Role of channels in the O(2) permeability of murine red blood cells II. Morphological and proteomic studies.

In this second of three papers, we examine red blood cell (RBC) morphometry and RBC-membrane proteomics from our laboratory mouse strain (C57BL/6(Case)). In paper #1, using stopped-flow absorbance spectroscopy to ascertain the rate constant for oxyhemoglobin (HbO(2)) deoxygenation (kHbO2) , we find substantial kHbO2 reductions with (1) membrane-protein inhibitors p-chloromercuribenzenesulfonate (pCMBS) or 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS); (2) knockouts of aquaporin-1 (AQP1-KO), or Rhesus blood-group-associated A-glycoprotein (RhAG-KO), or double knockouts (dKO); or (3) inhibitor+dKO. In paper #3, reaction-diffusion mathematical modeling/simulations reveal that kHbO2 could fall secondary to slowed intracellular O(2)/HbO(2)/Hb diffusion. Here in paper #2, blood smears as well as still/video images and imaging flow cytometry (IFC) of living RBCs show that ~97.5% to ~98.6% of control (not drug-treated) cells are biconcave disks (BCDs) across all genotypes. Pretreatment with pCMBS raises non-BCD abundance to ~8.7% for WT and ~5.7% for dKO; for DIDS pretreatment, the figures are ~41% and ~21%, respectively. Modeling (paper #3) accommodates for these shape changes. Light-scattering flow cytometry shows no significant difference in RBC size or shape among genotypes. IFC reveals minor differences among genotypes in RBC major diameter (∠Major) , which (along with mean corpuscular volume, paper #1) yields RBC thickness for simulations in paper #3. Label-free liquid chromatography/tandem mass spectrometry (LC/MS/MS) proteomic analyses of RBC plasma-membrane ghosts confirm the deletion of proteins targeted by our knockouts, and rule out changes in the 100 proteins of greatest inferred abundance. Thus, genetically induced changes in kHbO2 must reflect altered abundance of AQP1 and /or the Rh complex.