PIP2 hydrolysis stimulates the electrogenic Na+-bicarbonate cotransporter NBCe1-B and -C variants expressed in Xenopus laevis oocytes

Abstract

Electrogenic Na(+)-bicarbonate cotransporter NBCe1 variants contribute to pH(i) regulation, and promote ion reabsorption or secretion by many epithelia. Most Na(+)-coupled bicarbonate transporter (NCBT) families such as NBCe1 contain variants with differences primarily at the cytosolic N and/or C termini that are likely to impart on the transporters different modes of regulation. For example, N-terminal regions of NBCe1 autoregulate activity. Our group previously reported that cytosolic phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulates heterologously expressed rat NBCe1-A in inside-out macropatches excised from Xenopus laevis oocytes. In the current study on whole oocytes, we used the two-electrode voltage-clamp technique, as well as pH- and voltage-sensitive microelectrodes, to characterize the effect of injecting PIP(2) on the activity of heterologously expressed NBCe1-A, -B, or -C. Injecting PIP(2) (10 μM estimated final) into voltage-clamped oocytes stimulated NBC-mediated, HCO(3)(-)-induced outward currents by >100% for the B and C variants, but not for the A variant. The majority of this stimulation involved PIP(2) hydrolysis and endoplasmic reticulum (ER) Ca(2+) release. Stimulation by PIP(2) injection was mimicked by injecting IP(3), but inhibited by either applying the phospholipase C (PLC) inhibitor U73112 or depleting ER Ca(2+) with prolonged thapsigargin/EGTA treatment. Stimulating the activity of store-operated Ca(2+) channels (SOCCs) to trigger a Ca(2+) influx mimicked the PIP(2)/IP(3) stimulation of the B and C variants. Activating the endogenous G(q) protein-coupled receptor in oocytes with lysophosphatidic acid (LPA) also stimulated the B and C variants in a Ca(2+)-dependent manner, although via an increase in surface expression for the B variant. In simultaneous voltage-clamp and pH(i) studies on NBCe1-C-expressing oocytes, LPA increased the NBC-mediated pH(i)-recovery rate from a CO(2)-induced acid load by ∼80%. Finally, the general kinase inhibitor staurosporine completely inhibited the IP(3)-induced stimulation of NBCe1-C. In summary, injecting PIP(2) stimulates the activity of NBCe1-B and -C expressed in oocytes through an increase in IP(3)/Ca(2+) that involves a staurosporine-sensitive kinase. In conjunction with our previous macropatch findings, PIP(2) regulates NBCe1 through a dual pathway involving both a direct stimulatory effect of PIP(2) on at least NBCe1-A, as well as an indirect stimulatory effect of IP(3)/Ca(2+) on the B and C variants.

Figure 1. NBCe1-A, -B, and -C variants are identical except at the amino and/or carboxy termini

Eighty-five N-terminal residues of the B and C variants (grey) replace the unique 41 N-terminal residues of the A variant (black), and 61 C-terminal residues of the C variant (striped) replace the 46 C-terminal residues of the A and B variants (black). The transmembrane region denoted by the dotted lines contains as many as 14 transmembrane domains (Boron et al. 2009; Zhu et al. 2010).

Figure 2. Injecting PIP₂ stimulates the HCO₃⁻-induced outward currents of the B and C variants

A, switching from ND96 to CO₂/HCO₃⁻ elicited NBC-mediated, HCO₃⁻-induced outward currents (a and b) in an NBCe1-C-expressing oocyte voltage clamped at −60 mV. Injecting 100 μm PIP₂ (∼10 μm final concentration) increased the HCO₃⁻-induced outward currents ∼2-fold (c and d), and these currents were inhibited ∼80% by 200 μm DIDS (e). B, a similar experiment to that described in panel A was performed on an NBCe1-C-expressing oocyte, but H₂O instead of PIP₂ was injected. H₂O injection did not alter the HCO₃⁻-induced outward currents. C, in another control experiment on a H₂O-injected null oocyte, injecting PIP₂ did not appreciably stimulate endogenous HCO₃⁻-induced outward currents. D, summary data from panel A- and B-type experiments performed on all three variants, as well as a C variant missing its N-terminal 87 residues (C_ΔN87). ***P ≤ 0.001 for the mean current pre vs. post-injection. n ≥ 4 for each bar.

