Properties of channels from rat liver gap junction membrane fractions incorporated into planar lipid bilayers

Rat membrane fractions highly enriched for gap junctions can be incorporated into planar lipid bilayers exhibiting channel currents with both voltage-dependent and independent components. Voltage dependence, however, is only one of the characteristics of liver gap junction channels. Other features include poor ionic selectivity and sensitivity to calcium, pH, octanol and to some intracellularly applied antibodies. To further test the junctional nature of channels from membrane fractions highly enriched in gap junctions incorporated into lipid bilayers we studied the sensitivity of these channels to uncoupling agents and determined channel selectivity properties. We found the incorporated channels to be insensitive to calcium and octanol, and in most cases to pH in the range of 5-7, suggesting that either these agents do not interact directly with the junctional channels or that the corresponding gating regions are inactivated during the isolation and reconstitution procedures. Attempts to block channel activity using polyclonal and monoclonal connexin 32 antibodies were generally unsuccessful, although one antibody (a monoclonal directed against the carboxy terminus portion of connexin32) blocked channel activity. Selectivity measurements indicated that the incorporated channels were slightly cation selective (PNa=Pk > PCl) and were permeable to large ions. These results further support the idea that functional connexin32 gap junction channels are present in channel activity recorded from rat liver junctional membranes incorporated into planar bilayers

Glucose-6-phosphate phosphohydrolase activity in guinea pig liver microsomes is influenced by phosphatidylcholine. Interaction with cholesterol-enriched membranes.

Guinea pig liver microsomal membranes were cholesterol-enriched by feeding guinea pigs a high-cholesterol diet. Cholesterol enrichment as well as partial lipid removal of normal native microsomes by acetone-butanol extraction resulted in 40-50% loss in activity of the glucose-6-phosphate phosphohydrolase (G-6-Pase) (EC 3.1.3.9) enzyme system. The activity was restored by supplementation of microsomal total phospholipid (PL) and its phosphatidylcholine (PC) species but not with microsomal neutral lipids, cholesterol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin or diphosphatidylglycerol (cardiolipin). The activity was decreased by sodium deoxycholate but enhanced by dimethylsulfoxide. Egg-yolk PC and asolectin influenced the activity of the enzyme to the same extent as microsomal PC did. Lipid depletion and cholesterol produced an increase in Km while the Vmax was lowered. The non-linearity in the Arrhenius plot of the native microsomes was lost on lipid removal and cholesterol enrichment. The energy of activation (Ea) calculated from the continuous line was found to be lowered to the level that was observed above the break points in intact microsomes. Addition of microsomal PC to the assay system decreased the Km of the enzymatic reaction in native membranes, in partially lipid-depleted and cholesterol-enriched membranes, but did not alter the Vmax values and only marginally influenced the non-linear relationship of the Arrhenius expression of temperature dependence. The ability of immature rat liver phospholipid exchange protein to introduce alien PL into microsomal membrane was used to study the lipid dependence of G-6-Pase. Protein-catalyzed and detergent (cholate)-mediated membrane PL exchange for egg-yolk PC from the PC/cholesterol unilamellar liposomes resulted in substantial loss of enzyme activity. The discrepancies in the influence of PC on G-6-Pase were interpreted by assuming that the enzyme was a two-component system, a surface-located substrate transporter unit and a membrane integral catalytic phosphohydrolase unit. The lipid microenvironment and PL requirement in particular, could be different for the two components, although they represented a single functional unit at the time of enzymatic reaction.

Involvement of ATP-dependent aminophospholipid translocation in maintaining phospholipid asymmetry in diamide-treated human erythrocytes.

Crosslinking of membrane skeletal proteins such as spectrin by oxidation of their SH-groups can be provoked by treatment of intact erythrocytes with diamide. Shortly after exposure of human erythrocytes to diamide and despite the transverse destabilization of the lipid bilayer that was observed in these cells (Franck, P.F.H., Op den Kamp, J.A.F., Roelofsen, B. and Van Deenen, L.L.M. (1986) Biochim. Biophys. Acta 857, 127-130), no abnormalities could be detected regarding the asymmetric distribution of the phospholipids when probed by either the prothrombinase assay or brief exposure of the cells to a modified phospholipase A2 with enhanced membrane penetrating capacity. This asymmetry appeared to undergo dramatic changes however, when the ATP content of the cytosol had decreased to less than 10% of its original level during prolonged incubation of the treated cells. These observations indicate that the initial maintenance of phospholipid asymmetry in diamide-treated erythrocytes can be solely ascribed to the action of the ATP-dependent aminophospholipid translocase. This view is supported by experiments involving radiolabeled phospholipids of which trace amounts had been inserted into the outer membrane leaflet of diamide-treated red cells and which still showed a preferential translocation of both aminophospholipids in favour of the inner monolayer, be it that the efficiency of the translocase was found to be impaired when compared to control cells.

