Characterization of Sodium Bicarbonate Transporters NBCe1 and NBCn1 as CO 2 channels

The known “gas channels” are members of the AQP and Rh families, and appear to conduct CO 2 mainly through the stable hydrophobic central pores of these multimeric proteins. The present work helps to define a new class of gas‐conduction pathway: transporters that may conduct CO 2 through cracks that form transiently as the protein cycles through various conformations—much like H 2 O can leak through certain membrane proteins. To identify CO 2 channels, we developed a novel tool—expressing AQP5 in Xenopus oocytes, followed by injection of small amounts of carbonic anhydrase II (CAII), and then exposure of the cell to CO 2 /HCO 3 − solutions. Cytosolic CAII promotes the consumption of incoming CO 2 , thereby maintaining a low [CO 2 ] at the inner surface of the cell membrane, and maximizing the gradient for CO 2 influx. A semiquantitative measure of this CO 2 influx is the spiking increase in surface pH (DpH S ) that occurs as CO 2 enters the cell, lowers [CO 2 ] at the outer surface of the cell membrane, causes some HCO 3 − in the vicinity of the membrane to undergo the reaction HCO 3 − + H + ® H 2 O + CO 2 , and thereby replenishes some of the lost CO 2 . The pH S spike is greatest near time‐zero, when the inward CO 2 gradient—and thus the reaction HCO 3 − + H + ® H 2 O + CO 2 —is greatest, and then exponentially wanes as CO 2 equilibrates across the cell membrane. Oocytes expressing AQP5 and then injected with Tris buffer (but not CAII) have a much greater DpH S than controls injected with H 2 O (not cRNA) and Tris (not CAII), confirming that AQP5 is an excellent CO 2 channel. Injecting a small amount of CAII (dissolved in Tris) into oocytes not expressing AQP5 produces only a slight increase in DpH S because the rate‐limiting step is membrane CO 2 permeability. However, for AQP5‐expressing oocytes, injecting this same small amount of CAII produces a very large increase in DpH S . In other words, the CAII‐dependent change in spike height—D(DpH S )—is much greater in AQP5‐expressing oocytes than H 2 O‐injected controls. We conclude that the measured D(DpH S ) in ±CAII experiments is a powerful tool for assessing CO 2 conductance. We applied this method to measuring the CO 2 conductance of Na/HCO 3 co‐transporters, NBCe1‐A and NBCn1‐G. Preliminary data show that, in the presence of cytosol CAII, D(DpH S ) is >1.5‐fold greater in the oocytes expressing NBCe1‐A than the H 2 O‐injected controls, supporting the hypothesis that NBCe1‐A has a CO 2 conductance. Similarly, D(DpH S ) is >5‐fold greater in the oocytes expressing NBCn1‐G than the H 2 O‐injected controls, supporting the hypothesis that NBCn1‐G has a CO 2 conductance. Support or Funding Information Supported by grants GM111251 (the U grant) and N000141612535 (the MURI grant)