Figure 3. Injecting PIP₂ stimulates the voltage-dependent HCO₃⁻-induced currents of the B and C variants

A, the mean HCO₃⁻-dependent I–V plots from NBCe1-A-expressing oocytes were similar before injecting PIP₂ (diamonds) and after (squares). DIDS at 200 μm inhibited the currents (triangles). B, the mean HCO₃⁻-dependent currents from NBCe1-C-expressing oocytes at potentials more positive than ∼−100 mV were smaller before injecting PIP₂ (diamonds) than after (squares). DIDS at 200 μm inhibited the currents (triangles). C, the mean HCO₃⁻-dependent I–V plots for C_ΔN87 were similar to those for the A variant (panel A). D, negligible currents were observed in H₂O-injected null oocytes. The upward-shifted I–V plots with DIDS in panels A–C presumably reflect different baseline currents inherent in these experiments designed to minimize the delay between HCO₃⁻-induced currents ± DIDS after PIP₂ injection. For each panel, n = 3 from 1 batch of oocytes, and the data were repeated in a second batch.

Figure 4. Full stimulation by PIP₂ injection of the B and C variants requires PLC activity

A, PIP₂ injection elicited only a modest stimulation of the HCO₃⁻-induced outward current in an oocyte pre-incubated for 30 min in 10 μm of the PLC inhibitor U73122. B, summary data of the mean injected PIP₂-induced percentage NBC stimulation from panel A-type experiments on the three variants. For oocytes pre-incubated in the inactive analogue U73343, subsequent PIP₂ injection had little effect on the A variant, but stimulated the B and C variants by ∼100% (filled bars). Pre-incubating oocytes in U73122 reduced the stimulation by PIP₂ injection of the B and C variants to ∼35%, and increased the stimulation of the A variant by the same extent (open bars). H₂O injection failed to stimulate any of the variants (hatched bars). n ≥ 3 for each bar. Pretx, pretreatment.

Figure 5. Injecting IP₃—even at a low concentration— stimulates the HCO₃⁻-induced outward currents of the B and C variants

A, IP₃ injection stimulated the HCO₃⁻-induced outward currents by ∼3-fold in an NBCe1-C-expressing oocyte. B, summary data of the mean IP₃-induced percentage NBC stimulation from panel A-type experiments on the three variants, as well as C_ΔN87. ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05 for the mean current pre- vs. post-injection. n = 5 for each bar. C, summary data of the mean IP₃-induced percentage NBC stimulation from panel A-type experiments on NBCe1-C-expressing oocytes injected with one of three different concentrations of IP₃. These experiments with different IP₃ injections were performed in Ca²⁺-free solutions containing 1 mm EGTA. n ≥ 5 for each bar.

Figure 6. Co-expressing S68A IRBIT does not inhibit the IP₃-induced stimulation of NBCe1-C

A, as shown on the left, IP₃ injection stimulated the HCO₃⁻-induced outward current by ∼3-fold in an oocyte co-injected with equal amounts of NBCe1-C and S68A IRBIT cRNA. The break in the current trace at −400 nA denotes values below −400 nA not shown. As shown by an immunoblot on the right, an individual oocyte injected with NBCe1-C cRNA expressed ∼130 kDa NBCe1-C (left column), whereas an oocyte co-injected with both cRNAs expressed ∼130 kDa NBCe1-C and ∼60 kDa S68A IRBIT (right column). B, summary data of the mean IP₃-induced percentage NBC stimulation from panel A-type experiments on oocytes expressing NBCe1-C with or without S68A IRBIT (n = 7 for each bar).

Figure 7. Depletion of ER Ca²⁺ blocks the injected PIP₂- and IP₃-stimulated HCO₃⁻-induced currents of the B and C variants

A, pre-incubating an NBCe1-C-expressing oocyte in a 0 Ca²⁺/EGTA solution containing 10 μm thapsigargin (TG) for ∼4 h to deplete ER Ca²⁺ stores eliminated the injected-PIP₂ stimulation of the HCO₃⁻-induced outward current. B, summary data of the mean PIP₂-induced percentage NBC stimulation from panel A-type experiments on the three variants, as well as C_ΔN87 with the oocytes pre-incubated in 0 Ca²⁺/EGTA plus either DMSO (filled bars) or TG (open bars). n ≥ 3 for each bar. C, the 0 Ca²⁺/EGTA/TG pre-incubation also eliminated the injected-IP₃ stimulation of the HCO₃⁻-induced outward current of NBCe1-C. D, summary data of the mean IP₃-induced percentage NBC stimulation from panel C-type experiments on the variants with the oocytes pre-incubated in 0 Ca²⁺/EGTA plus either DMSO (filled bars) or TG (open bars). n ≥ 3 for each bar.

