Temperature dependence of water self-diffusion through lipid bilayers assessed by NMR

The temperature dependence of the coefficient of water self-diffusion across plane-parallel multib-ilayers of dioleoylphosphatidylcholine oriented on a glass support was studied in the 20–60°C range by pulsed field gradient NMR. The coefficient for transbilayer diffusion of water proved almost four orders of magnitude smaller than for bulk water, and 10 times smaller than that for lateral diffusion of lipid under the same conditions. The temperature dependence obeyed the Arrhenius law with apparent activation energy of 41 kJ/mol, much higher than that for bulk water (18 kJ/mol). The experimental data were analyzed using the “dissolution-diffusion” model, by simulating water passage through membrane channels, and by examining water exchange in states with different modes of translational mobility, including pore channels and bilayer defects. Each approach could take into account the role of bilayer permeability and assess the apparent activation energy for water diffusion in the hydrophobic part of the bilayer, which proved close to the value for bulk water. Estimates were obtained for water diffusion coefficients in the system, coefficients of bilayer permeability for water, and the influence of bilayer defects on the lateral and transverse diffusion coefficients.     

The effect of cholesterol on the lateral diffusion of phospholipids in oriented bilayers

Pulsed field gradient NMR was utilized to directly determine the lipid lateral diffusion coefficient for the following macroscopically aligned bilayers: dimyristoylphosphatidylcholine (DMPC), sphingomyelin (SM), palmitoyloleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC) with addition of cholesterol (CHOL) up to approximately 40 mol %. The observed effect of cholesterol on the lipid lateral diffusion is interpreted in terms of the different diffusion coefficients obtained in the liquid ordered (l(o)) and the liquid disordered (l(d)) phases occurring in the phase diagrams. Generally, the lipid lateral diffusion coefficient decreases linearly with increasing CHOL concentration in the l(d) phase for the PC-systems, while it is almost independent of CHOL for the SM-system. In this region the temperature dependence of the diffusion was always of the Arrhenius type with apparent activation energies (E(A)) in the range of 28-40 kJ/mol. The l(o) phase was characterized by smaller diffusion coefficients and weak or no dependence on the CHOL content. The E(A) for this phase was significantly larger (55-65 kJ/mol) than for the l(d) phase. The diffusion coefficients in the two-phase regions were compatible with a fast exchange between the l(d) and l(o) regions in the bilayer on the timescale of the NMR experiment (100 ms). Thus, strong evidence has been obtained that fluid domains (with size of micro m or less) with high molecular ordering are formed within a single lipid bilayer. These domains may play an important role for proteins involved in membrane functioning frequently discussed in the recent literature. The phase diagrams obtained from the analysis of the diffusion data are in qualitative agreement with earlier published ones for the SM/CHOL and DMPC/CHOL systems. For the DOPC/CHOL and the POPC/CHOL systems no two-phase behavior were observed, and the obtained E(A):s indicate that these systems are in the l(d) phase at all CHOL contents for temperatures above 25 degrees C.     

Lateral diffusion of cholesterol and dimyristoylphosphatidylcholine in a lipid bilayer measured by pulsed field gradient NMR spectroscopy

The pulsed field gradient NMR method for measuring self-diffusion has been used for a direct determination of the lateral diffusion coefficient of cholesterol, fluorine labeled at the 6-position, for an oriented lamellar liquid-crystalline phase of dimyristoylphosphatidylcholine (DMPC)/cholesterol/water. It is found that the diffusion coefficients of DMPC and cholesterol are equal over a large temperature interval. The apparent energy of activation for the diffusion process (58 kJ/mol) is about the same as for a lamellar phase of DMPC/water, whereas the phospholipid lateral diffusion coefficient is approximately four times smaller in the presence of cholesterol.     

