Phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) stimulates the electrogenic Na/HCO ₃ cotransporter NBCe1-A expressed in Xenopus oocytes

Abstract

Bicarbonate transporters are regulated by signaling molecules/ ions such as protein kinases, ATP, and Ca 2؉ . While phospholipids such as PIP2 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 (؊Vp ‫؍‬ ؊60 mV) containing a low-Na ؉ , nominally HCO 3 ؊ -free solution. Ϫ transporters play important roles in intracellular pH (pH i ) regulation and ion transport in many tissues. Since the expression cloning of the first cDNA encoding a cationcoupled HCO 3 Ϫ transporter (NBCe1) from salamander kidney (1), investigators have cloned by homology and characterized the function of additional electrogenic and electroneutral NBCs, cationcoupled anion exchangers, and associated splice variants (2). NBCe1 has 3 variants (A, B, and C) that differ at their amino and/or carboxyl termini. Na-coupled HCO 3 Ϫ transporters in conjunction with anion exchangers (AEs) are members of a superfamily of bicarbonate transporters (BTs).The pH field is now poised to address new questions regarding the physiologic importance of numerous BTs. We hypothesize that proper pH i regulation and ion transport require multiple BTseach one playing a specific role under different physiologic conditions or regulatory stimuli. Classic signaling molecules, such as PKA/cAMP, PKC, Ca 2ϩ , and ATP, can modulate heterologously expressed NBCe1. Investigators have reported that phosphorylation by activated PKA (3, 4) or an increase in cytosolic Ca 2ϩ (5) can change the Na:HCO 3 Ϫ stoichiometry of the transporter. In addition, both cAMP (3) and ATP (6) appear to stimulate an NBC-mediated current, whereas PKC isoforms inhibit transporter activity (7).Other binding proteins or NBC domains can regulate transporter function. For NBCe1, the unique amino terminus of the A variant stimulates activity, whereas the different amino terminus of the B and C variants inhibits activity (8). Such automodulation may involve additional binding proteins and signaling molecules. Indeed, Shirakabe et al. (9) reported that the IP 3 receptor binding protein released with IP 3 (IRBIT) can stimulate NBCe1-B co-expressed in oocytes by binding to the transporter's amino terminus and masking an autoinhibitory domain.Very little is known about the influence of phospholipids on the activity of BTs. PIP 2 is noteworthy because in addition to its classic role as a precursor of the Ca 2ϩ -mobilizing inositol triphosphate (IP 3 ) and the kinase-activating diacylglycerol (DAG), the phosphoinositide is a signaling molecule that can regulate solute movement (10). In pioneering work, Hilgemann and Ball (11) found that PIP 2 directly stimulates both the cardiac Na-Ca exchanger and K ATP channel. Phosphoino...

Regulators of Slc4 bicarbonate transporter activity

“…The rat-kidney NBCe1-A current displays rundown in a macropatch of Xenopus oocyte membrane that can be inhibited and reversed by directly applying a fast membrane incorporating short-chain PIP 2 (Wu et al, 2009). In addition, NBCe1-A current rundown is inhibited by vanadate and Mg 2+ -free solutions.…”

Molecular Mechanisms and Regulation of Urinary Acidification

“…Mg 2+ causes NBCe1-A rundown in Xenopus oocytes, that may involve a Mg 2+ -dependent phosphatase (5′-lipid phosphatase) which dephosphorylates PIP 2 (797). PIP 2 per se activates NBCe1-A, however the mechanism has not been determined (797). Various systemic and hormonal factors alter the expression and/or function of NBCe1-A.…”

Influence of lipids on protein-mediated transmembrane transport

“…+ /HCO − 3 cotransporter The rat bicarbonate transporter NBCe1-A, a homolog of human SLC4 transporters and member of the Anion Exchanger (AE) family 2.A.32 (Romero et al, 2004;Kurtz et al, 2004;Majumdar and Bevensee, 2010), was shown to be stimulated by PIP 2 (Wu et al, 2009). The mechanism of activation is not known and could either be by direct interaction with a stretch of lysines near the carboxyl terminus or via intermediate signalling molecules, perhaps similar to how inositol 1,4,5-trisphosphate indirectly activates pNBC1 (Shirakabe et al, 2006).…”