Zhao, P.; Moss, F. J.; Occhipinti, R.; Geyer, R. R.; Huffmann, D. E.; Meyerson, H. J.; Boron, W. F.

Many have believed that oxygen (O2) crosses red blood cell (RBC) membranes by dissolving in lipids that offer a finite resistance to diffusion or, alternatively, no resistance at all. However, analyses of hemoglobin (Hb) absorbance spectra during O2 off-loading from mouse RBCs indicate that RBC membranes do indeed offer resistance to O2 diffusion, and that the resistance would be far higher if not for the presence of O2-permeable channels. Two agents, both excluded from the RBC interior, markedly slow O2 off-loading: p-chloromercuribenzenesulfonate (pCMBS) reduces membrane O2 permeability (PM,O2 by ~82%, and 4,4\'-diisothiocyanatostilbene-2,2\'-disulfonate (DIDS) by ~56%. Because neither likely produces these effects via membrane lipids, we examined RBCs from mice genetically deficient in aquaporin-1 (AQP1), the Rh complex (RhCx = Rhesus blood group-associated A glycoprotein, RhAG + Rhesus blood group D antigen, RhD), or both. The double knockout (dKO) reduces PM,O2 by ~55%, and pCMBS+dKO, by ~91%. Proteomic analyses of RBC membranes, flow cytometry, hematology, and mathematical simulations rule out meaningful contributions from other membrane proteins, RBC geometry, or extracellular unconvected fluid (EUF). Our work represents a paradigm shift for O2 physiology by identifying the first two animal O2 channels, and points to the existence of at least a third, all of which could be subject to physiological regulation and pharmacological intervention.