Biology subjects

Boron, W. F.

Publications and source records attributed to Boron, W. F..

6 recordsLinked to original sources

bioRxiv2025

Synergistic roles of Aquaporin 5 and Intra- and Extracellular Carbonic Anhydrases In facilitating CO2 Diffusionacross the Xenopus Oocyte Plasma Membrane

Key PointsO_LIAccording to Ficks law, transmembrane CO2 flux (JCO2) is the product of membrane permeability (PM,CO2) and transmembrane concentration gradient ({Delta}[CO2]): JCO2=PM,CO2{Delta}[CO2]. Previous work separately showed that (1) human aquaporin-5 (hAQP5) enhances PM,CO2, and (2) intracellular and (3) extracellular carbonic anhydrases (CAs) enhance {Delta}[CO2] by consuming accumulated or replenishing lost CO2. We now examine interactio ns among #1-#3. C_LIO_LIWe assess CO2 fluxes--produced by addition/removal of extracellular CO2/[Formula]--using microelectrodes to monitor extracellular-surface pH (pHS) and intracellular pH (pHi) of Xenopus oocytes heterologously expressing hAQP5, injected with human CA II (hCA II), and/or exposed to extracellular bovine CA (bCA). C_LIO_LIEnhancing effects on CO2 fluxes are synergistic among hAQP5, hCA II, and bCA, any of which can become rate limiting, depending on the status of the other two. C_LIO_LICO2/[Formula] addition transiently increases pHS ({Delta}pHS), hCA II augments {Delta}pHS ({Delta}{Delta}pHS), and hAQP5 enhances {Delta}{Delta}pHS ({Delta}{Delta}{Delta}pHS)--a novel tool to assess potential CO2 channels. C_LI CO2 diffusion across plasma membranes depends on both membrane CO2 permeability (PM,CO2) and transmembrane CO2 concentration gradient ({Delta}[CO2])--Ficks law. Human aquaporin-5 (hAQP5) accelerates CO2 diffusion by increasing PM,CO2, whereas carbonic anhydrases (CAs) accelerate CO2 diffusion by enhancing CO2 consumption/production and thus {Delta}[CO2]. Here, we systematically assess functional interactions among a gas channel and intra-/extracellular CAs. On Day 1, we inject Xenopus oocytes with cRNA encoding hAQP5 (control: H2O). On Day 4, we inject hCA II protein in "Tris" buffer (control: "Tris"). We assess CO2 fluxes by introducing extracellular 1.5% CO2/10 mM [Formula] and using microelectrodes to measure (1) maximal extracellular-surface pH increase {Delta}pHS, (2) maximal rate of pHS relaxation (dpHS/dt)Max, and (3) maximal rate of intracellular-pH decrease (dpHi/dt)Max. By itself, hCA II minimally increases {Delta}pHS--measured "trans" to added cytosolic CA (CAi)--even at highest doses (100 ng/oocyte). However, hAQP5 alone triples {Delta}pHS, an effect further doubled by increasing hCA II. By itself, bovine erythrocyte CA (bCA) in the extracellular fluid doubles (dpHi/dt)Max magnitude--meas ured "trans" to added extracellular CA (CAo)--an effect further doubled by hAQP5. Note: pH measureme nts "cis" to added CAs--pHS for bCA, (dpHi/dt)Max for hCA II--are overwhelmed by enzymatical ly-produced/consumed H+, and cannot provide intuitive insight into CO2 fluxes. Our "trans" pH measurements: (1) confirm synergy between CAo and CAi; establish synergy between hAQP5 and both (2) CAo and (3) CAi; and show that enhancement of {Delta}pHS by CAi ({Delta}{Delta}pHS) is a useful tool for assessing CO2 permeability of membrane proteins (e.g., hAQP5).

physiology↗

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↗

bioRxiv2025

Role of channels in the O2 permeability of murine red blood cells. III. Mathematical modeling and simulations.

In this third of three papers, we develop a reaction-diffusion model for O2 offloading from a red blood cell (RBC), treated as a sphere with diameter approximating RBC thickness. Stopped-flow (SF) analysis (paper #1) of hemoglobin/oxyhemoglobin (Hb/HbO2) absorbance spectra during O2 efflux from intact murine RBCs show that membrane-impermeant inhibitor p-chloromercuribenzenesulfonate (pCMBS) reduces the HbO2-deoxygenation rate constant (kHbO2) by [~]61%. SF experiments show that kHbO2 falls by (1) 9% for aquaporin-1 knockouts (AQP1-KOs), (2) 17% for Rhesus A-glycoprotein knockouts (RhAG-KOs), (3) 30% for double knockouts (dKOs), and (4) [~]78% in dKOs/pCMBS. Here, we simulate HbO2 dissociation in the intact RBC (i.e., kHbO2); HbO2, Hb, and O2 diffusion through RBC cytosol; transmembrane O2 diffusion; and O2 diffusion through extracellular unconvected fluid (EUF) to bulk extracellular fluid. Informed by automated-hematology data (paper #1) and imaging-flow-cytometry data (paper #2), simulations predict that observed kHbO2 decreases cannot reflect changes only in RBC size/shape or [Hb/HbO2]. Instead, membrane O2 permeability (PM,O2) must fall by (1) 22% to account for AQP1-KO data, (2) 36% for RhAG-KOs, (3) 55% for dKOs, and (4) 91% for dKOs/pCMBS. Exploring predicted kHbO2 sensitivities to eight key parameters (e.g., [Hb/HbO2], diffusion constants, kHbO2[->]Hb+O2, thicknessEUF, diameterSphere) shows that no reasonable changes explain the kHbO2 data. We introduce a linear-combination approach to accommodate for the presence of poikilocytes. Finally, contrary to common beliefs, the model predicts that, in the absence of inhibitors, the RBC membrane represents >30% of total diffusive "resistance" to O2 offloading, even for a WT mouse. Key PointsO_LIIn this third of three papers, we develop a novel reaction-diffusion model for O2 offloading from a red blood cell (RBC), treated as a sphere with diameter approximating RBC thickness. C_LIO_LIUsing physical constants and parameter values from the literature and papers #1 and #2, we generate simulations that reproduce observed hemoglobin desaturation (rate constant, kHbO2) of intact RBCs from wild-type mice. C_LIO_LITo simulate kHbO2 decreases (9%, 17%, 30%) observed in RBCs from knockouts (KOs: aquaporin-1, Rhesus A-glycoprotein, both) we must decrease membrane O2 permeability (PM,O2) by far greater percentages (22%, 36%, 55%). C_LIO_LIAnalyses of simulated-kHbO2 sensitivity to kinetic and geometric parameters suggest that reasonable parameter-value changes cannot explain experimentally observed kHbO2 decreases in RBCs from KOs. Thus, experimentally observed kHbO2 decreases must reflect decreases in PM,O2. C_LIO_LITo accommodate for poikilocytes, we develop a linear-combination approach (poikilocytes + biconcave disks, BCDs) to extract the kHbO2 and PM,O2 of just BCDs. 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↗