Characterization of Sodium Bicarbonate Transporters NBCe1 and NBCn1 as CO2 channels

Abstract

The known “gas channels” are members of the AQP and Rh families, and appear to conduct CO2 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 CO2 through cracks that form transiently as the protein cycles through various conformations—much like H2O can leak through certain membrane proteins. To identify CO2 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 CO2/HCO3− solutions. Cytosolic CAII promotes the consumption of incoming CO2, thereby maintaining a low [CO2] at the inner surface of the cell membrane, and maximizing the gradient for CO2 influx. A semiquantitative measure of this CO2 influx is the spiking increase in surface pH (DpHS) that occurs as CO2 enters the cell, lowers [CO2] at the outer surface of the cell membrane, causes some HCO3− in the vicinity of the membrane to undergo the reaction HCO3− + H+ ® H2O + CO2, and thereby replenishes some of the lost CO2. The pHS spike is greatest near time‐zero, when the inward CO2 gradient—and thus the reaction HCO3− + H+ ® H2O + CO2—is greatest, and then exponentially wanes as CO2 equilibrates across the cell membrane. Oocytes expressing AQP5 and then injected with Tris buffer (but not CAII) have a much greater DpHS than controls injected with H2O (not cRNA) and Tris (not CAII), confirming that AQP5 is an excellent CO2 channel. Injecting a small amount of CAII (dissolved in Tris) into oocytes not expressing AQP5 produces only a slight increase in DpHS because the rate‐limiting step is membrane CO2 permeability. However, for AQP5‐expressing oocytes, injecting this same small amount of CAII produces a very large increase in DpHS. In other words, the CAII‐dependent change in spike height—D(DpHS)—is much greater in AQP5‐expressing oocytes than H2O‐injected controls. We conclude that the measured D(DpHS) in ±CAII experiments is a powerful tool for assessing CO2 conductance. We applied this method to measuring the CO2 conductance of Na/HCO3 co‐transporters, NBCe1‐A and NBCn1‐G. Preliminary data show that, in the presence of cytosol CAII, D(DpHS) is >1.5‐fold greater in the oocytes expressing NBCe1‐A than the H2O‐injected controls, supporting the hypothesis that NBCe1‐A has a CO2 conductance. Similarly, D(DpHS) is >5‐fold greater in the oocytes expressing NBCn1‐G than the H2O‐injected controls, supporting the hypothesis that NBCn1‐G has a CO2 conductance.Support or Funding InformationSupported by grants GM111251 (the U grant) and N000141612535 (the MURI grant)