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

<jats:sec><jats:title>Key points</jats:title><jats:p><jats:list list-type="bullet"> <jats:list-item><jats:p>We previously reported that the phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP<jats:sub>2</jats:sub>) directly stimulates heterologously expressed electrogenic Na<jats:sup>+</jats:sup>/bicarbonate cotransporter NBCe1‐A in an excised macropatch from the <jats:italic>Xenopus</jats:italic> oocyte, and indirectly stimulates NBCe1‐B and ‐C in the intact oocyte primarily through inositol 1,4,5‐trisphosphate/Ca<jats:sup>2+</jats:sup>.</jats:p></jats:list-item> <jats:list-item><jats:p>In the current study, we expand on a previous observation that PIP<jats:sub>2</jats:sub> may also directly stimulate NBCe1 in the intact oocyte.</jats:p></jats:list-item> <jats:list-item><jats:p>In this study on oocytes, we co‐expressed either NBCe1‐B or ‐C and a voltage‐sensitive phosphatase (VSP), which depletes PIP<jats:sub>2</jats:sub> without changing inositol 1,4,5‐trisphosphate, and monitored NBCe1‐mediated currents with the two‐electrode voltage‐clamp technique or pH<jats:sub>i</jats:sub> changes using <jats:italic>V</jats:italic><jats:sub>m</jats:sub>/pH‐sensitive microelectrodes.</jats:p></jats:list-item> <jats:list-item><jats:p>Activating VSP inhibited NBCe1‐B and ‐C outward currents and NBCe1‐mediated pH<jats:sub>i</jats:sub> increases, and changes in NBCe1 activity paralleled changes in surface PIP<jats:sub>2</jats:sub>.</jats:p></jats:list-item> <jats:list-item><jats:p>This study is a quantitative assessment of PIP<jats:sub>2</jats:sub> itself as a regulator of NBCe1‐B and ‐C in the intact cell, and represents the first use of VSP to characterize the PIP<jats:sub>2</jats:sub> sensitivity of a transporter.</jats:p></jats:list-item> <jats:list-item><jats:p>These data combined with our previous work demonstrate that NBCe1‐B and ‐C are regulated by two PIP<jats:sub>2</jats:sub>‐mediated signalling pathways. Specifically, a decrease in PIP<jats:sub>2</jats:sub> <jats:italic>per se</jats:italic> can inhibit NBCe1, whereas hydrolysis of PIP<jats:sub>2</jats:sub> to inositol 1,4,5‐trisphosphate/Ca<jats:sup>2+</jats:sup> can stimulate the transporter.</jats:p></jats:list-item> </jats:list></jats:p></jats:sec><jats:sec><jats:title>Abstract</jats:title><jats:p>The electrogenic Na<jats:sup>+</jats:sup>/bicarbonate cotransporter (NBCe1) of the <jats:italic>Slc4</jats:italic> gene family is a powerful regulator of intracellular pH (pH<jats:sub>i</jats:sub>) and extracellular pH (pH<jats:sub>o</jats:sub>), and contributes to solute reabsorption and secretion in many epithelia. Using <jats:italic>Xenopus laevis</jats:italic> oocytes expressing NBCe1 variants, we have previously reported that the phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP<jats:sub>2</jats:sub>) 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 (InsP<jats:sub>3</jats:sub>)/Ca<jats:sup>2+</jats:sup>. 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 PIP<jats:sub>2</jats:sub> to characterize the PIP<jats:sub>2</jats:sub> sensitivity of NBCe1‐B and ‐C in whole oocytes by co‐expressing a voltage‐sensitive phosphatase (VSP) that decreases PIP<jats:sub>2</jats:sub> and bypasses the InsP<jats:sub>3</jats:sub>/Ca<jats:sup>2+</jats:sup> pathway. An oocyte depolarization that activated VSP only transiently stimulated the NBCe1‐B/C current, consistent with an initial rapid depolarization‐induced NBCe1 activation, and then a subsequent slower VSP‐mediated NBCe1 inhibition. Upon repolarization, the NBCe1 current decreased, and then slowly recovered with an exponential time course that paralleled PIP<jats:sub>2</jats:sub> resynthesis as measured with a PIP<jats:sub>2</jats:sub>‐sensitive fluorophore and confocal imaging. A subthreshold depolarization that minimally activated VSP caused a more sustained increase in NBCe1 current, and did not lead to an exponential current recovery following repolarization. Similar results were obtained with oocytes expressing a catalytically dead VSP mutant at all depolarized potentials. Depleting endoplasmic reticulum Ca<jats:sup>2+</jats:sup> did not inhibit the NBCe1 current recovery following repolarization from VSP activation, demonstrating that changes in InsP<jats:sub>3</jats:sub>/Ca<jats:sup>2+</jats:sup> were not responsible. This study demonstrates for the first time that depleting PIP<jats:sub>2</jats:sub> <jats:italic>per se</jats:italic> inhibits NBCe1 activity. The data in conjunction with previous findings implicate a dual PIP<jats:sub>2</jats:sub> regulatory pathway for NBCe1 involving both PIP<jats:sub>2</jats:sub> itself and generated InsP<jats:sub>3</jats:sub>/Ca<jats:sup>2+</jats:sup>.</jats:p></jats:sec>