Quantitation of transmembrane O 2 flux via RhAG in a neutral‐buoyancy assay

Abstract

The central dogma of transmembrane gas flux had been that all gases cross all membranes by dissolving in the lipid phase of the membrane. Contradicting this simple view are three discoveries: (1) CO2-impermeable membranes; (2) CO2-permeable membrane proteins or “gas-channels”, namely, certain aquaporins (AQPs) and rhesus (Rh) proteins; and (3) a reduction in CO2-permeability when incorporating the CO2-impermeable Nicotiana tabacum AQP NtPIP2;1, into artificial membranes. The characterization of the CO2 and NH3 permeabilities of AQPs 0-9 and several rhesus (Rh) proteins shows that they can exhibit gas selectivity. Our laboratory has found that, during O2-offloading, ~55% of O2 exits mouse red blood cells (RBCs) via AQP1 and the Rh complex (i.e., RhAG + mRh). To characterize the mechanism by which RhAG and other proteins conduct O2, and to screen for novel O2 channels in a heterologous expression system, we have modified of 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 place into a saline-containing pressure-resistant tube. We then impose sufficient pressure in the air phase above the air-water interface to collapse the injected bubble enough to maintain the oocyte at a depth of 5 cm. As N2 gas dissolves in the cytosol and ultimately diffuses into the extracellular fluid (ECF), the bubble shrinks, cell density increases, and the oocyte sinks. A camera/computer combination detects the sinking and decreases air-phase pressure enough to maintain neutral buoyancy at 5 cm depth. Calibration exercises allow us to compute the time course of ΔnGas, and thus gas efflux. Here we modify the NBA to quantify gas influx. 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 now hold [N2]o constant at 2.06 mM but selectively increase [O2]o from 0.26 mM to 0.91 mM (pre-equilibrating saline with 78.96% N2/21% O2/0.04% CO2 at 3.5×ATA), nGas rises faster. Compared to control oocytes (injected with H2O rather than cRNA encoding RhAG), RhAG-expressing oocytes exhibit no significant difference in ΔnGas over 1000 s when [N2]o = 2.06 mM/[O2]o = 0.26 mM. Thus, RhAG appears not to be an N2 channel. However, when [N2]o = 2.06 mM/[O2]o = 0.91 mM, ΔnGas over 1000 s is significantly greater in RhAG vs control oocytes. Thus, RhAG is selective for O2 over N2. When we repeat the low-to-high [O2]o experiment with oocytes expressing NtPIP1;3 (reported to facilitate O2 flux in a spectrophotometric assay when expressed in yeast protoplasts), we also observe substantial increases in ΔnGas over 1000 s vs control oocytes. These data are consistent with our previous findings in RBCs that the Rh complex constitutes a major pathway for O2 flux across the plasma membrane, and also supports the NtPIP1;3 report from another laboratory. Although we specifically developed the NBA for N2 fluxes, here we demonstrate the versatility of the assay for measuring transmembrane fluxes of other gases.