Exploring the Intracellular‐pH Sensitivity of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1‐B when Co‐expressed with its Cytosolic Binding Partner IRBIT

The electrogenic Na/HCO 3 cotransporter (NBCe1), by transferring HCO 3 − across the cell plasma membrane, plays a major role in regulating intracellular pH (pH i )—a parameter that must be maintained within a tight range to support normal physiological functions. In addition, NBCe1 mediates HCO 3 − movement across epithelial cells. Because of its role in pH i regulation, we ask whether the activity of this transporter depends on pH i . Recently, we addressed this question by investigating the pH i ‐dependence of NBCe1‐A, the variant of NBCe1 that is most abundant in the kidney. Surprisingly, we found that, when expressed in oocytes, the activity of NBCe1‐A is not pH i sensitive in the pH range of 6.70–7.50 (Occhipinti and Boron, 2019 FASEB J , 33 (1): 544.2). Here, we extend the above study to NBCe1‐B, the variant of NBCe1 that is more widespread, but especially abundant in the pancreas. Because the activity of NBCe1‐B is naturally low when expressed by itself, we investigate the pH i ‐dependence of NBCe1‐B when co‐expressed with its cytosolic binding partner IRBIT [inositol trisphosphate (IP 3 )‐receptor binding protein released with IP 3 ], which is known to relieve autoinhibition by binding to the NH 2 ‐terminus. As done previously, we employ ion‐sensitive microelectrodes and two‐electrode voltage clamping to record pH i and NBCe1‐conductance ( G NBC ) on Xenopus oocytes injected with either H 2 O (control) or cRNA encoding NBCe1‐B with IRBIT or super‐IRBIT (which lacks the PP1 binding motif). Again, we use out‐of‐equilibrium (OOE) CO 2 /HCO 3 − solutions to control pH i as we vary bulk extracellular [CO 2 ] ([CO 2 ] o ) and keep [Na + ] o , pH o , and [HCO 3 − ] o constant. After exposing oocytes to ND96 (free of CO 2 and HCO 3 − ) and obtaining a first set of current‐voltage recordings (IV#1), we switch the superfusate to a “pure” HCO 3 − OOE solution (0% CO 2 /33 mM HCO 3 − /pH 7.50). In the case of NBCe1‐expressing oocytes, exposure to “pure” HCO 3 − leads to the entry of HCO 3 − into the cell, thereby raising pH i . Once pH i reaches ~7.50, we take a second set of current‐voltage recordings (IV#2). We then switch the bath solution to an OOE solution containing either 2.5% CO 2 or 20% CO 2 and, once pH i has reached its nadir (lower in the case of 20% CO 2 ), take a third set of current‐voltage recordings (IV#3). For each experiment, we compute G NBC as the slope of the I‐V curve in the presence of HCO 3 − minus the slope of the I‐V curve in the absence of HCO 3 − (i.e., in ND96). We compute G NBC between −20 and +20 mV, where the net NBCe1‐mediated HCO 3 − flux is strongly inward. To account for variability in protein expression levels, we normalize G NBC from IV#3 to G NBC from IV#2. Analysis of normalized G NBC reveals that the activity of NBCe1‐B, whether co‐expressed with IRBIT or with super‐IRBIT, does not change between pH i ~6.60 (20% CO 2 , apparent [HCO 3 − ] i = 16.6 mM) and ~7.60 (0% CO 2 , apparent [HCO 3 − ] i = 0 mM). Assuming that NBCe1‐B is insensitive to CO 2 and (while operating in the inward direction) insensitive to [HCO 3 − ] i , we reach the surprising conclusion that NBCe1‐B—when activated by IRBIT or super‐IRBIT—is independent of pH i . Support or Funding Information Supported by NIH K01‐DK107787 to RO and NIH R01‐DK113197, ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFB