Phosphatidylinositol 4,5‐bisphosphate degradation inhibits the Na+/bicarbonate cotransporter NBCe1‐B and ‐C variants expressed in Xenopus oocytes

• Published in: The Journal of physiology 2015-02, Vol.593 (3), p.541-558
• Main Authors: Thornell, Ian M., Bevensee, Mark O.
• Format: Article
• Language: English
• Subjects: Animals; Hydrolysis; Molecular and Cellular; Phosphatase; Phosphatidylinositol 4,5-Diphosphate - metabolism; Protein Isoforms - metabolism; Sodium-Bicarbonate Symporters - metabolism; Xenopus
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2014.284307

Key points We previously reported that the phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP2) directly stimulates heterologously expressed electrogenic Na+/bicarbonate cotransporter NBCe1‐A in an excised macropatch from the Xenopus oocyte, and indirectly stimulates NBCe1‐B and ‐C in the intact oocyte primarily through inositol 1,4,5‐trisphosphate/Ca2+. In the current study, we expand on a previous observation that PIP2 may also directly stimulate NBCe1 in the intact oocyte. In this study on oocytes, we co‐expressed either NBCe1‐B or ‐C and a voltage‐sensitive phosphatase (VSP), which depletes PIP2 without changing inositol 1,4,5‐trisphosphate, and monitored NBCe1‐mediated currents with the two‐electrode voltage‐clamp technique or pHi changes using Vm/pH‐sensitive microelectrodes. Activating VSP inhibited NBCe1‐B and ‐C outward currents and NBCe1‐mediated pHi increases, and changes in NBCe1 activity paralleled changes in surface PIP2. This study is a quantitative assessment of PIP2 itself as a regulator of NBCe1‐B and ‐C in the intact cell, and represents the first use of VSP to characterize the PIP2 sensitivity of a transporter. These data combined with our previous work demonstrate that NBCe1‐B and ‐C are regulated by two PIP2‐mediated signalling pathways. Specifically, a decrease in PIP2 per se can inhibit NBCe1, whereas hydrolysis of PIP2 to inositol 1,4,5‐trisphosphate/Ca2+ can stimulate the transporter. The electrogenic Na+/bicarbonate cotransporter (NBCe1) of the Slc4 gene family is a powerful regulator of intracellular pH (pHi) and extracellular pH (pHo), and contributes to solute reabsorption and secretion in many epithelia. Using Xenopus laevis oocytes expressing NBCe1 variants, we have previously reported that the phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP2) directly stimulates NBCe1‐A in an excised macropatch, and indirectly stimulates NBCe1‐B and ‐C in the intact oocyte primarily through inositol 1,4,5‐trisphosphate (InsP3)/Ca2+. In the current study, we used the two‐electrode voltage‐clamp technique alone or in combination with pH/voltage‐sensitive microelectrodes or confocal fluorescence imaging of plasma membrane PIP2 to characterize the PIP2 sensitivity of NBCe1‐B and ‐C in whole oocytes by co‐expressing a voltage‐sensitive phosphatase (VSP) that decreases PIP2 and bypasses the InsP3/Ca2+ pathway. An oocyte depolarization that activated VSP only transiently stimulated the NBCe1‐B/C current, consistent with an initial rap