Fabian Itel

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

Monolayer interactions between lipids and amphiphilic block copolymers.

Interactions in binary mixed monolayers from lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and amphiphilic poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline) block copolymers were studied by using the Langmuir balance technique and Brewster angle microscopy. It is shown that monolayers from the saturated lipid (DPPC) are more sensitive to the presence of polymers in the film, resulting in phase separation and the formation of pure lipid domains at high surface pressure. The morphology and composition of such phase-separated lipid-polymer films were studied by fluorescence microscopy and ToF-SIMS. In contrast, in DOPC-containing monolayers, the polymers tend to phase-separate at low surface pressures only and homogeneous films are obtained upon further compression, due to higher lipid fluidity. The analysis of excess energy of mixing shows that while the separation effect in densely packed DPPC-containing films is strongly dependent on the polymer size (with the larger polymer having a much stronger influence), in the case of monolayers with DOPC much smaller effects are observed. The results are discussed in terms of the monolayer composition, lipid fluidity, and polymer size.

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.

Gas-tight triblock-copolymer membranes are converted to CO2 permeable by insertion of plant aquaporins

We demonstrate that membranes consisting of certain triblock-copolymers were tight for CO2. Using a novel approach, we provide evidence for aquaporin facilitated CO2 diffusion. Plant aquaporins obtained from heterologous expression were inserted into triblock copolymer membranes. These were employed to separate a chamber with a solution maintaining high CO2 concentrations from one with depleted CO2 concentrations. CO2 diffusion was detected by measuring the pH change resulting from membrane CO2 diffusion from one chamber to the other. An up to 21 fold increase in diffusion rate was determined. Besides the supply of this proof of principle, we could provide additional arguments in favour of protein facilitated CO2 diffusion to the vivid on-going debate about the principles of membrane gas diffusion in living cells.

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

Does Membrane Thickness Affect the Transport of Selective Ions Mediated by Ionophores in Synthetic Membranes

Biomimetic polymer nanocompartments (polymersomes) with preserved architecture and ion-selective membrane permeability represent cutting-edge mimics of cellular compartmentalization. Here it is studied whether the membrane thickness affects the functionality of ionophores in respect to the transport of Ca2+ ions in synthetic membranes of polymersomes, which are up to 2.6 times thicker than lipid membranes (5 nm). Selective permeability toward calcium ions is achieved by proper insertion of ionomycin, and demonstrated by using specific fluorescence markers encapsulated in their inner cavities. Preservation of polymersome architecture is shown by a combination of light scattering, transmission electron microscopy, and fluorescence spectroscopy. By using a combination of stopped-flow and fluorescence spectroscopy, it is shown that ionomycin can function and transport calcium ions across polymer membranes with thicknesses in the range 10.7-13.4 nm (7.1-8.9 times larger than the size of the ionophore). Thicker membranes induce a decrease in transport, but do not block it due to the intrinsic flexibility of these synthetic membranes. The design of ion selective biomimetic nanocompartments represents a new path toward the development of cellular ion nanosensors and nano-reactors, in which calcium sensitive biomacromolecules can be triggered for specific biological functions.