"Adaptive" behavior of ligand-gated ion channels: constraints by thermodynamics.

The calcium-induced calcium release channel of the cardiac sarcoplasmic reticulum has been reported to inactivate in a novel manner (termed "adaptation"), which permits reactivation by exposure to successively higher concentrations of calcium. I examined the limitations placed by thermodynamics on the possible kinetic mechanisms for such behavior. The mechanism suggested by Gyorke and Fill, in which the affinity of a calcium-binding site decreases during adaptation, is not thermodynamically feasible for a passive system, but requires an external input of free energy. Possible sources of such energy are 1) metabolic energy, which is excluded by the fact that adaptation was observed in isolated channels in the absence of ATP, or 2) coupling of ion permeation to gating, for which there is currently no evidence. I derived a general limit on the thermodynamic feasibility of a sequence of channel activations and adaptations, irrespective of channel kinetics, from the requirement that the free energy must decrease during the spontaneous evolution of the system from the state existing immediately after a step increase in [Ca2+] to the state of maximum open probability that follows. The opening of the channel must involve an increase in free energy, which must be compensated by the free energy released by the incremental binding of calcium. This requirement leads to a complicated system of inequalities, which was simplified and manipulated algebraically into the form of a linear programming problem. Numerical solution of this problem showed that the sequence of adaptations of the SR channel observed by Gyorke and Fill requires the presence of at least 10 calcium-binding sites on the channel if it is to occur in the absence of exogenous sources of free energy. This indicates either that a large number of calcium-binding sites participate in the regulation of the SR calcium release channel, or that the existing data are significantly flawed with respect to the low open probability in the resting state, the importance of "calcium spike" artifacts from flash photolysis, or both.     

Activation of a cGMP-sensitive calcium-dependent chloride channel may cause transition from calcium waves to whole cell oscillations in smooth muscle cells

In vitro, alpha-adrenoreceptor stimulation of rat mesenteric small arteries often leads to a rhythmic change in wall tension, i.e., vasomotion. Within the individual smooth muscle cells of the vascular wall, vasomotion is often preceded by a period of asynchronous calcium waves. Abruptly, these low-frequency waves may transform into high-frequency whole cell calcium oscillations. Simultaneously, multiple cells synchronize, leading to rhythmic generation of tension. We present a mathematical model of vascular smooth muscle cells that aims at characterizing this sudden transition. Simulations show calcium waves sweeping through the cytoplasm when the sarcoplasmic reticulum (SR) is stimulated to release calcium. A rise in cGMP leads to the experimentally observed transition from waves to whole cell calcium oscillations. At the same time, membrane potential starts to oscillate and the frequency approximately doubles. In this transition, the simulated results point to a key role for a recently discovered cGMP-sensitive calcium-dependent chloride channel. This channel depolarizes the membrane in response to calcium released from the SR. In turn, depolarization causes a uniform opening of L-type calcium channels on the cell surface, stimulating a synchronized release of SR calcium and inducing the shift from waves to whole cell oscillations. The effect of the channel is therefore to couple the processes of the SR with those of the membrane. We hypothesize that the shift in oscillatory mode and the associated onset of oscillations in membrane potential within the individual cell may underlie sudden intercellular synchronization and the appearance of vasomotion.     

荔枝雌蕊发育过程中钙分布变化与细胞程序性死亡

应用焦锑酸钾沉淀法研究了荔枝雌花和雄花雌蕊发育过程中钙的分布变化。在大孢子母细胞阶段,雌花近珠孔内珠被细胞和花柱细胞的钙沉淀颗粒主要分布在细胞壁和细胞间隙,少部分在液泡;雌花花柱维管细胞中含有很多的钙沉淀颗粒;在雄花的近珠孔内珠被细胞钙沉淀颗粒大多在液泡中;雄花花柱细胞和维管细胞中钙沉淀颗粒很少。大孢子母细胞减数分裂后,雌花雌蕊继续发育,雄花雌蕊败育。雌花维管中的钙沉淀颗粒数量减少,可能被转运到将要发生花粉萌发和受精的部位。雌花近珠孔内珠被细胞壁的钙沉淀颗粒分布增加,花柱细胞从上（近柱头）到下（近子房）钙沉淀颗粒量递增。雄花近珠孔内珠被细胞发生程序性死亡：液泡中的钙进入细胞核启动细胞程序性死亡,核周隙与质膜腔形成连续的通道,钙在核与细胞质之间的流动不受限制;在特定的时间段,钙沉淀颗粒出现在线粒体、过氧化物体和线型内质网的外膜上。钙在细胞中重新分布可能触发和调节细胞程序性死亡的进程。缺乏钙沉淀颗粒的雄花花柱细胞迅速解体。     

Minimum energy analysis of membrane deformation applied to pipet aspiration and surface adhesion of red blood cells.

