Oxygen-offloading rate from mouse Red Blood Cells of genetically diverse strains

Abstract

O2 diffusion across red blood cell (RBC) membranes is a critically important physiological process for the maintenance of life. Our previous work on murine RBCs ( bioRxiv. doi: 10.1101/2020.08.28.265066 ) showed that (a) the genetic deletion of aquaporin-1 (AQP1) and the Rh complex (mainly RhAG) together reduce O2 permeability of RBC membranes ( P M, O2) by ~55%, and (b) the double knockout (dKO) of AQP1 and RhAG plus pCMBS (sulfhydryl reagent and nonspecific inhibitor of membrane proteins that is nonetheless excluded from the RBC interior) reduces P M, O2 by ~91%. In order to identify the unknown membrane protein(s) that may function as O2 channels and contribute to the missing 91% – 55% = ~36% of P M, O2, we use comparative physiology. Using stopped-flow absorbance spectroscopy to monitor the hemoglobin (Hb) absorbance spectrum, we studied the oxygen-offloading rate from hemoglobin ( k HbO2) of RBCs from genetically diverse mouse strains. We compared two different mouse strains purchased from Jackson Labs—C57BL/6J and BALB/cJ with our standard mouse strain that ultimately derives from mice received from UCSF—C57BL/6Case. We found that, compared with C57BL/6Case, C57BL/6J mice have a k HbO2 for intact RBCs that is ~22% higher (N=18, 9 male plus 9 female, p<0.01), and a k HbO2 for pure hemolysate that is ~7% lower (N=12, 6 male plus 6 female, p<0.01). Comparing with C57BL/6Case, BALB/cJ mice have a k HbO2 for intact RBCs that is ~9% lower (N=18, 9 male plus 9 female, p<0.01), and a k HbO2 of pure hemolysate that is ~10% higher (N=12, 6 male plus 6 female, p<0.01). Because changes in k HbO2 of pure hemolysate are opposite in direction to changes in k HbO2 of intact RBCs, differences in Hb cannot contribute to the observed differences in O2-offloading rates in the two Jackson strains. Likewise, changes of mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), together, would only produce minor percent changes of k HbO2 in either C57BL/6J or BALB/cJ mice. Thus, it is likely that the differences in k HbO2 for intact RBCs among the three mouse strains reflect major differences in the O2 permeabilities of the RBC membranes. Planned comparative analyses of morphology (e.g., major diameter of biconcave discs), proteomics, lipidomics, and genomics (for deduced amino-acid sequences of proteins) will provide valuable insight into the contribution of specific membrane proteins and lipids to the relatively large differences in k HbO2 of intact RBCs between C57BL/6J and BALB/cJ mice. Office of Naval Research (N00014-16-1-2535 and N00014-21-1-2366), and NIH research grant (1RO1HL 160857-01) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.