pH-Induced Proton Permeability Changes of Plasma Membrane Vesicles

In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied ΔpH the proton fluxes and proton permeability coefficients (P_H+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external pH on P_H+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero ΔpH, P_H+ increased from 65 × 10⁻⁸ at pH 8.5 to 10⁻¹ m/sec at pH 11 and the conductance from 13 × 10⁻⁶ to 30 × 10⁻⁴ S/m². At a constant pH _i of 8 and a pH _o going from 8.5 to 11, P_H+ increased more than tenfold from 2 to 26 × 10⁻⁶ m/sec. The calculated values of P_H+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed. Received: 5 April 1995/Revised: 28 March 1996     

Electrokinetic Properties of the Sarcoplasmic Reticulum Membrane Obtained from Reconstitution Studies

Electrophoretic mobility data of SR vesicles reconstituted with uncharged and two mixtures of charged and uncharged lipids (Brethes, D., Dulon, D., Johannin, G., Arrio, B., Gulik-Krzywicki, T., Chevallier, J. 1986. Study of the electrokinetic properties of reconstituted sarcoplasmic reticulum vesicles. Arch. Biochem. Biophys. 246:355–356) were analyzed in terms of four models of the membrane-water interface: (I) a smooth, negatively charged surface; (II) a negatively charged surface of lipid bilayer covered with an electrically neutral surface frictional layer; (III) an electrically neutral lipid bilayer covered with a neutral frictional layer containing a sheet of negative charge at some distance above the surface of the bilayer; (IV) an electrically neutral lipid bilayer covered with a homogeneously charged frictional layer. The electrophoretic mobility was predicted from the numerical integration of Poisson-Boltzmann and Navier-Stokes equations. Experimental results were consistent only with predictions based on Model-III with charged sheet about 4 nm above the bilayer and frictional layer about 10 nm thick. Assuming that the charge of the SR membrane is solely due to that on Ca⁺⁺-ATPase pumps, the dominant SR protein, the mobility data of SR and reconstituted SR vesicles are consistent with 12 electron charges/ATPase. This value compares well to the net charge of the cytoplasmic portion of ATPase estimated from the amino acid sequence (-11e). The position of the charged sheet suggests that the charge on the ATPase is concentrated in the middle of the cytoplasmic portion. The frictional layer of SR can be also assigned to the cytoplasmic portion of Ca⁺⁺-ATPase. The layer has been characterized with hydrodynamic shielding length of 1.1 nm. Its thickness is comparable to the height of the cytoplasmic portion of Ca⁺⁺-ATPase. Received: 15 June 1998/Revised: 8 October 1998     

Monitoring of the Membrane Potential in Proteoliposomes with Incorporated Cytochrome-c Oxidase Using the Fluorescent Dye Indocyanine

A method has been developed to monitor changes of the membrane potential across vesicle membranes in real time. Using the potential-sensitive fluorescent dye indocyanine and on the basis of a water/lipid redistribution model, a calculation procedure has been introduced to estimate the membrane potential in vesicles with incorporated cytochrome-c oxidase. Physical parameters, such as vesicle size distribution and density of the lipid bilayer were estimated and used as calculation parameters. By extrapolation of the transient potential change to zero time, the initial rate of the potential change (dU/dt) could be calculated. It is also shown, that the initial potential change (dU/dt) may be used to study the proton/electron stoichiometry of cytochrome-c oxidase incorporated in the vesicles. Received: 28 September 1995/Revised: 6 February 1996     

Directly Observed Membrane Fusion Between Oppositely Charged Phospholipid Bilayers