An experimental procedure is demonstrated which can be used to determine the interfacial free energy density for red cell membrane adhesion and membrane elastic properties. The experiment involves micropipet aspiration of a flaccid red blood cell and manipulation of the cell proximal to a surface where adhesion occurs. A minimum free energy method is developed to model the equilibrium contour of unsupported membrane regions and to evaluate the partial derivatives of the total free energy, which correspond to the micropipet suction force and the interfacial free energy density of adhesion. It is shown that the bending elasticity of the red cell membrane does not contribute significantly to the pressure required to aspirate a flaccid red cell. Based on experimental evidence, the upper bound for the bending or curvature elastic modulus of the red cell membranes is 10-12 ergs (dyn-cm). Analysis of the adhesion experiment shows that interfacial free energy densities for red cell adhesion can be measured from a lower limit of 10-4 ergs/cm2 to an upper limit established by the membrane tension for lysis of 5-10 ergs/cm2.     

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.     

An aromatic heptaene antibiotic levorin and its derivatives in muscle activity]

The review is concerned with the outlooks for the use of levorin, a membrane active and channel forming polyene antibiotic, and its alkyl derivatives in muscle activity. In complex with cholesterol and ergosterol, the aromatic heptaene antibiotic levorin forms structural ionic channels of the molecular size in the lipid and cell membranes. Levorin increases the membrane permeability for monosucrose and other neutral molecules as follows: H2O > urea > acetamide > glycerine > ribose > arabinose > glucose > saccharose. As a channel forming compound, levorin is able to induce in the cell membranes of the muscle fibres formation of additional channels permeable for the cations and to increase the flow of the energy dependent substrates to the cells and the outburst of the metabolites from them during intensive muscle activity. Levorin several times decreases the surface tension of aqueous solutions. In some models of experimental animals levorin promoted an increase of the blood fluidity and accelerated the blood stream in the blood vessels both in rest and in muscle activity. Physical load in a high power zone increases the intensity of lipid peroxidation that results in fatigue and lower physical efficiency. Possible prevention of an increase of the rate of free radical reactions by levorin and its alkyl derivatives providing higher antioxidant protection is discussed.     

Thermodynamic and mechanical properties of model mitochondrial membranes

Cardiolipin is a unique four-tailed, doubly negatively charged lipid found predominantly within the inner mitochondrial membrane, and is thought to be influential in determining membrane potential and permeability. To determine the role of cardiolipin in modulating the properties of membranes, this study investigates the thermodynamics of mixed cardiolipin and phosphatidylcholine monolayers and bilayers. Gibbs free energy analysis of mixed monolayers indicates that at low cardiolipin concentrations (5-10 mol%), there is a positive deviation from ideality on a pure water subphase, while at physiological salt concentrations a negative deviation from ideality is observed. The mechanical properties of bilayers containing cardiolipin were measured using micropipette aspiration. Both apparent area compressibility modulus, as well as lysis tension, decrease with increasing cardiolipin content. This destabilization indicates a decrease in the cohesive energy of the membrane. This interplay between interactions of lipids in monolayers and bilayers, suggests cardiolipin plays a dual role in modulating membrane properties. Cardiolipin enhances lateral interactions between lipids within monolayer leaflets, while simultaneously decreasing the cohesive energy of membranes at physiologically relevant concentrations. Taken together, these findings correlate with the decreased permeability and creation of folds in the inner mitochondrial membrane.     

Membrane tension, lipid adaptation, conformational changes, and energetics in MscL gating

This study aims to explore gating mechanisms of mechanosensitive channels in terms of membrane tension, membrane adaptation, protein conformation, and energetics. The large conductance mechanosensitive channel from Mycobacterium tuberculosis (Tb-MscL) is used as a model system; Tb-MscL acts as a safety valve by releasing small osmolytes through the channel opening under extreme hypoosmotic conditions. Based on the assumption that the channel gating involves tilting of the transmembrane (TM) helices, we have performed free energy simulations of Tb-MscL as a function of TM helix tilt angle in a dimyristoylphosphatidylcholine bilayer. Based on the change in system dimensions, TM helix tilting is shown to be essentially equivalent to applying an excess surface tension to the membrane, causing channel expansion, lipid adaptation, and membrane thinning. Such equivalence is further corroborated by the observation that the free energy cost of Tb-MscL channel expansion is comparable to the work done by the excess surface tension. Tb-MscL TM helix tilting results in an expanded water-conducting channel of an outer dimension similar to the proposed fully open MscL structure. The free energy decomposition indicates a possible expansion mechanism in which tilting and expanding of TM2 facilitates the iris-like motion of TM1, producing an expanded Tb-MscL.     

