Investigation of the Intracellular‐pH (pHi) Dependence of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1‐A
Authors: Occhipinti Rossana , Boron Walter F. 
Published: June 2021

Maintaining intracellular pH (pHi) within a narrow range is essential for many cellular processes to occur. The fine‐tuning of pHi is the result of the simultaneous action of a variety of cellular pH buffers, that tend to minimize changes in pHi, and transporters that adjust their rates to move acid‐base equivalents across the plasma membrane, tending to stabilize pHi. The electrogenic Na/HCO3 cotransporter (NBCe1), by moving HCO3− into or out of a cell, is a major player in pHi regulation. In addition, NBCe1 often plays a central role in transepithelial HCO3− transport. The purpose of the present study is to investigate the pHi‐dependence of NBCe1‐A, the variant of NBCe1 mainly expressed in the renal proximal tubule. We inject Xenopus oocytes with H2O (control) or cRNA encoding NBCe1‐A. A few days later, we use ion‐selective microelectrodes and two‐electrode‐voltage clamping to simultaneously monitor pHi and the ionic current carried by NBCe1‐A (INBC). We take advantage of out‐of‐equilibrium (OOE) CO2/HCO3− solutions to keep extracellular [Na+] ([Na+]o), pHo, and [HCO3−]o fixed as we change [CO2]o from (a) 0% (“pure” HCO3−) to (b) 2.5% (‘OOE1’), or (c) 5% (standard equilibrated solution, ‘EQ’), or (d) 10% (‘OOE2’), or (e) 20% (‘OOE3’). The use of OOE solutions containing different [CO2]o allows us to manipulate the nadir value that pHi reaches after CO2 equilibration (i.e., the higher the [CO2]o, the lower the pHi after CO2 equilibration). Our experimental protocol is the following. After obtaining a first set of voltage‐clamp recordings (IV#1) in the continuous presence of ND96 (CO2/HCO3− free), we switch the superfusion solution from ND96 to “pure” HCO3− (0% CO2/33 mM HCO3−/pH 7.50). For an oocyte expressing NBCe1‐A, the entry of HCO3− into the cell via NBCe1‐A causes pHi to rise and the membrane potential (Vm) to become more negative. As pHi reaches ~7.50, we obtain a second set of voltage‐clamp recordings (IV#2). Following IV#2, we switch the superfusion solution from “pure” HCO3− to a CO2‐containing solution (‘OOE1’, ‘EQ’, ‘OOE2’ or ‘OOE3’). After ~5 min, once pHi reaches its nadir and before the typical pHi recovery caused by HCO3− entry into the cell, we obtain a third set of voltage‐clamp recordings (IV#3). For a H2O‐injected oocyte, which has no appreciable pathways for HCO3− movements through the plasma membrane, exposure to “pure” HCO3− causes only a very slow and tiny fall in pHi, due to a tiny amount of contaminating CO2 in the “pure” HCO3− solution, and no hyperpolarization; exposure to a CO2‐containing solution causes pHi to decay. For each experiment, we calculate the HCO3−‐dependent conductance (G) as an index of NBCe1‐A activity. We account for different NBCe1‐A expression levels, by normalizing G in CO2‐containing solution (G from IV#3) to G in “pure” HCO3− (G from IV#2). Analysis of normalized G versus pHi shows no correlation between these two quantities. Thus, we reach the surprising conclusion that NBCe1‐A is not pHi sensitive in the pH range of our experiments (i.e., 6.70–7.50).Support or Funding InformationSupported by NIH K01‐DK107787 to RO and NIH R01‐DK113197, ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFBThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.