A novel method was developed for the direct examination of pairwise encounters between positively and negatively charged phospholipid bilayer vesicles. Giant bilayer vesicles (unilamellar, 4–20 μm in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with individual anionic phospholipid vesicles. Fluorescence video microscopy revealed that such vesicles commonly underwent fusion within milliseconds (1 video field) after contact, without leakage. Fusion occurred at constant volume and, since flaccid vesicles were rare, the excess membrane was not available after fusion. Hemifusion (the outer monolayers of each vesicle fused while the inner monolayers remained intact) was inferred from membrane-bound dye transfer and a change in the contact area. Hemifusion was observed as a final stable state and as an intermediate to fusion of vesicles composed of charged phospholipids plus zwitterionic phospholipids. Hemifusion occurred in one of three ways following adhesion: either delayed with an abrupt increase in area of contact, immediately with a gradual increase in area of contact, or with retraction during which adherent vesicles dissociated from a flat contact to a point contact. Phosphatidylethanolamine strongly promoted immediate hemifusion; the resultant hemifused state was stable and seldom underwent complete fusion. Although sometimes single contacts between vesicles led to rupture of both, in other cases, a single vesicle underwent multiple fusion events. Direct observation has unequivocally demonstrated the fusion of two, isolated bilayer-bounded bodies to yield a stable, non-leaky product, as occurs in cells, in the absence of proteins. Received: 25 November 1998/Revised: 23 March 1999     

Inositol 1,4,5-Trisphosphate But Not Ryanodine-Receptor Agonists Induces Calcium Release from Rat Liver Golgi Apparatus Membrane Vesicles

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptor agonists on Ca²⁺ release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with ⁴⁵Ca²⁺. Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP₃ at concentrations ranging from 100 nm to 100 μm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases, promoted a rapid decrease in the Ca²⁺ content of GA vesicles. The amount of Ca²⁺ released from the vesicles was a function of IP₃ concentration, reaching about 60% in both GA and ER fractions at 100 μm IP₃. Calcium release was inhibited by heparin, an antagonist of IP₃ receptors. Calcium exhibited a bell-shaped effect on IP₃-dependent Ca²⁺ released from GA vesicles: it activated Ca²⁺ release at concentrations up to 1 μm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca²⁺ release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP₃-sensitive Ca²⁺ release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca²⁺ release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane. Received: 13 March 2000/Revised: 13 July 2000     

Kinetics of Chloride-Bicarbonate Exchange Across the Human Red Blood Cell Membrane

We had previously shown that an influx of extracellular Ca²⁺ (Ca²⁺ _e ), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca²⁺ _e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ``ghosts' — a process difficult to analyze by any other method or in any other system. Maximal exocytotic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca<sup>2+</sup>] <sub>e</sub> ≥ 5 × 10<sup>−6</sup> m, while normally a [Ca<sup>2+</sup>] <sub>e</sub> = 0.5 mm is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca<sup>2+</sup>] <sub>e</sub> even beyond levels usually available to cells. Quenching of [Ca<sup>2+</sup>] <sub>e</sub> by EGTA application to levels of resting [Ca<sup>2+</sup>] <sub>i</sub> or slightly below does reduce (by ∼50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden either by increasing stimulation time or by readdition of an excess of Ca<sup>2+</sup> <sub>e</sub> . Our data are compatible with the assumption that normally exocytotic membrane fusion will include a step of rapid Ca<sup>2+</sup>-mobilization from subplasmalemmal pools (``alveolar sacs') and, as a superimposed step, a Ca²⁺-influx, since exocytotic membrane fusion can occur at [Ca²⁺] _e even slightly below resting [Ca²⁺] _i . The other important conclusion is that increasing [Ca²⁺] _e facilitates exocytotic and endocytotic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca²⁺] _e also drives detachment of ``ghosts' — a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca²⁺, orders of magnitude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle. Received: 27 September 1996/Revised: 11 February 1997     

Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers

Summary Addition of fragmented sarcoplasmic reticulum (SR) vesicles to the aqueous phase of a black lipid membrane (BLM) causes a large increase in BLM conductance within 10 min. The conductance increase is absolutely dependent on three conditions: The presence of at least 0.5mm Ca⁺⁺, an acidic phospholipid such as phosphatidylserine or diphosphatidylglycerol in the BLM phospholipid mixture, and an osmotic gradient across the SR vesicle membrane, with the internal osmolarity greater than the external. These requirements are identical to conditions under which the fusion of phospholipid vesicles occurs.When the early part of the time course of conductance rise is examined at high sensitivity, the conductance is seen to increase in discrete steps. The probability of a step increases with the concentration of Ca⁺⁺ in the medium, with the fraction of acidic phospholipid in the BLM, and with the size of the osmotic gradient across the SR vesicle membrane. On the other hand, the average conductance change per step is independent of the above parameters, but varies with the type and concentration of ions present in the aqueous phase. For a given ion, the mean specific conductance per step is independent of the ion's concentration between 10 and 100mm.The probability distribution of the step-conductances agrees well with the distribution of SR vesicle surface areas, both before and after sonication of the vesicles.The evidence indicates that SR vesicles fuse with the BLM, thereby inserting SR membrane conductance pathways into it. Each discrete conductance jump appears to be the result of the fusion of a single SR vesicle with the BLM. This technique may serve as a general method for inserting membrane vesicles into an electrically accessible system.     