Migration of zebrafish primordial germ cells: a role for myosin contraction and cytoplasmic flow

The molecular and cellular mechanisms governing cell motility and directed migration in response to the chemokine SDF-1 are largely unknown. Here, we demonstrate that zebrafish primordial germ cells whose migration is guided by SDF-1 generate bleb-like protrusions that are powered by cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where activation of myosin contraction occurs. Separation of the acto-myosin cortex from the plasma membrane at these sites is followed by a flow of cytoplasm into the forming bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in free calcium that in turn activates myosin contraction in the part of the cell responding to higher levels of the ligand SDF-1. The biased formation of new protrusions in a particular region of the cell in response to SDF-1 defines the leading edge and the direction of cell migration.     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.     

Influence of ion occupancy and membrane deformation on gramicidin A channel stability in lipid membranes.

The average lifetime of gramicidin A channels in monoolein/decane bilayer membranes was measured. The results support the hypothesis of channel stabilization by ion occupancy. The effects of electric field and salt concentration are consistent with the expected effects on both occupancy and membrane compression. The lifetime in asymmetric solutions with divalent cation blockers on one side of the membrane shows a voltage dependence such that the lifetime decreases for positive voltages applied from the blocking side and increases for negative voltages. This result strongly supports the occupancy hypothesis. The lifetime increases with permeant ion concentration, and at the one molar level it also increases with voltage. The voltage dependence of lifetime for a low concentration of permeant ion depends on the total salt level. The results for these conditions are consistent with the assumption that membrane compression also influences the lifetime, even for the "soft" solvent-containing membrane considered here. It is proposed that the channel nearest neighbor lipids need not be fixed in a plane at the channel end. Using a liquid crystal model it may then be shown that surface tension is the major component of the membrane deformation free energy, which may explain the significant effects of the membrane compression on the lifetime.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

THE DEFORMABILITY AND THE WETTING PROPERTIES OF LEUCOCYTES AND ERYTHROCYTES

The resistance to deformation of polymorphonuclear neutrophile leucocytes under the conditions of our observations has been shown to be on the average considerably less than the resistance to deformation of large mononuclear leucocytes. It is recognized of course that the viscosity of leucocytes, as of other cells, may be markedly influenced by osmotic conditions (17), by the reaction of the suspending medium (18, 19), by temperature, or by injury (20, 21). Although the conditions of our observations were quite different from those of the body, they were nevertheless closely similar to those of simultaneous phagocytosis experiments in which the cells functioned exceedingly well (3). Moreover E. R. and E. L. Clark (22) have noted that polymorphonuclear leucocytes in the tails of living tadpoles were more fluid than the macrophages. And Goss (23) in microdissecting human polymorphonuclear neutrophiles reports that they are more fluid than the clasmatocytes and monocytes studied by Chambers and Borquist (24). Other types of leucocytes have in our experience seemed to fall between the large mononuclear and the polymorphonuclear leucocytes in their average resistance to the interfacial tensions. The leucocyte of each type studied is surrounded by an exceedingly delicate membrane. This membrane appears under the dark-field microscope as a pale, silvery line not distinguishable by inspection alone from a simple phase boundary between two immiscible liquids. That this is a membrane, however, and not a mere interface between immiscible phases, seems certain. In the first place the cell cytoplasm and the suspending medium are not immiscible. When the cell organization is broken down by the interfacial tension the greater part of the cell contents is immediately dissolved or dispersed. Goss (23) has noted that when the membrane is torn with a microdissection needle disintegration at once spreads over the membrane and the cytoplasm undergoes profound change. Moreover it is improbable that a simple phase boundary could exist in the presence of so much protein, lipoid, and other surface active materials as are present in protoplasm; the tendency of these substances to lower the free interfacial energy must necessarily tend to their adsorption in the interface until, if sufficient material is available at the interface, an adsorption film or membrane may be formed. Kite (25), in a pioneer microdissection study, described the polymorphonuclear leucocyte as "naked" protoplasm. The contradiction between this statement and those just made is more apparent than real. For the capacity swiftly to form a limiting membrane between itself and other liquids is an attribute of "naked" protoplasm, as has been shown by the beautiful experiments of Chambers (20). The present study of the wetting properties of leucocytes shows that their external membranes are hydrophilic, a character suggesting a surface in which proteins, probably bound water and salts (27), possibly the polar radicles of soaps or fatty acids, rather than non-polar lipoid groupings, are predominantly exposed. This makes it the more remarkable that a cell of such fluidity as for instance the polymorphonuclear leucocyte, composed largely of water and of water-soluble materials, should maintain its integrity in an aqueous medium with the aid of a membrane so delicate and so mobile. The mobility of the membrane, frequently extended in forming new pseudopodia or spreading over the surface of particles being ingested, must require constant entrance into and exit from the membrane of component materials, and their constant reorganization there. The limiting factors in the reformation of such a membrane would be the amounts of adsorbable materials available and their rates of movement up to the surface rather than the time required for orientation there, since the latter phenomenon is exceedingly rapid. Harkins (29), for instance has calculated that at a water-water vapor interface at 20°C., from the area occupied by one molecule of water, a molecule would jump out into the vapor and a vapor molecule would fall into this area of the surface 7,000,000 times in one second; the time of orientation of the water molecule he estimates to be of the order of 1/100,000,000 second or less. The mammalian erythrocyte possesses a surface membrane capable of being folded and of withstanding tension in the interface. This has also been stretched by microdissection needles (21). The surface of the erythrocyte, as evidenced by its wetting properties, is relatively hydrophobic, relatively non-polar in character, as compared with the leucocyte. Evidence indicating that the erythrocyte surface contains both lipoid and protein components has been summarized in earlier papers (8, 30). We have little to add here other than to point out that the wetting properties of the chicken erythrocyte surface are similar to those fully described for the mammal. A serious source of error in certain isoelectric point determinations is discussed.     