Theory of protein-induced lateral phase separation in lipid membranes.

An account is given of the current status of theoretical modeling of the phase equilibria in lipid membranes with intrinsic proteins. Special attention is paid to the description of lateral phase separation, which is important for membrane function since it may lead to biologically differentiated regions. We discuss in particular the mattress-model approach by Mouritsen and Bloom, who take matching between protein and lipid hydrophobic thicknesses as a determining factor for the phase behavior. The model has been developed in the framework of phenomenological thermodynamic solution theory. The predictions of the theory are compared to a variety of experimental measurements, including those of membrane recombinants of the protein content of the reaction center and antenna protein of the bacterial photosynthetic apparatus as well as the erythrocyte band 3 protein. The physical effects of lipid-protein interactions are contrasted to those of lipid-cholesterol interactions. The concept of hydrophobic matching is then used as a basis for discussing a possible relationship between membrane thickness and physiological function.

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers.

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.

Gating properties of channels formed by Colicin Ia in planar lipid bilayer membranes.

Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (greater than +10 mV) are applied to the cis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant (tau) of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric around Vo (voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction in n, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be it cis or trans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change in n. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open, F(v). For a given voltage, F(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino- acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel.

Ion transport mediated by copolymers composed of polyoxyethylene and polyoxypropylene.

Block copolymers composed of polyoxyethylene and polyoxypropylene were found to increase the influx of Na+ and the efflux of K+ from human erythrocytes. They were, however, ineffective at promoting the transport of Ca2+. The size of the ion fluxes induced by the copolymers correlated with their efficacy in stimulating inflammation. These compounds were also found to induce conductance increases in planar lipid bilayers in a nonvoltage dependent and nonstepwise manner. In both experimental systems, ion transport was facilitated only under temperature and ionic-strength conditions in which the polymers form aggregates in aqueous solution. In neither system did the concentration dependence of transport activity exhibit a pronounced cooperativity. These observations are consistent with the view that aqueous monomers of these surface active agents partition into the membrane, where they facilitate the conductive movement of monovalent cations by means of a carrier type mechanism. As a novel class of ionophores, these substances are of practical interest because they can be water soluble and are potentially reversible.

Biomembrane structure and function: recent studies and new techniques.

The consensus view of biomembrane structure is outlined. The present model is built upon a fluid lipid matrix, usually two molecules in length, into which the proteins are embedded. The lipid bilayer organization is discussed, such as their phase transition and fluid character and the effect of cholesterol upon the chain organization. The non-lamellar arrangement which some lipids adopt is described. The use of new physical techniques for obtaining information about the structure and dynamics of membrane proteins are described. These techniques include electron diffraction, electron microscopy and FTIR spectroscopy. Models of the structures of the Ca2+-ATPase and the glucose transporter from erythrocytes are shown, indicating the putative helices embedded in the lipid bilayer and the groups of amino acids in the aqueous environment. These models are based upon biochemical methods to obtain amino acid sequences using DNA cloning techniques. Finally, an experimental method using triplet probes is described for the study of the rotational dynamics of membrane proteins. Labelled monoclonal antibodies for studying the dynamics of the glucose transporter have been used.

Electrical injury mechanisms: electrical breakdown of cell membranes.

Electric fields are capable of damaging cells through both thermal and nonthermal mechanisms. While joule heating is generally recognized to mediate tissue injury in electrical trauma, the possible role of electrical breakdown of cell membranes has not been thoroughly considered. Evidence is presented suggestive that in many instances of electrical trauma the local electrical field is of sufficient magnitude to cause electrical breakdown of cell membranes and cell lysis. In theory, large cells such as muscle and nerve cells are more vulnerable to electrical breakdown. To illustrate the significance of cell size and orientation, a geometrically simple model of an elongated cell is analyzed.