Osmotic gradient-induced water permeation across the sarcolemma of rabbit ventricular myocytes

The mechanism of water permeation across the sarcolemma was characterized by examining the kinetics and temperature dependence of osmotic swelling and shrinkage of rabbit ventricular myocytes. The magnitude of swelling and the kinetics of swelling and shrinkage were temperature dependent, but the magnitude of shrinkage was very similar at 6 degrees, 22 degrees, and 37 degrees C. Membrane hydraulic conductivity, Lp, was approximately 1.2 x 10(-10) liter.N-1.s-1 at 22 degrees C, corresponding to an osmotic permeability coefficient, Pf, of 16 microns.s-1, and was independent of the direction of water flux, the magnitude of the imposed osmotic gradient (35-165 mosm/liter), and the initial cell volume. This value of Lp represents an upper limit because the membrane was assumed to be a smooth surface. Based on capacitive membrane area, Lp was 0.7 to 0.9 x 10(-10) liter.N-1.s-1. Nevertheless, estimates of Lp in ventricle are 15 to 25 times lower than those in human erythrocytes and are in the range of values reported for protein- free lipid bilayers and biological membranes without functioning water channels (aquaporin). Evaluation of the effect of unstirred layers showed that in the worst case they decrease Lp by < or = 2.3%. Analysis of the temperature dependence of Lp indicated that its apparent Arrhenius activation energy, Ea', was 11.7 +/- 0.9 kcal/mol between 6 degrees and 22 degrees C and 9.2 +/- 0.9 kcal/mol between 22 degrees and 37 degrees C. These values are significantly greater than that typically found for water flow through water-filled pores, approximately 4 kcal/mol, and are in the range reported for artificial and natural membranes without functioning water channels. Taken together, these data strongly argue that the vast majority of osmotic water flux in ventricular myocytes penetrates the lipid bilayer itself rather than passing through water-filled pores.     

Effect of temperature on water transport through aquaporins

The mean effective water self-diffusion coefficient in maize root segments under the effect of aquaporin blocker (mercuric chloride, 0.1 mM) was measured using the spin-echo NMR method with pulsed magnetic field gradient within the temperature range from 10 to 35 °C. HgCl₂ caused the reduction in water diffusion by 30 % as compared to the control samples. Temperature dependences of water self-diffusion coefficients showed two linear regions with different values of Q₁₀ and activation energy, E_a. As the temperature reduced from 20 to 10 °C, E_a values calculated from the Arrhenius plots were close to those of bulk water (20 ± 3 kJ mol⁻¹) and slightly changed for the sample pretreated HgCl₂. Within the temperature range from 25 to 35 °C the slope of temperature dependences became steeper and E_a values were 31 ± 3 kJ mol⁻¹ for the control and 40 ± 4 kJ mol⁻¹ for the treated sample. In the vicinity of 20 °C, the temperature dependence of water diffusion via the mercury-sensitive water channels showed extreme value. In the region, the specific area of the mercury-sensitive aquaporins was 0.004 % of the total cell surface area. The data indicate that water transfer via aquaporins is sensitive to temperature, and the contributions of the transmembrane pathways (aquaporins, lipid bilayer) differ in different temperature ranges.     

Effect of atrial natriuretic factor on the water permeability of endothelial cells.

Atrial natriuretic factor increases the water permeability of the whole endothelium. This study investigates how it would affect the transcellular osmotic water permeability of bovine artery endothelial cells. The cyclic-GMP production by the isolated cells was maximal for 10(-6)M atrial natriuretic factor within 30 minutes at 37 degrees C. The cyclic-GMP protein kinase cell concentration was 1.87 +/- 0.15 ng/mg protein. The control apparent water permeability of the cells measured by light scattering was 195 +/- 11 microns/sec (n = 5). Membrane folding revealed by light and scanning electron microscopy indicated that their true water permeability values would be close to 20-40 microns/sec, similar to the values for lipid membranes. The energy activation calculated from the temperature dependence of water permeability between 15 degrees C and 37 degrees C was 9.3 kcal/mol. This value suggests water movement through the lipid bilayer and not through water channels. Atrial natriuretic factor 10(-6)M did not significantly increase the water permeability of the cells. Hence, atrial natriuretic factor-stimulated increase in water permeability of the endothelium is more related to changes in paracellular water pathways than in transcellular water flux.     

