Samer Al-Samir

CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels

Recent observations that some membrane proteins act as gas channels seem surprising in view of the classical concept that membranes generally are highly permeable to gases. Here, we study the gas permeability of membranes for the case of CO2, using a previously established mass spectrometric technique. We first show that biological membranes lacking protein gas channels but containing normal amounts of cholesterol (30–50 mol% of total lipid), e.g., MDCK and tsA201 cells, in fact possess an unexpectedly low CO2 permeability (PCO2) of ~0.01 cm/s, which is 2 orders of magnitude lower than the PCO2 of pure planar phospholipid bilayers (~1 cm/s). Phospholipid vesicles enriched with similar amounts of cholesterol also exhibit PCO2 ≈.01 cm/s, identifying cholesterol as the major determinant of membrane PCO2. This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in PCO2, respectively. We demonstrate, furthermore, that reconstitution of human AQP‐1 into cholesterol‐containing vesicles, as well as expression of human AQP‐1 in MDCK cells, leads to drastic increases in PCO2, indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability.—Itel, F., Al‐Samir, S., Öberg, F., Chami, M., Kumar, M., Supuran, C. T., Deen, P. M. T., Meier, W., Hedfalk, K., Gros, G., Endeward, V. CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels. FASEB J. 26, 5182–5191 (2012). www.fasebj.org

How does carbon dioxide permeate cell membranes? A discussion of concepts, results and methods

We review briefly how the thinking about the permeation of gases, especially CO2, across cell and artificial lipid membranes has evolved during the last 100 years. We then describe how the recent finding of a drastic effect of cholesterol on CO2 permeability of both biological and artificial membranes fundamentally alters the long-standing idea that CO2—as well as other gases—permeates all membranes with great ease. This requires revision of the widely accepted paradigm that membranes never offer a serious diffusion resistance to CO2 or other gases. Earlier observations of “CO2-impermeable membranes” can now be explained by the high cholesterol content of some membranes. Thus, cholesterol is a membrane component that nature can use to adapt membrane CO2 permeability to the functional needs of the cell. Since cholesterol serves many other cellular functions, it cannot be reduced indefinitely. We show, however, that cells that possess a high metabolic rate and/or a high rate of O2 and CO2 exchange, do require very high CO2 permeabilities that may not be achievable merely by reduction of membrane cholesterol. The article then discusses the alternative possibility of raising the CO2 permeability of a membrane by incorporating protein CO2 channels. The highly controversial issue of gas and CO2 channels is systematically and critically reviewed. It is concluded that a majority of the results considered to be reliable, is in favor of the concept of existence and functional relevance of protein gas channels. The effect of intracellular carbonic anhydrase, which has recently been proposed as an alternative mechanism to a membrane CO2 channel, is analysed quantitatively and the idea considered untenable. After a brief review of the knowledge on permeation of O2 and NO through membranes, we present a summary of the 18O method used to measure the CO2 permeability of membranes and discuss quantitatively critical questions that may be addressed to this method.

Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1

•  Controversial results have been reported on the hypothesis that the cytosolic carbonic anhydrase II (CAII) of the red cell is largely bound to the cells Cl−/HCO3− exchanger AE1, forming a ‘metabolon complex’ that greatly enhances transport activity of AE1. •  In examining so far untested aspects of this hypothesis, we report that fluorophore‐labelled AE1 and CAII proteins, expressed in tsA201 cells, neither colocalize at the cell membrane nor show close proximity by Förster resonance emission spectroscopy. •  Antibody against Flag‐tagged AE1 expressed in tsA201 cells co‐immunoprecipitates coexpressed ankyrin but not CAII. •  CAII‐deficient human red blood cells with substantial CAI activity exhibit HCO3− permeabilities identical to those of normal red cells. •  A mathematical model of CO2/HCO3− transport of red cells indicates that this process occurs more rapidly when the CA of the cell is distributed homogeneously across the cytoplasm rather than being bound to the membrane interior.

