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

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