Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates the electrogenic Na/HCO3 cotransporter NBCe1-A expressed in Xenopus oocytes.
Bicarbonate transporters are regulated by signaling molecules/ions such as protein kinases, ATP, and Ca(2+). While phospholipids such as PIP(2) can stimulate Na-H exchanger activity, little is known about phospholipid regulation of bicarbonate transporters. We used the patch-clamp technique to study the function and regulation of heterologously expressed rat NBCe1-A in excised macropatches from Xenopus laevis oocytes. Exposing the cytosolic side of inside-out macropatches to a 5% CO(2)/33 mM HCO(3)(-) solution elicited a mean inward current of 14 pA in 74% of macropatches attached to pipettes (-V(p) = -60 mV) containing a low-Na(+), nominally HCO(3)(-)-free solution. The current was 80-90% smaller in the absence of Na(+), approximately 75% smaller in the presence of 200 microM DIDS, and absent in macropatches from H(2)O-injected oocytes. NBCe1-A currents exhibited time-dependent rundown that was inhibited by removing Mg(2+) in the presence or absence of vanadate and F(-) to reduce general phosphatase activity. Applying 5 or 10 microM PIP(2) (diC8) in the presence of HCO(3)(-) induced an inward current in 54% of macropatches from NBC-expressing, but not H(2)O-injected oocytes. PIP(2)-induced currents were HCO(3)(-)-dependent and somewhat larger following more NBCe1-A rundown, 62% smaller in the absence of Na(+), and 90% smaller in the presence of 200 microM DIDS. The polycation neomycin (250-500 microM) reduced the PIP(2)-induced inward current by 69%; spermine (100 microM) reduced the current by 97%. Spermine, poly-D-lysine, and neomycin all reduced the baseline HCO(3)(-)-induced inward currents by as much as 85%. In summary, PIP(2) stimulates NBCe1-A activity, and phosphoinositides are regulators of bicarbonate transporters.
Wu
Jianping
J
Department of Physiology and Biophysics, Nephrology Research and Training Center, Center of Glial Biology in Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
McNicholas
Carmel M
CM
Bevensee
Mark O
MO
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D001692:Biological Transport; D003600:Cytosol / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods; D005260:Female; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D009865:Oocytes / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism / Q000502:physiology*; D029867:Xenopus Proteins / Q000378:metabolism / Q000502:physiology*; D014982:Xenopus laevis / Q000378:metabolism*
D001639:Bicarbonates; D019269:Phosphatidylinositol 4,5-Diphosphate; D010743:Phospholipids; C522009:SLC4A4 protein, Xenopus; D027982:Sodium-Bicarbonate Symporters; D029867:Xenopus Proteins