Effect of grafted polyethylene glycol (PEG) on the size, encapsulation efficiency and permeability of vesicles

Liposomes have been prepared by the vesicle extrusion method (VETs) from mixtures of dipalmitoylphosphatidylcholine (DPPC), phosphatidylinositol (PI) and dipalmitoylphosphatidylethanolamine with covalently linked poly(ethylene glycol) molecular mass 5000 and 2000 (DPPE-PEG 5000 and DPPE-PEG 2000) covering a range of 0-7.5 mole%. The encapsulation of D-glucose has been studied and found to be markedly dependent on the mole% DPPE-PEG. The permeability of the liposomes to D-glucose has been measured both as a function of temperature and liposome composition. The permeability coefficients for D-glucose increase with mole% DPPE-PEG 5000 and with temperature over the range 25-50 degrees C. The activation energies for glucose permeability range from 90 to 23 kJ mol(-1). The decrease in activation energy with increasing temperature is attributed to an increasing number of bilayer defects as the liposome content of PEG-grafted lipid is increased. The dependence of D-glucose encapsulation as a function of PEG-grafted lipid content is discussed in terms of the conformation of the PEG molecules on the inner surface of the bilayer. For liposomes containing DPPE-PEG 5000 the relative percentage encapsulation of glucose, assuming that the PEG surface layer excludes glucose, is comparable to that predicted from the mushroom and brush conformational models.     

Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation

The molecular motions of membrane proteins in liquid-crystalline lipid bilayers lie at the interface between motions in isotropic liquids and in solids. Specifically, membrane proteins can undergo whole-body uniaxial diffusion on the microsecond time scale. In this work, we investigate the ¹H rotating-frame spin-lattice relaxation (T _1ρ) caused by the uniaxial diffusion of the influenza A M2 transmembrane peptide (M2TMP), which forms a tetrameric proton channel in lipid bilayers. This uniaxial diffusion was proved before by ²H, ¹⁵N and ¹³C NMR lineshapes of M2TMP in DLPC bilayers. When bound to an inhibitor, amantadine, the protein exhibits significantly narrower linewidths at physiological temperature. We now investigate the origin of this line narrowing through temperature-dependent ¹H T _1ρ relaxation times in the absence and presence of amantadine. Analysis of the temperature dependence indicates that amantadine decreases the correlation time of motion from 2.8 ± 0.9 μs for the apo peptide to 0.89 ± 0.41 μs for the bound peptide at 313 K. Thus the line narrowing of the bound peptide is due to better avoidance of the NMR time scale and suppression of intermediate time scale broadening. The faster diffusion of the bound peptide is due to the higher attempt rate of motion, suggesting that amantadine creates better-packed and more cohesive helical bundles. Analysis of the temperature dependence of $ { \ln }\left( {T_{1\rho }^{ - 1} } \right) $ indicates that the activation energy of motion increased from 14.0 ± 4.0 kJ/mol for the apo peptide to 23.3 ± 6.2 kJ/mol for the bound peptide. This higher activation energy indicates that excess amantadine outside the protein channel in the lipid bilayer increases the membrane viscosity. Thus, the protein-bound amantadine speeds up the diffusion of the helical bundles while the excess amantadine in the bilayer increases the membrane viscosity.     

Effects of diffusion limitation on immobilized nitrifying microorganisms at low temperatures

Activation energies of suspended and immobilized nitrifying bacteria were determined and compared to determine if diffusion limitation results in decreased sensitivity for temperature. The activation energy for the respiration activity of suspended Nitrosomonas europaea and Nitrobacter agilis was found to be 86.4 and 58.4 kJ mol(-1), respectively. The activation energy for oxygen diffusion in the support material, kappa-carrageenan, determined from the effect of temperature on the effective diffusion coefficient (D), was 17.2 kJ mol(-1). Consequently, the apparent actvation energy of diffusion limited cells should be lower. It was indeed shown that due to the effect of diffusion limitation and to temperature effects on the Monod constant K(s), the immobilized-cell activity was less sensitive to temperature. The apparent activation energy for immobilized Ns. europaea was between 28.6 and 94.2 kJ mol(-1) and for immobilized Nb. agilis between 1.4 and 72.9 kJ mol(-1), depending on the oxygen concentration and temperature. (c) 1995 John Wiley & Sons, Inc.     