Evidence for high affinity binding-protein dependent transport systems in gram-positive bacteria and in Mycoplasma

Gram-negative bacteria are surrounded by two membranes. In these bacteria, a class of high affinity transport systems for concentrating substrates from the medium into the cell, involves a binding protein located between the outer and inner membranes, in the periplasmic region. These 'periplasmic binding-proteins' are thought to bind the substrate in the vicinity of the inner membrane, and to transfer it to a complex of inner membrane proteins for concentration into the cytoplasm. We report evidence leading us to propose that a Gram-positive bacterium, Streptococcus pneumoniae, and a mycoplasma, Mycoplasma hyorhinis, which are surrounded by a single membrane and have therefore no periplasmic region, possess an equivalent to the high affinity periplasmic binding-protein dependent transport systems, i.e. extra-cytoplasmic binding lipoprotein dependent transport systems. The 'binding lipoproteins' would be maintained at proximity of the inner membrane by insertion of their N-terminal glyceride-cysteine into this membrane.     

Outer and inner membrane preparations of extreme thermophile and their physico-chemical properties

Outer and inner (cytoplasmic) membranes were partially purified from the gram negative extremely thermophilic bacteria, Thermus thermophilus HB-8 by sucrose density gradient centrifugation. In spite of our efforts to separate them, the inner membrane fraction contained some outer membrane components as determined by enzyme assay and electrophoresis. When studied by 5DS spin labeling, the outer membranes showed a larger 2T11 value (lower fluidity) than the inner membranes, although the fatty acid compositions were similar. The inner membranes of the cells cultured at higher temperature showed a larger 2T11 value than the cells cultured at lower temperature. A similar phenomenon was observed with the TEMPO parameter of liposomal membranes. The upper break point (Th) of the inner membranes observed by spin labeling was slightly lower than the culture temperature of the cells, and the lower break point (T1) corresponded well to the lowest temperature limit of growth. The calorimetric heating curve of the inner membranes had a broader temperature range of transition than that of the liposomal membranes. The transition temperature observed by calorimetry seems to reflect the melting properties of the membrane lipids, while fatty acid spin probe probably reports the local environment of the membrane, which is more directly related to its biological function.     