The Role of MIP in Lens Fiber Cell Membrane Transport

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in Cat ^Fr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (p _H2O ) on the order of 1 μm/sec whereas normal fiber cell membrane p _H2O was 17 μm/sec frog, 32 μm/sec rabbit and 43 μm/sec mouse. Cat ^Fr mouse lens fiber cell p _H2O was reduced by 13 μm/sec for heterozygous and 30 μm/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the p _H2O conferred by MIP is not sensitive to Hg²⁺ whereas that of CHIP28 (AQP1) is blocked by Hg²⁺. The fiber cell membrane p _H2O was also not sensitive to Hg²⁺ whereas lens epithelial cell p _H2O (136 μm/sec in rabbit) was blocked by Hg²⁺. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous Cat ^Fr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses. Received: 17 February 1999/Revised: 16 April 1999     

Studies on membrane fusion. II. Induction of fusion in pure phospholipid membranes by calcium ions and other divalent metals

The effect of divalent metals on the interaction and mixing of membrane components in vesicles prepared from acidic phospholipids has been examined using freeze-fracture electron microscopy and differential scanning calorimetry. Ca²⁺, and to a certain extent Mg²⁺, induce extensive mixing of vesicle membrane components and drastic structural rearrangements to form new membranous structures. In contrast to the mixing of vesicle membrane components in the absence of Ca²⁺ described in the accompanying paper which occurs via diffusion of lipid molecules between vesicles, mixing of membrane components induced by Ca²⁺ or Mg²⁺ results from true fusion of entire vesicles. There appears to be a “threshold” concentration at which Ca²⁺ and Mg²⁺ become effective in inducing vesicle fusion and the threshold concentration varies for different acidic phospholipid species. Different phospholipids also vary markedly in their relative responsiveness to Ca²⁺ and Mg²⁺, with certain phospholipids being much more susceptible to fusion by Ca²⁺ than Mg²⁺. Vesicle fusion induced by divalent cations also requires that the lipids of the interacting membranes be in a “fluid” state ($\text{T > T}{}_{\mathrm{c}}$). Fusion of vesicle membranes by Ca²⁺ and Mg²⁺ does not appear to be due to simple electrostatic charge neutralization. Rather the action of these cations in inducing fusion is related to their ability to induce isothermal phase transitions and phase separations in phospholipid membranes. It is suggested that under these conditions membranes become transiently susceptible to fusion as a result of changes in molecular packing and creation of new phase boundaries induced by Ca²⁺ (or Mg²⁺).     

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg²⁺ transport across the epithelium involves a transcellular, Na⁺- and Na⁺/K⁺-ATPase dependent pathway. In our search for the Mg²⁺ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na⁺/Mg²⁺ antiport or ATP-driven transport. Evidence is provided, however, that Mg²⁺ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl⁻ gradient stimulated Mg²⁺ uptake (as followed with the radionuclide ²⁷Mg) twofold. As Cl⁻-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg²⁺ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg²⁺ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl⁻-stimulated Mg²⁺ uptake yielded a K _m (Mg²⁺) of 6.08 ± 1.29 mmol · l⁻¹ and a K _m (Cl⁻) of 26.5 ± 6.5 mmol · l⁻¹, compatible with transport activity at intracellular Mg²⁺- and Cl⁻-levels. We propose that Mg²⁺ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996     