Low CO2 permeability of cholesterol-containing liposomes detected by stopped-flow fluorescence spectroscopy

Here we ask the following: 1) what is the CO2 permeability (Pco2) of unilamellar liposomes composed of L‐α‐phosphatidylcholine (PC)/L‐α‐phosphatidylserine (PS) = 4:1 and containing cholesterol (Chol) at levels often occurring in biologic membranes (50 mol%), and 2) does incorporation of the CO2 channel aquaporin (AQP)1 cause a significant increase in membrane Pco2? Presently, a drastic discrepancy exists between the answers to these two questions obtained from mass‐spectrometric 18O‐exchange measurements (Chol reduces Pco2 100‐fold, AQP1 increases Pco2 10‐fold) vs. from stopped‐flow approaches observing CO2 uptake (no effects of either Chol or AQP1). A novel theory of CO2 uptake by vesicles predicts that in a stopped‐flow apparatus this fast process can only be resolved temporally and interpreted quantitatively, if 1) a very low CO2 partial pressure (pCO2) is used (e.g., 18 mmHg), and 2) intravesicular carbonic anhydrase (CA) activity is precisely known. With these prerequisites fulfilled, we find by stopped‐flow that 1) Chol‐containing vesicles possess a Pco2 = 0.01cm/s, and Chol‐free vesicles exhibit ~1 cm/s, and 2) the Pco2 of 0.01 cm/s is increased ≥ 10‐fold by AQP1. Both results agree with previous mass‐spectrometric results and thus resolve the apparent discrepancy between the two techniques. We confirm that biologic membranes have an intrinsically low Pco2 that can be raised when functionally necessary by incorporating protein‐gas channels such as AQP1.—Tsiavaliaris, G., Itel, F., Hedfalk, K., Al‐Samir, S., Meier, W., Gros, G., Endeward, V. Low CO2 permeability of cholesterol‐containing liposomes detected by stopped‐flow fluorescence spectroscopy. FASEB J. 29, 1780‐1793 (2015). www.fasebj.org

CO2 permeability and carbonic anhydrase activity of rat cardiomyocytes

To determine the CO2 permeability (PCO2) of plasma membranes of cardiomyocytes. These cells were chosen because heart possesses the highest rate of O2 consumption/CO2 production in the body.

Maximal Oxygen Consumption Is Reduced in Aquaporin-1 Knockout Mice

We have measured maximal oxygen consumption (V˙O2,max) of mice lacking one or two of the established mouse red-cell CO2 channels AQP1, AQP9, and Rhag. We intended to study whether these proteins, by acting as channels for O2, determine O2 exchange in the lung and in the periphery. We found that V˙O2,max as determined by the Helox technique is reduced by ~16%, when AQP1 is knocked out, but not when AQP9 or Rhag are lacking. This figure holds for animals respiring normoxic as well as hypoxic gas mixtures. To see whether the reduction of V˙O2,max is due to impaired O2 uptake in the lung, we measured carotid arterial O2 saturation (SO2) by pulse oximetry. Neither under normoxic (inspiratory O2 21%) nor under hypoxic conditions (11% O2) is there a difference in SO2 between AQP1null and WT mice, suggesting that AQP1 is not critical for O2 uptake in the lung. The fact that the % reduction of V˙O2,max is identical in normoxia and hypoxia indicates moreover that the limitation of V˙O2,max is not due to an O2 diffusion problem, neither in the lung nor in the periphery. Instead, it appears likely that AQP1null animals exhibit a reduced V˙O2,max due to the reduced wall thickness and muscle mass of the left ventricles of their hearts, as reported previously. We conclude that very likely the properties of the hearts of AQP1 knockout mice cause a reduced maximal cardiac output and thus cause a reduced V˙O2,max, which constitutes a new phenotype of these mice.

CO2 Permeability of Biological Membranes and Role of CO2 Channels

We summarize here, mainly for mammalian systems, the present knowledge of (a) the membrane CO2 permeabilities in various tissues; (b) the physiological significance of the value of the CO2 permeability; (c) the mechanisms by which membrane CO2 permeability is modulated; (d) the role of the intracellular diffusivity of CO2 for the quantitative significance of cell membrane CO2 permeability; (e) the available evidence for the existence of CO2 channels in mammalian and artificial systems, with a brief view on CO2 channels in fishes and plants; and, (f) the possible significance of CO2 channels in mammalian systems.

Cholesterol is the main regulator of the carbon dioxide permeability of biological membranes

We present here a compilation of membrane CO2 permeabilities (Pco2) for various cell types from the literature. Pco2 values vary over more than two orders of magnitude. Relating Pco2 to the cholesterol content of the membranes shows that, with the exception of red blood cells, it is essentially membrane cholesterol that determines the value of Pco2. Thus, the observed strong modulation of Pco2 in the majority of membranes is caused by cholesterol rather than gas channels.