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

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.