Fluidity-dependence of membrane adhesiveness can be explained by thermotropic shifts in surface potential.

It is demonstrated that the transition of both dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) from the gel to liquid-crystalline phase is paralleled by a pronounced increase in the negative surface potential of liposomes composed of either lipid. The Derjaguin-Landau-Verwey-Overbeek (DLVO) theory is applied to show that this phenomenon can serve as a simple explanation of diverse adhesive properties of solid and fluid lipid bilayers.

Is flexoelectricity the coupling factor between chemical energy and osmotic work in the pump? A model of pump.

The following pump model is proposed. A gate is responsible for pump specificity. The actual driving force of the transport of ions against the electrochemical potential gradient is the electric field originating from an altered curvature of the phospholipid bilayer around the pump. The physical origin of this curvature-induced electric field arises from a basic liquid crystal property of lipid bilayers called flexoelectricity. Alterations occurring in phospholipid bilayer arrangement are due to changed conformation of protein; the main energy source of this change is ATP. Consequently, the energy of ATP is transformed, in our pump model, into osmotic work in following steps: ATP + protein (conformation I)----protein (conformation II)----alterations in phospholipid bilayer arrangement----electric field----active transport of ions. This model is the most simple one. In Na, K-pump there is a bidirectional ion transport. In our model of Na, K-pump three conformational states of pump proteins and two different electric fields formed sequentially in opposite directions are supposed.

Changes in the organization of the lipid bilayer of the plasma membrane during spermatogenesis and epididymal maturation.

Ram, bull, and mouse sperm cells were stained with several fluorescent membrane probes. In contrast to nonspecific probes, merocyanine 540 (MC540), which displays preferential binding to loosely packed phospholipids in model membranes, was specifically localized to the anterior portion of the head and the midpiece of mature sperm. To establish when during development this distinctive staining pattern was acquired, germ cells from prepubescent and adult mouse testes as well as sperm from the caput, corpus, and cauda epididymides were isolated and examined. Localized staining with MC540 was not observed until sperm reached the corpus epididymidis, where those cells with a completely translocated (i.e., distally located) cytoplasmic droplet fluoresced. Likewise, when sperm were stained with fluoresceinated concanavalin A (fl-ConA), a localized pattern of fluorescence with lectin restricted to the anterior portion of the head was not observed until the corpus epididymidis was reached. However, in contrast to MC540 staining, only a fraction of sperm with completely translocated droplets exhibited this localized staining with fl-ConA, the remainder exhibiting diffuse fluorescence over the entire cell as seen on caput epididymal sperm. These developmental changes in staining patterns are specific to murine cells, since no change in the pattern of staining by either MC540 or fl-ConA was seen on epididymal sperm of the ram. These results are discussed with respect to: 1) species-to-species differences in sperm membrane features; and 2) the hypothesis that domains of loosely packed lipids may be involved in the regionalization of membrane proteins that occurs during sperm development.

Single-channel activity of bilayers derived from sea urchin sperm plasma membranes at the tip of a patch-clamp electrode.

Changes in the plasma membrane permeability of echinoderm sperm play a fundamental role in the acrosome reaction. During the reaction there is an increase in intracellular Ca2+ and Na+ and an efflux of H+ and K+. We have formed bilayers at the tip of patch pipets from a mixture of lipid vesicles and sea urchin sperm plasma membranes (12-50 microgram protein/ml). We observed three types of K+ channels (conductances: 22, 46, and 82 pS), two of which are partially blocked by TEA, and one Cl- channel (148 pS). The presence of K+ channels in sperm plasma membranes is consistent with the inhibition by TEA of the acrosome reaction in whole sperm and the membrane potential change that occurs during the reaction.

A three state model for alamethicin conductance in bilayer membranes.