Structural requirement for the rapid movement of charged molecules across membranes. Experiments with tetraphenylborate analogues.

Charge-pulse experiments were performed in the presence of structural analogues of tetraphenylborate (TPB) on membranes made of dioleoyl phosphatidylethanolamine and dioleoyl phosphatidylcholine. The analysis of the experimental results using a previously proposed model allowed the calculation of the partition coefficient, beta, and of the translocation rate constant, kappa i. The temperature dependence of the partition coefficients was used to calculate the thermodynamics of the adsorption of the lipophilic ions to the membranes. The analysis of the translocation rate constants obtained at different temperatures yielded detailed information on the free energy of the TPB-analogues within artificial lipid bilayer membranes, and on the activation energy of the translocation rate constants. The adsorption of the different TPB-analogues to the membranes was only slightly affected by their structure, whereas a dramatic influence of the structure on the free energy of the lipophilic ions within the membranes was observed. The free energy of the ions in the membranes decreased from triphenylcyanoborate (TPCB) to tetrakis(3-trifluoromethylphenyl)borate (TTFPB) by more than 31 kJ/mol (7.4 kcal/mol). This could be concluded from the observed increase in the translocation rate constant by almost six orders of magnitude. The change of the free energy in the membrane was used for the estimation of an effective radius of the TPB-analogues with respect to TPB.     

The water permeability of the monoolein/triolein bilayer membrane

The water permeability of the lipid bilayer can be used as a probe of membrane structure. A simple model of the bilayer, the liquid hydrocarbon model, views the membrane as a thin slice of bulk hydrocarbon liquid. A previous study (Petersen, D. (1980) Biochim. Biophys. Acta 600, 666–677) showed that this model does not accurately predict the water permeability of the monoolein/n-hexadecane bilayer: the measured activation energy for water permeation is 50% above the predicted value. From this it was inferred that the hydrocarbon chains in the lipid bilayer are more ordered than in the bulk hydrocarbon liquid. The present study tests the liquid hydrocarbon model for the monoolein/triolein bilayer, which has been shown to contain very little triolein in the plane of the membrane (Waldbillig, R.C. and Szabo, G. (1979) Biochim. Biophys. Acta 557, 295–305). Measurements of the water permeability coefficient of the bilayer are compared with predictions of the liquid hydrocarbon model based on measurements of the water permeability coefficient of bulk 8-heptadecene. The predicted and measured values agree quite closely over the temperature range studied (15–35°C): the predicted activation energy is 11.1±0.2 kcal/mol, whereas the measured activation energy for the bilayer is 9.8±0.7 kcal/mol. This close agreement is in contrast with the monoolein/n-hexadecane results and suggests that, insofar as water permeation is concerned, the liquid hydrocarbon model quite closely represents the monoolein/triolein bilayer.     

Autooxidation and oxygenation of human hemoglobin]

The processes of reversible oxygen binding and nonreversible autoxidation of human hemoglobin were studied. The activation energy of the oxygen binding, as determined by the temperature dependence of the P50 parameter, was 26 +/- 4 kJ/mol, the activation energy of the autoxidation, as determined by the temperature dependence of the apparent rate constant of autoxidation, was 120 +/- 15 kJ/mol. Pyridoxal phosphate decreased the oxygen affinity of hemoglobin, slightly diminished the cooperativity of the oxygenation process and unaffected the activation energy of the oxygen binding. Pyridoxal phosphate slightly reduced the Bohr coefficient value from 0.70 to 0.65. Pyridoxal phosphate, but not pyridoxal, raised the apparent rate constant of autoxidation reaction. The rate of autoxidation significantly increased as the pH value of the medium decreased, reflecting, probably, protonation of the distal histidine of the hemoglobin. The activation energy of autoxidation was independent of pH. Aliphatic alcohols also increased the rate of the autoxidation process, probably, either by stabilization of the hemoglobin T-state, or by direct nucleophilic displacement of the oxygen molecule.     

