Quantitation of transmembrane CO₂ flux via AQP5 and NBCe1 in a neutral‐buoyancy assay

Abstract

The discoveries of CO₂-impermeable membranes in gastric glands; the CO₂-permeability of the membrane protein aquaporin 1 (AQP1), the first “gas channel”; and of “CO₂-blocking” proteins incorporated into artificial membranes show that Overton's rule—namely, membrane permeability of solute X depends uniquely on the lipid solubility of X—is not universal. Various AQPs conduct CO₂ in differing proportions, in part through their hydrophilic monomeric pores, and in part via the hydrophobic central pore of the tetramer. Rhesus (Rh) proteins also can conduct CO₂. Our laboratory has identified a 3rd class of CO₂-conducting proteins that lack a classical pore. NBCe1-A (SLC4A4) cotransports Na+ and CO3=. Using out-of-equilibrium CO₂/HCO₃⁻ solutions and electrophysiological approaches with Xenopus oocytes, we previously reported that NBCe1-A also conducts CO₂, but only in the presence of HCO₃⁻. We speculate that transient pathways within the NBCe1 molecule arise during Na+/CO3= cotransport, allowing CO₂ to pass through the protein. To elucidate the mechanism of transmembrane CO₂ flux via AQPs and NBCe1-A, we modified our Neutral Buoyancy Assay (NBA), originally developed to assess transmembrane N₂ fluxes. We inject a precise volume of N₂ gas (number of gas molecules = nGas) into a Xenopus oocyte, which we transfer to a saline-containing tube. We then increase the pressure (PNB) in the air phase above the air-water interface so that it is just sufficient to collapse the injected bubble enough to make the oocyte neutrally buoyant, 5 cm depth below the meniscus. As N₂ exits the bubble and ultimately diffuses into the extracellular fluid (ECF), the bubble shrinks, cell density increases, and the oocyte sinks. A camera/computer system detects this sinking and decreases PNB to re-establish neutral buoyancy at 5 cm depth. PNB decays exponentially over 1000 s. Calibrations allow us to compute the ΔnGas time course, and thus gas efflux. We modified the NBA to quantify gas influx by measuring bubble inflation. When we raise [N₂] in the ECF from [N₂]o = 0.56 mM (room air at 1×ATA) to [N₂]o = 2.06 mM (pre-equilibrating saline with 93% N₂/7% O₂ at 3×ATA) at constant [O₂]o = 0.26 mM, N₂ enters the cell during the NBA and nGas rises. If we further increase [N₂]o to 2.81 mM at fixed [O₂]o (pre-equilibrating saline with 95% N₂/5% O₂ at 4×ATA), ΔnGas over 1000 s increases more. To perform the inflation assay with CO₂ (the high solubility of which translates to a low tendency to inflate the bubble), we expose the oocyte to 100% CO₂/95 mM HCO₃⁻/pHo 6.66. nGas increases rapidly as CO₂ rushes into the cell, and peaks at <200 s, after which N₂ exit dominates and nGas decreases. Compared to control oocytes (injected with H2O rather than cRNA), those expressing AQP5 or NBCe1-A have a significantly greater maximal rate of nGas increase (i.e., CO₂ influx). The peak ΔnGas from AQP5 or NBCe1-A oocytes is also larger and more delayed. Thus, we identify a novel approach that confirms that both AQP5 and NBCe1-A conduct CO₂. Whereas we developed the NBA specifically to measure N₂ fluxes, here we demonstrate the adaptability of this method for measuring transmembrane fluxes of other gases.