Figure 8. Activating store-operate Ca²⁺ channels stimulates the HCO₃⁻-induced outward currents of the B and C variants

A, the NBCe1-C-expressing oocyte was pre-incubated for ∼4 h in a 0 Ca²⁺/EGTA solution containing 10 μm thapsigargin (TG) and maintained in the 0 Ca²⁺/ETGA solution at the beginning of the experiment. Subsequently returning stimulated the HCO₃⁻-induced outward currents by ∼3-fold compared to the currents in the absence of . These stimulated currents were reversed by removing . B, summary data of the mean percentage NBC stimulation from panel A-type experiments on the three variants, as well as C_ΔN87. ***P ≤ 0.001 and **P ≤ 0.01 for the mean current pre- vs. post-injection. n ≥ 3 for each bar. C, summary of mean single-oocyte chemiluminescence (SOC) from oocytes first pretreated with the 0 Ca²⁺/EGTA/TG solution, and then either mainta-ined in 0 Ca²⁺ (filled bars) or re-exposed to for 5 min (open bars). n ≥ 20 (2 oocyte batches) for each bar.

Figure 9. Staurosporine blocks the IP₃-induced stimulation of NBCe1-C

A, pre-incubating an NBCe1-C-expressing oocyte in ND96 containing 20 μm staurosporine for ∼4 h to inhibit kinase activity eliminated the injected-IP₃ stimulation of the HCO₃⁻-induced outward current. B, summary data of the mean IP₃-induced percentage NBC stimulation from panel A-type experiments from oocytes pretreated with DMSO (filled bar) or staurosporine (open bar). n ≥ 5 for each bar.

Figure 10. Lysophosphatidic acid (LPA) stimulates the HCO₃⁻-induced outward currents of the B and C variants

A, transiently exposing an NBCe1-C-expressing oocyte to 1 μm LPA for ∼15 s stimulated the HCO₃⁻-induced outward currents by ∼2.3-fold. B, summary data of the normalized HCO₃⁻-induced outward currents for variant-expressing oocytes pre-incubated for 5–7 h in DMSO (filled bars) vs. 10 μm BAPTA-AM (open bars). n ≥ 4 for each bar. C, BAPTA-AM pre-incubation inhibited the LPA-stimulated, HCO₃⁻-induced currents in an NBCe1-C-expressing oocyte. D, summary data of the mean LPA-induced percentage NBC stimulation from panel A-type experiments on oocytes pretreated with DMSO (filled bars) or BAPTA-AM (open bars). n ≥ 4 for each bar.

Figure 11. LPA stimulates both a HCO₃⁻-dependent pH_i recovery and outward current in an NBCe1-C-expressing oocyte voltage clamped at −60 mV

A, as shown in the top panel, switching from ND96 to CO₂/HCO₃⁻ elicited a pH_i decrease (a–b) due to CO₂ influx, followed by a recovery (b–c) due to NBC activity. Applying 1 μm LPA for ∼15 s increased the pH_i recovery rate shortly thereafter (c–d) consistent with NBC stimulation. Removing and returning external Na⁺ caused pH_i to decrease and then increase again (d–e–f). Switching back to ND96 caused pH_i to increase (f–g). In the simultaneous current recording, the HCO₃⁻ solution elicited the characteristic NBC-mediated outward current (a′–b′–c′). LPA induced a transient inward current presumably due to Ca²⁺-activated Cl⁻ channel activity, followed by a pronounced outward current (c′–d′) due to NBC stimulation. Removing and returning external Na⁺ reversed and then reinstated the outward current (d′–e′–f′). Switching back to ND96 eliminated the outward current (f′–g′). B, a similar experiment was performed on a H₂O-injected null oocyte. As shown in the upper panel, the CO₂/HCO₃⁻ solution elicited the expected decrease in pH_i (a–b), but no subsequent pH_i recovery (b–c). Subsequent experimental manoeuvres had little effect on pH_i (c–f). As shown in the lower panel, the solutions had little effect on current (a′–g′). C, left pair of bars summarizes the mean dpH_i/dt from segment b–c pH_i recoveries for oocytes expressing NBCe1-C (filled bar) or injected with H₂O (open bar). Right pair of bars summarizes the mean dpH_i/dt obtained 5 min after applying LPA to oocytes expressing NBCe1-C (filled bar) or injected with H₂O (open bar). n ≥ 4 for each bar. ***P < .001, **P < .01, NS = not significant. D, LPA stimulated the NBCe1-C-mediated dpH_i/dt (calculated from panel C data) by ∼80% and current by ∼40%. The downward pH_i spikes elicited by LPA in panels A and B are truncated at the lowest pH_i shown on each y-axis.