Effect of the lipid phase transition on the kinetics of H+/OH− diffusion across phosphatidic acid bilayers

The kinetics of H⁺/OH^? diffusion across dimyristoyl phosphatidic acid bilayer membranes was measured by following the absorbance of the pH-sensitive indicator Cresol red ($\text{o-}\text{cresolsulfonphthalein}$) entrapped in single lamellar vesicles after rapidly changing the external pH in a stopped-flow apparatus. The H⁺/OH^?-permeability coefficient was found to be in the 10^?5 to 10^?3 cm·s^?1 range. The lipid phase transition has a strong influence on the permeation kinetics as the permeability coefficients in the liquid-crystalline phase are drastically higher. The permeability shows no maximum at the phase transition temperature as is the case for other ions, but displays a similar temperature dependence as water permeation. This is also reflected in the high activation energy of approx. 20 kcal/mol and supports the hypothesis (Nichols, J.W. and Deamer, D.W. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2038–2042) of H⁺/OH^? permeation via hydrogen bonded water molecules. A second slower kinetic phase is also observed, where the permeation is obviously controlled by counterion diffusion. The temperature dependence of this slow process displays the for ion diffusion characteristic maximum in the permeability at the phase-transition temperature.     

The conductance of lecithin bilayers: The dependence upon temperature

The temperature dependence of the area-specific conductance of egg-lecithin/cholesterol bilayers formed with n-hexadecane in 1 mM KCl has been studied. From Arrhenius plots the activation energy for conduction was measured as 35 ± 2 kJ/mol. Comparison of this value with those predicted by various mechanisms whereby charge could be translocated through a bilayer indicate that it is extremely unlikely that ions pass directly through the hydrophobic interior. It is possible however, that ions are translocated across the bilayer through aqueous pores (with radius > 1 nm) which are an intrinsic, if fluctuating, part of the bilayer structure.     

The influence of temperature on the flocculation rate of renneted casein micelles

The flocculation rate constant of completely renneted casein micelles in milk ultrafiltrate was measured by Rayleigh light scattering between 20 and 35 degrees C. In this temperature range an apparent energy of activation of 103 kJ mol (+/-11 kJ mol : n = 50) was measured. At 15 degrees C clotting was not longer perceptible. The activation of the flocculation between 20 and 35 degrees C is explained not so much by the height of the energy barrier separating the clotting micelles, as by the very negative temperature coefficient of that barrier. In line with this conclusion it is suggested that renneted micelles adhere through hydrophobic bonding. The flocculation rate constant of renneted casein micelles is independent of micelle size at the four temperature levels studied.     

Water and nonelectrolyte permeability of lipid bilayer membranes

Both the permeability coefficients (Pd's) through lipid bilayer membranes of varying composition (lecithin [L], lecithin:cholesterol [LC], and spingomyelin:cholesterol [SC]) and the n-hexadecane:water partition coefficients (Knc's) of H2O and seven nonelectrolytes (1,6 hexanediol, 1,4 butanediol, n-butyramide, isobutyramide, acetamide, formamide, and urea) were measured. For a given membrane compositiin, Pd/DKnc (where D is the diffusion constant in water) is the same for most of the molecules tested. There is no extraordinary dependence of Pd on molecular weight; thus, given Pd(acetamide), Pd(1,6 hexanediol) is correctly predicted from the Knc and D values for the two molecules. The major exceptions are H2O, whose value of Pd/DKnc is about 10-fold larger, and urea, whose value is about 5-fold smaller than the general average. In a "tight" membrane such as SC, Pd(n- butyramide)/Pd(isobutyramide)=2.5; thus this bilayer manifests the same sort of discrimination between branched and straight chain molecules as occurs in many plasma membranes. Although the absolute values of the Pd's change by more than a factor of 100 in going from the tightest membrane (SC) to the loosest (L), the relative values remain approximately constant. The general conclusion of this study is that H2O and nonelectrolytes cross lipid bilayer membranes by a solubility- diffusion mechanism, and that the bilayer interior is much more like an oil (a la Overton) than a rubber-like polymer (a la Lieb and Stein).     

