The First N2 Channels Identified by a Novel Technique for Assessing Nitrogen Gas Efflux from Oocytes

Abstract

Decompression illness (DCI) is a serious problem for both divers and pilots. Divers must schedule their ascents to minimize the risk of nitrogen (N2) bubble formation as the partial pressure of N2 (PN2) decreases. Pilots can experience the same effects via decreases in cabin pressure. In order to approach management or even prevention of DCI, it would be beneficial to understand the mechanism of N2 transport across cell membranes. Specific aquaporins and rhesus (Rh) proteins can conduct CO2, NH3, NO, and probably O2 across membranes. Certain solute transporters (e.g., NBCe1) may also be able to conduct gases. Whereas several assays exist for CO2, O2 and NH3 permeability, an effective assay for N2 movements across membranes has been elusive. Here we present the first such assay, based on maintaining the neutral buoyancy of a Xenopus oocyte previously injected with a 200‐nl air bubble (79% N2, 21% O2). We apply pressure to the air above a saline solution, causing the bubble to constrict sufficiently that the oocyte falls to a depth of ~5 cm. As air molecules dissolve in and diffuses through the cytoplasm, and eventually exit the cell, the bubble tends to collapse. A feedback system (webcam, computer, digitally controlled pressure regulator) reacts by lowering the neutral‐buoyancy air pressure (PNB) to maintain the oocyte at a 5‐cm depth. The pressure inside the bubble (PBubble > PNB by fixed amount) falls proportionally with the decreasing number of air molecules. The rate at which PNB falls thus reflects gas efflux from the bubble. For control oocytes injected with H2O rather than cRNA, PNB falls slowly, reflecting a slow exit of N2 (+ O2) from the bubble (kPNB = 1.16 × 10−3 ± 0.02 s−1, n=47). We estimate that 10–50% of kPNB represents N2 dissolving in cellular water/lipids. For oocytes injected with cRNA to express NOD26—a plant AQP in cell membranes of root nodules of the N2‐fixing soybean plant, Glycine max—PNB falls 13.0% more rapidly (kPNB = 1.31 × 10−3 ± 0.04 s−1, p=0.018, n=5), presumably reflecting an increased N2 (+ O2) efflux across the oocyte membrane. kPNB for oocytes expressing human AQP5 or the plant AQP, AtPIP 2;1—AQPs known to function as CO2 channels—were not significantly different from H2O‐injected control oocytes (n=20 & 5, respectively). However, kPNB was 8.6% greater in oocytes expressing human RhAG (kPNB = 1.26 × 10−3 ± 0.03 s−1, p=0.003, n=26) vs. H2O‐injected control oocytes. 200 mM 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) decreased the RhAG‐dependent component of kPNB by 89% (kPNB = 1.17 × 10−3 ± 0.03 s−1, p=0.04, n=12). DIDS had no effect on H2O‐injected controls (p=0.65, n=11). Together these experiments demonstrate a simple and robust assay for the efflux of N2 across a biological membrane, as well as the first examples of N2 channels in a biological membrane.Support or Funding InformationFunding: Office of Naval Research (N00014‐15‐1‐2060 & N00014‐16‐1‐2535)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.