Effect of inhibitors on oxygen permeability of wild type and knockout mouse red blood cells

We now recognize that the paradigm that all gases cross red blood cell (RBC) membranes by dissolving in and diffusing through the lipid phase is not correct. Work on human RBCs genetically deficient in aquaporin‐1 (AQP1) or the Rh complex (mainly RhAG) showed that these two proteins account for ~90% of membrane CO 2 permeability ( P M,CO2 ), and that 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) reduces P M,CO2 . We used stopped‐flow (SF) analysis of hemoglobin (Hb) absorbance spectra as we mixed oxygenated RBCs with an extracellular O 2 scavenger, and determined the rate constant of Hb deoxygenation ( k HbO2 ). Because k HbO2 depends on Hb kinetics as well as diffusion of O 2 to the extracellular space, we used a mathematical model to estimate effects on membrane O 2 permeability ( P M,O2 ). We examined the effects of DIDS and chloromercuribenzenesulfonate (pCMBS), agents that decrease P M,CO2 . We find that, in RBCs from wild type (WT) mice, pCMBS reduces k HbO2 by 64% (est. P M,O2 by ~84%) and DIDS, by 41% (est. P M,O2 by ~67%). Because pCMBS and DIDS likely act by targeting membrane proteins (rather than lipids), we hypothesized that O 2 , at least in part, crosses RBC membranes via the same membrane proteins that conduct CO 2 . Therefore, we examined RBCs from mice genetically deficient in AQP1, RhAG, or both, ± inhibitors. Deletion of AQP1 lowers k HbO2 by 11% (est. P M,O2 by ~27%); RhAG, by 16% (est. P M,O2 by ~35%); the double knockout (dKO), by 33% (est. P M,O2 by ~59%). The combination dKO + pCMBS lowers k HbO2 (vs. WT without inhibitors) by 77% (est. P M,O2 by ~91%), whereas dKO + DIDS lowers k HbO2 by 52% (est. P M,O2 by ~76%). The effects of pCMBS on estimated P M,O2 are very similar when we compare dKO vs. RhAG −/− , or when we compare WT vs. AQP1 −/− . Thus, the deletion of AQP1 may have little effect on the sensitivity of P M,O2 to pCMBS—consistent with the hypothesis that AQP1 conducts O 2 only via pathways other than the mercury‐sensitive monomeric pores (e.g., via the hydrophobic central pore). In summary, our data suggest that AQP1 and RhAG account for nearly 60% of RBC O 2 permeability, and point towards the existence of other O 2 channels that account for at least 30%. Support or Funding Information Supported by ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFB and NIH K01‐DK107787 to RO This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .