Role of channels in the oxygen permeability of red blood cells

Many have believed that oxygen (O 2 ) crosses red blood cell (RBC) membranes by dissolving in lipids that offer no resistance to diffusion. However, using stopped-flow (SF) analyses of hemoglobin (Hb) absorbance spectra during O 2 off-loading from mouse RBCs, we now report that most O 2 traverses membrane-protein channels. Two agents excluded from the RBC interior markedly slow O 2 off-loading: p-chloromercuribenzenesulfonate (pCMBS) reduces inferred membrane O 2 permeability ( P Membrane ) 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 (i.e., rhesus proteins RhAG + mRh), or both. The double knockout (dKO) reduces P Membrane by ∼55%, and pCMBS+dKO, by ∼91%. Proteomic analyses of RBC membranes, flow cytometry, hematology, and mathematical simulations rule out explanations involving other membrane proteins, RBC geometry, or extracellular unconvected fluid (EUF). By identifying the first two O 2 channels and pointing to the existence of other O 2 channel(s), all of which could be subject to physiological regulation and pharmacological intervention, our work represents a paradigm shift for O 2 handling.