{HCO}}_3^{-}$. In panel F, we also present a second application of 50 µM EIPA to demonstrate that the increase in [Na⁺]_i on starting the second CO₂-induced acidification is still attributable to XL-NHE. G, Both hCA II and bCA significantly enhance (dpH_i/dt)_max during CO₂-induced acidifications but the magnitude of the enhancement is not significantly different between oocytes injected with either 50 ng or 300 ng of hCA II or bCA (Statistics Table 16G). H, Both hCA II and bCA enhance (d[Na⁺]_i/dt)_max in a concentration dependent manner. The Na⁺-influx could be completely inhibited by 50 µM EIPA in all cases. For panels G & H, † indicates that the measured means are significantly different between H₂O or Tris injected oocytes vs. 50 ng hCA II and 300 ng hCA II injected oocytes, or vs. 50 ng bCA and 300 ng bCA respectively as determined by an unpaired t-test with Welch’s correction. ‡ indicates a significant difference between the mean for 50 ng vs. 300 ng from hCA II or bCA injected oocytes. Holm-Bonferroni analyses determine significance (α = 0.05, m = 3, Statistics Table 16H part I). For panel H, paired-samples t-test determine the significance of the differences between mean (d[Na⁺]_i/dt)_max pre- and post EIPA perfusion in each injection. * denotes a significant difference as determined by the Holm-Bonferroni method (α = 0.05, m = 6, Statistics Table 16H part II). The numbers in parentheses in panel G and H indicate the number of oocytes for each group. Bar values are mean ± SD in panel G & H with the individual points used to calculate each mean represented by solid symbols for +Tris or +H₂O oocytes and open symbols for +hCA II or + bCA oocytes.

Figure 17:. Inhibition of the Xenopus oocyte Na-H exchanger (XL-NHE) eliminates bCA dependent changes in NBCe1-A current

e1 oocytes are injected with 50 ng bCA and data acquired according to the protocol described in the legend for Fig. 7 and in addition, 50 μM EIPA is continuously perfused to inhibit endogenous XL-NHE activity during data acquisition. A, Representative pH_i, V_m, I_m, and [Na⁺]_i traces from a single oocyte. B, Steady-state initial pH_i returns to a significantly more alkaline pH prior to the initiation of the 2^nd CO₂/${\text{HCO}}_3^{-}$ exposure (a₁ vs. a₂). After the second CO₂/${\text{HCO}}_3^{-}$ exposure and a 10 min incubation in 10 μM EZA, the steady-state initial pH_i in e1+bCA oocytes settles at value significantly lower than prior to the second CO₂/${\text{HCO}}_3^{-}$ exposure (a₂ vs. a₃). C, Perfusion with 10 μM EZA also significantly inhibits the activity of the injected bCA as reported by the mean rate of CO₂-induced acidification, but no significant bCA-mediated differences in initial D, [Na⁺]_i E, d[Na⁺]_i/dt or F, ΔI_m are recorded. G, I_m−V_m relationships (legend key indicates symbols used to plot data from IV_#1, IV_#2 and IV_EZA recorded in each bath solution) determined that no significant differences in H, G_m or I, G_NBC are recorded pre- and post-bCA inhibition by the EZA. Paired-samples t-tests compare the differences between each successive CO₂/${\text{HCO}}_3^{-}$ exposure in panels B–F, H and I. * denotes a significant difference from point a₂ or period #2. Holm-Bonferroni analyses determine significance (α = 0.05, m = 2). Statistics Table 17B–F, H & I present the unadjusted p-values and adjusted α values for all analyses. The numbers in parentheses in panels B–F, H and I indicate the number of oocytes for each group. Values are mean ± SD in panels B–I.