Biology subjects

Geyer, R. R.

Publications and source records attributed to Geyer, R. R..

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

Mechanism of CO2 and NH3 Transport through Human Aquaporin 1: Evidence for Parallel CO2 Pathways

The traditional view had been that dissolved gases cross membranes simply by dissolving in and diffusing through membrane lipid. However, some membranes are impermeable to CO2 and NH3, whereas some aquaporin (AQP) water channels--tetramers with hydrophobic central pores--are permeable to CO2, NH3, or both. Nevertheless, we understand neither the routes that CO2 and NH3 take through AQP tetramers, nor the basis of CO2/NH3 selectivity. Here, we show-- for human AQP1 (hAQP1)--that virtually all NH3 and H2O pass through the hydrophilic, monomeric pores. However CO2 passes both through the monomeric pores and another pathway. We expressed hAQP1 in Xenopus oocytes and used microelectrodes to monitor the maximal surface-pH transient ({Delta}pHS) caused by CO2 or NH3 influxes. We found that p-chloromercuribenzene sulfonate (pCMBS)--which reacts with C189 in the monomeric pore--eliminates the entire hAQP1-dependent (*) NH3 signal ({Delta}pHS*)NH3, but only half of the signals for CO2 ({Delta}pHS*)CO2 or osmotic water permeability Pf*. 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS), eliminates the remaining ({Delta}pHS*)CO2 but has no effect on ({Delta}pHS*)NH3 or Pf*. Together, the two drugs completely eliminate the CO2 permeability of hAQP1. When we express hAQP1 in Pichia pastoris, treat spheroplasts with DIDS, and examine hAQP1 by SDS-PAGE, reactivity with an anti-DIDS antibody shows that DIDS crosslinks hAQP1 monomers. Our results provide the first evidence that a molecule can move through an AQP via a route other than the monomeric pore, and raise the possibility that selectivity depends on the extent to which CO2/NH3 move through monomeric pores vs. an alternate pathway (e.g., the central pore). Key PointsO_LISome membranes have little or no CO2 permeability, absent protein channels like aquaporin-1 (AQP1). C_LIO_LIWe confirm that, during CO2 influx, heterologous expression of human AQP1 (hAQP1) in Xenopus oocytes increases the magnitude of the transient surface-pH increase by an amount ({Delta}pHS*)CO2, measured with microelectrodes. During NH3 influx, hAQP1 expression increases the magnitude of the transient pHS decrease by ({Delta}pHS*)NH3. C_LIO_LIp-chloromercuribenzene sulfonate (pCMBS), which reacts with C189 in the monomeric pore, reduces ({Delta}pHS*)CO2 by; ({Delta}pHS*)NH3, to zero; and AQP1-dependent osmotic water permeability (Pf*), by half. C_LIO_LI4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS) reduces ({Delta}pHS*)CO2 by half, but has no effect on ({Delta}pHS*)NH3 or Pf*. DIDS crosslinks AQP1 monomers expressed in Pichia pastoris. C_LIO_LITogether, pCMBS+DIDS reduces ({Delta}pHS*)CO2 to zero. The C189S mutation of AQP1 eliminates the effects of pCMBS, but not DIDS. Our results thus show that CO2 traverses AQP1 via the monomeric pore plus a novel, DIDS-sensitive route that may be the central pore. 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↗