Investigation of the Intracellular‐pH (pH i ) Dependence of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1‐A

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