Alamethicin is an antibiotic which produces voltage gated channels in lipid bilayer membranes. Recently completed studies of the pressure dependence of alamethicin conductance have shown that its onset following application of a suprathreshold voltage step at a pressure of 100 MPa (1000 atm) is markedly slowed relative to that observed at ambient pressure. Furthermore, the time course of the onset of conductance becomes distinctly sigmoidal at elevated pressure, a condition which is not evident at atmospheric pressure. The decay of alamethicin conductance upon removal of suprathreshold applied voltage is also slowed by application of hydrostatic pressure, but it follows a single exponential time course at all pressures. In addition, kinetic parameters characterizing the onset and decay of conductance show distinctly different pressure dependences. These observations cannot be explained by a two state model in which alamethicin moves reversibly between nonconducting and conducting states. Therefore we re-examine critically a hypothesis made by previous workers, namely that alamethicin, in monomeric or aggregate form, moves upon application of suprathreshold voltage first from a nonconducting surface state to a nonconducting preassembly or precursor state, and then finally into a conducting state. Parameters of this three state model are related to a geometric factor which measures the degree of sigmoidal conductance response and which can be evaluated directly from experimental data. An alternative aggregation-type analysis, equivalent to that applied by Hodgkin & Huxley to the potassium conductance in squid axon, is also considered in the context of this same geometric factor. The possibility of distinguishing between these analyses on the basis of experimental data is discussed.

Pressure effects on mechanisms of charge transport across bilayer membranes.

Ion transport across diphytanoylphosphatidylcholine/decane bilayer membranes was measured as a function of hydrostatic pressure over the range 0.1-100 MPa (1-1000 atm). Carrier-mediated K+ conductance decreased with increasing pressure, yielding positive activation volumes of 45 A3 per complex for valinomycin mediated transport, and 74 A3 per complex in the case of nonactin. Comparison with the known pressure dependence of the viscosity of bulk alkane liquids supports the view that the rate limiting step for carrier-mediated transport is the translocation of the carrier-cation complex across an essentially fluid hydrocarbon membrane core. The parameters characterizing transient conductance by the hydrophobic anions, dipicrylaminate and tetraphenylborate, by contrast, were found to be insensitive to pressure over the range available. This was also the case for the steady-state conductance observed at elevated concentrations of both tetraphenylborate and the hydrophobic cation, tetraphenylarsonium. The quasi-stationary conductance observed at elevated concentrations of dipicrylaminate did, however, decrease significantly with increasing pressure, indicating a positive activation volume of 20 A3 per ion. Alternative explanations of this more complex response of hydrophobic ions to pressure are considered. Ancillary measurements of specific membrane capacitance revealed an increase of about 10% with an increase of pressure to 100 MPa, yielding an estimated membrane compressibility on the order of 10(-9) m2 X N-1, comparable to that of bulk liquid hydrocarbons.

Calcium depletion in rabbit myocardium. Ultrastructure of the sarcolemma and correlation with the calcium paradox.

The ultrastructure of the Ca-depleted myocardial sarcolemma (via Ca-free and Ca-free plus EGTA perfusion at 28 degree C and 37 degree C) was studied in the vascularly perfused interventricular septum of the rabbit. Thin-section and freeze-fracture electron microscopy was used. Two major structural defects in the sarcolemma were found. (1) Ninety percent of the Ca-depleted cells have between 30 and 40% of their glycocalyx separated from the bilayer. With tannic acid staining, the separation is seen to occur between the external lamina and the surface coat. (2) Freeze-fracture data showed an apparent decrease in intramembrane particles on the P face of unidirectionally shadowed replicas. Quantitation of rotary-shadowed replicas showed no decrease in density of intramembrane particles. It was concluded from this that there was no loss of intramembrane particles, but rather a reorientation in the plane of the bilayer after Ca depletion. Both glycocalyx and bilayer changes were present after perfusion of the heart for only 5 minutes (37 degree C) with Ca-free perfusate. With low temperature and Cd substitution, separation of the glycocalyx occurred in less than 1% of the cells. After Ca depletion at 18 degree C, the density of intramembrane particles on the P face was not significantly different from controls. Cd substitution did not prevent the decrease total intramembrane particles per square micron, but the larger intramembrane particles had similar densities (154/micrometer2) as control (181/micrometer2), and as Ca-depletion with hypothermia (180/micrometer2). These findings indicate that structural changes in the glycocalyx and the bilayer can be totally prevented by hypothermia. Cd, on the other hand, prevents glycocalyx separation and affords protection only to the large intramembrane particles. Upon reperfusion with Ca, the intramembrane particles undergo the further alteration of aggregation, while numerous vesicles can be seen in the fracture plane of the membrane.