The State of Water in the Isolated Toad Bladder in the Presence and Absence of Vasopressin

An attempt has been made to assess the validity of applying the frictional and viscous coefficients of bulk water to the movement of water and solutes through the urinary bladder of the toad. The temperature dependence of diffusion of THO, C¹⁴-urea, C¹⁴-thiourea, and net water transfer across the bladder was determined in the presence and absence of vasopressin. The activation energy for diffusion of THO was 9.8 kcal per mole in the absence of vasopressin and 4.1 kcal per mole with the hormone present. Activation energies simultaneously determined following vasopressin for diffusion and net transfers of water were similar, and in the same range as known activation energies for diffusion and viscous flow in water. Urea had activation energies for diffusion of 4.1 and 3.9 kcal per mole in the absence and presence of vasopressin, respectively. Thiourea had a high activation energy for diffusion of 6.3 kcal per mole, which was unchanged, 6.6 kcal per mole, following hormone. These findings suggest that in its rate-limiting permeability barrier, water is present in a structured state, offering a high resistance to penetration by water. Vasopressin enlarges the aqueous channels so that the core of water they contain possesses the physical properties of ordinary bulk water. Urea penetrates the tissue via these aqueous channels while thiourea is limited by some other permeability barrier.     

Penetration of ascorbic acid into bilayer phospholipid membranes

Using the EPR method, the temperature dependencies of the rates of ascorbic acid-induced reduction of nitroxyl radicals carrying the nitroxyl fragment in different positions of the fatty acid chain [N(4-methylidene++-1-oxyl-2,2,5,5-tetramethyl-3-imidazolidine hydrazine)]myristic acid (I) and 1-oxyl-2,2-dimethyloxazolidine derivatives of 5-ketostearic (II) and 12-ketostearic (III) acids incorporated into egg phosphatidylcholine liposomal membranes were studied. The reduction rates, activation energy and shape of kinetic curves were found to be dependent on the mode of liposome preparation (ultrasonication or reverse phase evaporation), label type and chemical composition of the membrane (with regard to the presence or absence of stearic acid). The coefficients of partition and diffusion of ascorbic acid through the membrane lipid bilayer were calculated from the rates of transbilayer (flip-flop) diffusion of I and ascorbate penetration inside the liposomes containing Fremi salt nitroxyl radical. The experimental results formed the basis for a hypothesis on the dependence of the rate of membrane-embedded spin probe reduction on the ascorbate distribution pattern inside the lipid bilayer.     

Extrinsic charge movement in the squid axon membrane

The absorption of the lipophilic anions dipycrilamine (DPA^-) and tetraphenylborate (TPhB^-) by the lipid matrix of the squid axon membrane, and the kinetics of their translocation, were studied by the charge pulse relaxation technique. The axons were treated with tetrodotoxin (TTX) and 4-aminopyridine to block the ionic currents responsible for nerve excitation. At high enough concentrations of absorbed ions ( 10^-12 mol/cm²) the membrane voltage relaxation following a brief current pulse consisted mainly of two exponential components, whose time constants and relative amplitudes were used for estimating the translocation rate constant, K, and the density of absorbed ions, N. These measurements were performed at different hydrostatic pressures in the range 1–100 MPa ( 1,000 atm), and at different temperatures in the range 5° C–20° C. Both K and N were found to be little affected by pressure. The pressure dependence of K indicated that the translocation of lipophilic ions across the nerve membrane involves activation volumes of the order of 5 cm³/mol. In all experiments the passive membrane resistance was little affected by pressures up to 80 MPa. However, above 100 MPa it fell dramatically to low values, presumably because of phase separation phenomena between the membrane components. The temperature dependence of K, both for DPa^- and TPhB^-, implied an activation energy for ion translocation of the order of 60 kJ/mol, close to that measured in artificial lipid bilayers.It is concluded that the lipid bilayer structure of the nerve membrane is not modified by pressures below 80 MPa and that the intramembrane movements of relatively small charged groups cannot account for the large activation volumes involved in the gating of ionic channels.