Cation Permeability and Selectivity of a Root Plasma Membrane Calcium Channel

Calcium channels in the plasma membrane of root cells fulfill both nutritional and signaling roles. The permeability of these channels to different cations determines the magnitude of their cation conductances, their effects on cell membrane potential and their contribution to cation toxicities. The selectivity of the rca channel, a Ca²⁺-permeable channel from the plasma membrane of wheat (Triticum aestivum L.) roots, was studied following its incorporation into planar lipid bilayers. The permeation of K⁺, Na⁺, Ca²⁺ and Mg²⁺ through the pore of the rca channel was modeled. It was assumed that cations permeated in single file through a pore with three energy barriers and two ion-binding sites. Differences in permeation between divalent and monovalent cations were attributed largely to the affinity of the ion binding sites. The model suggested that significant negative surface charge was present in the vestibules to the pore and that the pore could accommodate two cations simultaneously, which repelled each other strongly. The pore structure of the rca channel appeared to differ from that of L-type calcium channels from animal cell membranes since its ion binding sites had a lower affinity for divalent cations. The model adequately accounted for the diverse permeation phenomena observed for the rca channel. It described the apparent submillimolar K _m for the relationship between unitary conductance and Ca²⁺ activity, the differences in selectivity sequences obtained from measurements of conductance and permeability ratios, the changes in relative cation permeabilities with solution ionic composition, and the complex effects of Ca²⁺ on K⁺ and Na⁺ currents through the channel. Having established the adequacy of the model, it was used to predict the unitary currents that would be observed under the ionic conditions employed in patch-clamp experiments and to demonstrate the high selectivity of the rca channel for Ca²⁺ influx under physiological conditions. Received: 23 August 1999/Revised: 12 November 1999     

Muscarinic Activation of BK Channels Induces Membrane Oscillations in Glioma Cells and Leads to Inhibition of Cell Migration

Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca²⁺] _i ) associated with periodic Ca²⁺ oscillations during prolonged ACh applications. The early transient rise in [Ca²⁺] _i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca²⁺ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca²⁺] _i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca²⁺-sensitive K⁺ currents. These K⁺ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca²⁺ from intracellular stores which in turn activate Ca²⁺-dependent (BK-type) K⁺ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration. Received: 17 December/Revised: 17 March 2000     

Fluid Flow Modulates Calcium Entry and Activates Membrane Currents in Cultured Human Aortic Endothelial Cells

Human aortic endothelial cells (HAEC) respond to flow with Ca²⁺ entry, activation of a nonselective cation channel, activation of a chloride channel, and activation of a calcium-activated potassium channel. Conversely, human capillary endothelial cells were unaffected by similar flow rates. In HAEC the flow induced cytosolic free calcium increase ([Ca²⁺] _i ) and the ionic currents associated with it were sustained for up to 15 min after perfusion was stopped. In the absence of extracellular Ca²⁺, fluid flow was unable to evoke the [Ca²⁺] _i increase or the increase in membrane currents but the response could be restored by addition of extracellular Ca²⁺. Surprisingly, the flow response was inhibited in 50% of the cells by inhibitors of nitric oxide production. The results suggest that the sustained flow response in HAEC may be partially mediated by nitric oxide production and release. Received: 29 January 1999/Revised: 2 June 1999     

Transient and Permanent Fusion of Vesicles in Zea mays Coleoptile Protoplasts Measured in the Cell-attached Configuration

Exocytosis in protoplasts from Zea mays L. coleoptiles was studied using patch-clamp techniques. Fusion of individual vesicles with the plasma membrane was monitored as a step increase of the membrane capacitance (C _m ). Vesicle fusion was observed as (i) An irreversible step increase in C _m . (ii) Occasionally, irreversible C _m steps were preceded by transient changes in C _m , suggesting that the electrical connection between the vesicle with the plasma membrane opens and closes reversibly before full connection is achieved. (iii) Most frequently, however, stepwise transient changes in C _m did not lead to an irreversible C _m step. Within one patch of membrane capacitance steps due to transient and irreversible fusions were of similar amplitude. This suggests that the exocytosis events do not result from the fusion of vesicles with different sizes but are due to kinetically different states in a fusion process of the same vesicle type. The dwell time histogram of the transient fusion events peaked at about 100 msec. Fusion can be described with a circular three-state model for the fusion process of two fused states and one nonfused state. It predicts that energy input is required to drive the system into a prevailing direction. Received: 27 August 1999/Revised: 28 October 1999     

