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 CO(2), 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 CO(2) permeability (P(CO2)) of ∼0.01 cm/s, which is 2 orders of magnitude lower than the P(CO2) of pure planar phospholipid bilayers (∼1 cm/s). Phospholipid vesicles enriched with similar amounts of cholesterol also exhibit P(CO2) ≈ 0.01 cm/s, identifying cholesterol as the major determinant of membrane P(CO2). This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in P(CO2), 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 P(CO2), indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability.

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.

Key points• Controversial results have been reported on the hypothesis that the cytosolic carbonic anhydrase II (CAII) of the red cell is largely bound to the cell's Cl − /HCO 3 − 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 HCO 3 − permeabilities identical to those of normal red cells.• A mathematical model of CO 2 /HCO 3 − 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. AbstractWe have investigated the previously published 'metabolon hypothesis' postulating that a close association of the anion exchanger 1 (AE1) and cytosolic carbonic anhydrase II (CAII) exists that greatly increases the transport activity of AE1. We study whether there is a physical association of and direct functional interaction between CAII and AE1 in the native human red cell and in tsA201 cells coexpressing heterologous fluorescent fusion proteins CAII-CyPet and YPet-AE1. In these doubly transfected tsA201 cells, YPet-AE1 is clearly associated with the cell membrane, whereas CAII-CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. Förster resonance energy transfer measurements fail to detect close proximity of YPet-AE1 and CAII-CyPet. The absence of an association of AE1 and CAII is supported by immunoprecipitation experiments using Flag-antibody against Flag-tagged AE1 expressed in tsA201 cells, which does not co-precipitate native CAII but co-precipitates coexpressed ankyrin. Both the CAII and the AE1 fusion proteins are fully functional in tsA201 cells as judged by CA activity and by cellular HCO 3 − permeability (P HCO 3 − ) sensitive to inhibition by 4,4 -Diisothiocyano-2,2 -stilbenedisulfonic acid. Expression of the non-catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of total intracellular CA activity. Overexpression of an N-terminally truncated CAII lacking the proposed site of interaction with the C-terminal cytoplasmic tail of AE1 substantially increases intracellular CA activity, as does overexpression of wild-type CAII. These variously co-transfected tsA201 cells exhibit a positive correlation between cellular P HCO 3 − and intracellular CA activity. The relationship reflects that expected from changes in cytoplasmic CA activity improving substrate supply to or removal from AE1, without requirement for a CAII-AE1 metabolon involving physical interaction. A functional contribution of the hypothesized CAII-AE1 m...

Here we ask the following: 1) what is the CO 2 permeability (PCO 2 ) of unilamellar liposomes composed of L-a-phosphatidylcholine (PC)/L-a-phosphatidylserine (PS) = 4:1 and containing cholesterol (Chol) at levels often occurring in biologic membranes (50 mol%), and 2) does incorporation of the CO 2 channel aquaporin (AQP)1 cause a significant increase in membrane PCO 2 ? Presently, a drastic discrepancy exists between the answers to these two questions obtained from mass-spectrometric 18 O-exchange measurements (Chol reduces PCO 2 100-fold, AQP1 increases PCO 2 10-fold) vs. from stopped-flow approaches observing CO 2 uptake (no effects of either Chol or AQP1). A novel theory of CO 2 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 CO 2 partial pressure (pCO 2 ) 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 PCO 2 = 0.01cm/s, and Chol-free vesicles exhibit ∼1 cm/s, and 2) the PCO 2 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 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.

In comparison with the P of other cells, this value is quite high and about identical to that of the human red cell membrane. As no major protein CO channels such as aquaporins 1 and 4 are present in rat cardiac sarcolemma, the high P of this membrane is likely due to its low cholesterol content of about 0.2 (mol cholesterol)·(mol total membrane lipids) . Previous work predicted a P of ≥0.1 cm s from this level of cholesterol. We conclude that the low cholesterol establishes a P high enough to render the membrane resistance to CO diffusion almost negligible, even under conditions of maximal O consumption of the heart.

We have studied cardiac and respiratory functions of aquaporin-1-deficient mice by the Pressure-Volume-loop technique and by blood gas analysis. In addition, the morphological properties of the animals' hearts were analyzed. In anesthesia under maximal dobutamine stimulation, the mice exhibit a moderately elevated heart rate of < 600 min−1 and an O2 consumption of ~0.6 ml/min/g, which is about twice the basal rate. In this state, which is similar to the resting state of the conscious animal, all cardiac functions including stroke volume and cardiac output exhibited resting values and were identical between deficient and wildtype animals. Likewise, pulmonary and peripheral exchange of O2 and CO2 were normal. In contrast, several morphological parameters of the heart tissue of deficient mice were altered: (1) left ventricular wall thickness was reduced by 12%, (2) left ventricular mass, normalized to tibia length, was reduced by 10–20%, (3) cardiac muscle fiber cross sectional area was decreased by 17%, and (4) capillary density was diminished by 10%. As the P-V-loop technique yielded normal end-diastolic and end-systolic left ventricular volumes, the deficient hearts are characterized by thin ventricular walls in combination with normal intraventricular volumes. The aquaporin-1-deficient heart thus seems to be at a disadvantage compared to the wild-type heart by a reduced left-ventricular wall thickness and an increased diffusion distance between blood capillaries and muscle mitochondria. While under the present quasi-resting conditions these morphological alterations have no consequences for cardiac function, we expect that the deficient hearts will show a reduced maximal cardiac output.

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this posttranslational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl-https://www.jbc.org/cgi/