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

Key points The Na+bicarbonate cotransporter NBCe1 regulates cell and tissue pH, as well as ion movement across cell layers in organs such as kidney, gut, and pancreas. We previously showed that the signalling molecule PIP2 stimulates the cloned A variant of NBCe1 in a patch of biological membrane. In the current study, we characterize the effect of injecting PIP2 into intact oocytes expressing an NBCe1 variant (A, B, or C). PIP2 stimulates the B and C variants, but not the A variant, through hydrolysis to IP3. Stimulation requires an intracellular Ca2+ store and kinase activity. The results will contribute to our understanding of multiple HCO3--dependent transporters with different modes of regulation, as well as how molecules that stimulate specific membrane receptors lead to changes in cell/tissue pH, and perhaps how pathologies such as stroke and ischaemia that lead to energy deficiency cause tissue acidosis. Abstract Electrogenic Na+bicarbonate cotransporter NBCe1 variants contribute to pHi 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 (PIP2) 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 PIP2 on the activity of heterologously expressed NBCe1-A, -B, or -C. Injecting PIP2 (10 mu m estimated final) into voltage-clamped oocytes stimulated NBC-mediated, HCO3--induced outward currents by >100% for the B and C variants, but not for the A variant. The majority of this stimulation involved PIP2 hydrolysis and endoplasmic reticulum (ER) Ca2+ release. Stimulation by PIP2 injection was mimicked by injecting IP3, but inhibited by either applying the phospholipase C (PLC) inhibitor U73112 or depleting ER Ca2+ with prolonged thapsigargin/EGTA treatment. Stimulating the activity of store-operated Ca2+ channels (SOCCs) to trigger a Ca2+ influx mimicked the PIP2/IP3 stimulation of the B and C variants. Activating the endogenous Gq protein-coupled receptor in oocytes with lysophosphatidic acid (LPA) also stimulated the B and C variants in a Ca2+-dependent manner, although via an increase in surface expression for the B variant. In simultaneous voltage-clamp and pHi studies on NBCe1-C-expressing oocytes, LPA increased the NBC-mediated pHi-recovery rate from a CO2-induced acid load by 80%. Finally, the general kinase inhibitor staurosporine completely inhibited the IP3-induced stimulation of NBCe1-C. In summary, injecting PIP2 stimulates the activity of NBCe1-B and -C expressed in oocytes through an increase in IP3/Ca2+ that involves a staurosporine-sensitive kinase. In conjunction with our previous macropatch findings, PIP2 regulates NBCe1 through a dual pathway involving both a direct stimulatory effect of PIP2 on at least NBCe1-A, as well as an indirect stimulatory effect of IP3/Ca2+ on the B and C variants.