Membrane Potential Mediates H+-ATPase Dependence of ``Degradative Pathway' Endosomal Fusion

In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as ``degradative endocytosis.' The endosomal pathways of the renal proximal tubule and the visceral yolk sac share highly specialized structures for ``degradative endocytosis.' These endosomal pathways also have a unique distribution of their H⁺-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H⁺-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H⁺-ATPase dependent in baby hamster kidney cells, and H⁺-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic endosomal fusion is H⁺-ATPase dependent, we inhibited v-type H⁺-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited by the v-type H⁺-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H⁺-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assay this data provides the first direct evidence for a role of v-type H⁺-ATPase in mammalian homotypic endosomal fusion. Received: 29 October 1996/Revised: 8 December 1997     

Single vesicle assaying of SNARE-synaptotagmin-driven fusion reveals fast and slow modes of both docking and fusion and intrasample heterogeneity

Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca²⁺ dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca²⁺ accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca²⁺ accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t_mix < 1 s) while an ensemble assay revealed two slow mixing processes with t_mix ∼ 1 min and t_mix ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition.     

Studies on membrane fusion. III. The role of calcium-induced phase changes

The interaction of phosphatidylserine vesicles with Ca²⁺ and Mg²⁺ has been examined by several techniques to study the mechanism of membrane fusion. Data are presented on the effects of Ca²⁺ and Mg²⁺ on vesicle permeability, thermotropic phase transitions and morphology determined by differential scanning calorimetry, X-ray diffraction, and freeze-fracture electron microscopy. These data are discussed in relation to information concerning Ca²⁺ binding, charge neutralization, molecular packing, vesicle aggregation, phase transitions, phase separations and vesicle fusion.The results indicate that at Ca²⁺ concentrations of 1.0–2.0 mM, a highly cooperative phenomenon occurs which results in increased vesicle permeability, aggregation and fusion of the vesicles. Under these conditions the hydrocarbon chains of the lipid bilayers undergo a phase change from a fluid to a crystalline state. The aggregation of vesicles that is observed during fusion is not sufficient in itself to induce fusion without a concomitant phase change. Mg²⁺ in the range of 2.0–5.0 mM induces aggregation of phosphatidylserine vesicles but no significant fusion nor a phase change.From the effect of variations in pH, temperature, Ca²⁺ and Mg²⁺ concentration on the fusion of vesicles, it is concluded that the key event leading to vesicle membrane fusion is the isothermic phase change induced by the bivalent metals. It is proposed that this phase change induces a transient destabilization of the bilayer membranes that become susceptible to fusion at domain boundaries.     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

Bacillus thuringiensis CrylAa δ-Endotoxin Affects the K+/Amino Acid Symport in Bombyx mori Larval Midgut

The mechanical properties of brush border membrane vesicles, BBMV, from rabbit kidney proximal tubule cells, were studied by measuring the initial and final equilibrium volumes of vesicles subjected to different osmotic shocks, using cellobiose as the impermeant solute in the preparation buffer. An elevated intracellular hydrostatic pressure was inferred from osmotic balance requirements in dilute solutions. For vesicles prepared in 18 and 85 mosm solutions, these pressures are close to 17 mosm (290 mm Hg). The corresponding membrane surface tension is 6.0 × 10⁻⁵ N cm⁻¹ while the membrane surface area is expanded by at least 2.2%. When these vesicles are exposed to very dilute solutions the internal hydrostatic pressure rises to an estimated 84 mosm (1444 mm Hg) just prior to lysis. The corresponding maximal surface tension (pre-lysis) is 18.7 × 10⁻⁵ N cm⁻¹, and the maximal expansion of membrane area is 6.8%. The calculated area compressibility elastic modulus was 2.8 × 10⁻³ N cm⁻¹. Received: 8 August 1996/Revised: 4 March 1997     

Membrane Bending Energy and Fusion Pore Kinetics in Ca-Triggered Exocytosis

A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca²⁺-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca²⁺-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca²⁺-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape.