Role of AQP1 and RhAG proteins in the deoxygenation of mouse red blood cells during aging

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The traditional view had been that all O2 moves through red blood cell (RBC) membranes by dissolving in and diffusing through the membrane lipid. In a previous abstract, we reported data obtained using stopped‐flow analysis of hemoglobin (Hb) absorbance spectra on RBCs from wild‐type (WT) vs. double‐knockout (dKO) mice genetically deficient in both aquaporin‐1 (AQP1) and the rhesus (Rh) protein RhAG. We found that the rate constant of Hb deoxygenation (kHbO2)—which includes intra/extracellular diffusion and membrane permeability—was ~31% lower for RBCs from dKO than for WT mice. From these data, we computed that, together, AQP1 and the Rh complex account for ~60% of membrane O2 permeability. We now examine the effect of aging on kHbO2 of RBCs from WT vs. dKO mice, studying mice at six ages (2–3 months, 6 months, 12 months, 18 months, 24 months, and 29 months). The kHbO2 of RBCs from WT mice are indistinguishable from age 2–3 months through 12 months. However, compared to 2–3 months, kHbO2 is 9% lower at 18 months (p<0.05, N = 6), 12% lower at 24 months (p<0.05, N = 5), and 13% lower at 29 months (p<0.05, N = 3). For RBCs from dKO mice, kHbO2 starts off ~30% lower than that of WT mice at 2–3 months (see above), and remains at this same depressed level through 29 months (N = 5 to 6 at 6–24 months, 4 at 29 months). In the case of WT mice at 24 months, our hematology data—which show decreases for both mean corpuscular volume and mean corpuscular hemoglobin—cannot explain the observed decreases in kHbO2. DIC microscopy of living RBCs and microscopic observation of stained blood smears with RBCs from 3 months and 29 months with both WT and dKO groups confirm that RBCs are normal biconcave disks. Preliminary data from mass spectroscopy of RBC ghosts from WT mice (N = 1 each for 2–3 months and 29 months) suggests that, compared to 2–3 months, membrane protein levels at 29 months are lower by 11% for AQP1, 19% for RhAG, 27% for mRh, and 22% for AE1. We conclude from our dKO data that the non‐AQP1/RhAG component of RBC membrane O2 permeability is independent of aging. We hypothesize that the 12–13% decrease of kHbO2 at 24–29 months for WT mice is due in part to decreased expression of AQP1 and the Rh complex (i.e., the AQP1 and RhAG components of O2 membrane permeability decline with aging).Support or Funding InformationN00014‐15‐1‐2060 Gas Channels, ONRand N00014‐16‐1‐2535 ONR‐MURI