Biology subjects

Meyerson, H. J.

Publications and source records attributed to Meyerson, H. J..

3 recordsLinked to original sources

bioRxiv2025

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

physiology↗

bioRxiv2025

Role of channels in the O2 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/6Case). In paper #1, using stopped-flow absorbance spectroscopy to ascertain the rate constant for oxyhemoglobin (HbO2) 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 O2/HbO2/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 (OMajor), 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. Key PointsO_LIO2-offloading from red blood cells (RBCs) depends not only on membrane O2 permeability and oxyhemoglobin dissociation, but also on RBC size and shape. In this second of three papers, we use blood smears, still/video images of living RBCs, and imaging flow cytometry to examine morphometry of RBCs from paper #1. C_LIO_LIWe find that mouse RBCs of all genotypes--wild-type, aquaporin-1 knockout (AQP1-KO), Rhesus blood group-associated A-glycoprotein knockout (RhAG-KO), and double knockout--are dominantly biconcave discs, with [~]1.4% to [~]2.5% poikilocytosis (shape change, SC). Drug pre-treatment increases %SC. C_LIO_LIUsing label-free liquid chromatography/tandem mass spectrometry to assess apparent abundance of RBC-ghost proteins, we find no significant differences among genotypes for any of the [~]100 most abundant protein species except, as appropriate, AQP1, RhAG, or Rhesus blood group D antigen. C_LIO_LIThus, the substantial effects observed in paper #1 cannot be attributed to differences in morphometry or protein content. C_LI

physiology↗

bioRxiv2020

Role of channels in the oxygen permeability of red blood cells

Many have believed that oxygen (O2) 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 O2 off-loading from mouse RBCs, we now report that most O2 traverses membrane-protein channels. Two agents excluded from the RBC interior markedly slow O2 off-loading: p-chloromercuribenzenesulfonate (pCMBS) reduces inferred membrane O2 permeability (PMembrane) 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 PMembrane 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 O2 channels and pointing to the existence of other O2 channel(s), all of which could be subject to physiological regulation and pharmacological intervention, our work represents a paradigm shift for O2 handling.

physiology↗