Influx of Ca2+ via Cav1.3 calcium channels in satellite cells of muscle fibers in rats

Voltage-dependent L-type Ca_v1.3 channels have been detected in satellite cells localized to muscle fibers. It was established that the action of carbachol, which activates nicotinic acetylcholine receptors and causes cell membrane to depolarize, resulted in the activation of these channels. In addition, verapamil and amlodipine, selective L-type calcium channel blockers, suppressed extracellular calcium influx into the cytoplasm. It was noted that in a calcium-free medium, carbachol had no influence on the concentration of calcium in the cytoplasm of satellite cells, whereas adrenaline induced calcium efflux from intracellular stores. In addition, calcium influx into the cytoplasm was not suppressed by verapamil and amlodipine under the action of adrenaline and noradrenalin in a medium with calcium, and an ICI-118551 blocker of β₂-adrenoreceptros significantly decreased the increase in the concentration of calcium in the cytoplasm.     

Effects of chlorpromazine HCl on the structural parameters of bovine brain membranes

Fluorescence probes located in different membrane regions were used to evaluate the effects of chlorpromazine .HCl on structural parameters (transbilayer lateral mobility, annular lipid fluidity, protein distribution, and lipid bilayer thickness) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. The experimental procedure was based on the selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, radiationless energy transfer from the tryptophan of membrane proteins to Py-3-Py, and energy transfer from Py-3-Py monomers to 1-anilinonaphthalene-8-sulfonic acid (ANS). In this study, chlorpromazine .HCl decreased the lateral mobility of Py-3-Py in a concentration dependent-manner, showed a greater ordering effect on the inner monolayer than on the outer monolayer, decreased annular lipid fluidity in a dose dependent-manner, and contracted the membrane lipid bilayer. Furthermore, the drug was found to have a clustering effect on membrane proteins.     

Deformation free energy of bilayer membrane and its effect on gramicidin channel lifetime.

The deformation free energy of a lipid bilayer is presented based on the principle of a continuum theory. For small deformations, the free energy consists of a layer-compression term, a splay-distortion term, and a surface-tension term, equivalent to the elastic free energy of a two-layer smectic liquid crystal with surface tension. Minimization of the free energy leads to a differential equation that, with boundary conditions, determines the elastic deformation of a bilayer membrane. When a dimeric gramicidin channel is formed in a membrane of thickness greater than the length of the channel, the membrane deformation reduces the stability of the channel. Previously this effect was studied by comparing the variation of channel lifetime with the surface tension of bilayers (Elliott, J. R., D. Needham, J. P. Dilger, and D. A. Hayden, 1983, Biochim. Biophys. Acta, 735:95-103). The tension was assumed to pull a dimer for a distance z before the channel loses ion conductivity. To account for the data, z was found to be 18 A. With the deformation free energy, the data can be accounted for with z less than or approximately to 1 A, which is consistent with the breaking of hydrogen bonds in a dimer dissociation. Increasing the strength of lipid-protein interactions is not the only consequence of the complete free energy compared with the previous discussions. It also changes the shape of membrane deformation around an embedded channel from convex to concave, and increases the range of deformation from less than 10 A to greater than 20 A. Clearly these will be important factors in the general considerations of lipid-protein interactions and membrane-mediated interactions between proteins. In addition, thermal fluctuations of a membrane are calculated; in particular, we calculate the relations between the intrinsic thickness and the experimentally measured values. The experimental parameters of monoolein-squalene membranes are used for quantitative analyses.     

Ionic selectivity, saturation and block in gramicidin A channels: I. Theory for the electrical properties of ion selective channels having two pairs of binding sites and multiple conductance states.

A model for the gramicidin A channel is proposed which extends existing models by adding a specific cationic binding site at each entrance to the channel. The binding of ions to these outer channel sites is assumed to shift the energy levels of the inner sites and barriers and thereby alter the channel conductance. The resulting properties are analyzed theoretically for the simplest case of two inner sites and a single energy barrier. This for-site model (two outer and two inner) predicts that the membrane potential at zero current (Uo) should be a Goldman-Hodgkin-Katz equation with concentration-dependent permeability ratios. The coefficients of the concentration-dependent terms are shown to be related to the peak energy shifts of the barrier and to the binding constants of the outer sites. The thory also predicts the channel conductance in symmetrical solutions to exhibit three limiting behaviors, from which the properties of the outer and inner sites can be characterized. In two-cation symmetrical mixtures the conductance as a function of mole fraction is shown to have a minimum, and the related phenomenon of inhibition and block exerted by one ion on the other is explained explicitly by the theory. These various phenomena, having ion interactions in a multiply occupied channel as a common physical basis, are all related (by the theory) through a set of measurable parameters describing the properties of the system.     

Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity.

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanide-sensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 microM and 20 microM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured. Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity. The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.