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

Academic Article

Abstract

  • Electrogenic Na -bicarbonate cotransporter NBCe1 variants contribute to pH 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 ) 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 on the activity of heterologously expressed NBCe1-A, -B, or -C. Injecting PIP (10 μm estimated final) into voltage-clamped oocytes stimulated NBC-mediated, HCO -induced outward currents by >100% for the B and C variants, but not for the A variant. The majority of this stimulation involved PIP hydrolysis and endoplasmic reticulum (ER) Ca release. Stimulation by PIP injection was mimicked by injecting IP , but inhibited by either applying the phospholipase C (PLC) inhibitor U73112 or depleting ER Ca with prolonged thapsigargin/EGTA treatment. Stimulating the activity of store-operated Ca channels (SOCCs) to trigger a Ca influx mimicked the PIP /IP stimulation of the B and C variants. Activating the endogenous G protein-coupled receptor in oocytes with lysophosphatidic acid (LPA) also stimulated the B and C variants in a Ca -dependent manner, although via an increase in surface expression for the B variant. In simultaneous voltage-clamp and pH studies on NBCe1-C-expressing oocytes, LPA increased the NBC-mediated pH -recovery rate from a CO -induced acid load by ∼80%. Finally, the general kinase inhibitor staurosporine completely inhibited the IP -induced stimulation of NBCe1-C. In summary, injecting PIP stimulates the activity of NBCe1-B and -C expressed in oocytes through an increase in IP /Ca that involves a staurosporine-sensitive kinase. In conjunction with our previous macropatch findings, PIP regulates NBCe1 through a dual pathway involving both a direct stimulatory effect of PIP on at least NBCe1-A, as well as an indirect stimulatory effect of IP /Ca on the B and C variants. © 2012 The Physiological Society. + + - 2+ 2+ 2+ 2+ 2+ 2+ 2+ i 2 2 2 3 2 2 3 2 3 q i i 2 3 2 3 2 2 3
  • Authors

    Digital Object Identifier (doi)

    Author List

  • Thornell IM; Wu J; Liu X; Bevensee MO
  • Start Page

  • 5993
  • End Page

  • 6011
  • Volume

  • 590
  • Issue

  • 23