Role of channels in the O2 permeabilityof murine red blood cells. I. Stopped-flow and hematological studies

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. In this first in a series of three interrelated papers, we examine these idea in murine RBCs. In this first paper, 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 reduce the rate constant for O2 off-loading (kHbO2): p-chloromercuribenzenesulfonate (pCMBS) reduces membrane O2 permeability (PM,O2 by [~]82% (computed from kHbO2 in paper #3), 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 (see paper #2), and mathematical simulations (paper #3) 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. Key PointsO_LISome have held that red-blood-cell (RBC) membranes have a finite O2 permeability, governed by O2 solubility in membrane lipids; others, that membranes offer no resistance whatsoever to O2 diffusion. C_LIO_LIThe present interdisciplinary study comprises three papers. This first paper describes stopped-flow absorbance spectroscopy in which we examine the rate constant of O2 offloading from intact RBCs (kHbO2) from wild-type (WT) mice or those lacking AQP1 and/or RhAG, sometimes treated with p-chloromercuribenzenesulfonate (pCMBS) or 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS). C_LIO_LIThe second paper describes RBC morphometry and membrane proteomics. The third introduces a mathematical reaction-diffusion model that generates simulations comporting with physiological data. C_LIO_LIThis first paper shows that pCMBS or DIDS treatments, or deletion of AQP1 and/or RhAG, or combinations thereof, substantially reduce kHbO2. C_LIO_LIThe three papers show that WT RBC membranes offer considerable resistance to O2 diffusion. AQP1 ([~]22%), Rh ([~]36%), and an unknown pCMBS-sensitive protein account for [~]91% of membrane O2 permeability. C_LI