Fusion of a single influenza virion particle with BLM

One of the key stages of cell infection with influenza virus is the enveloped virus fusion with the cell endosome membrane. To study fusion of single fluorescently-labeled influenza virions with a model bilayer membrane (BLM), a special model system was developed. A small patch of BLM with several adsorbed virions was localized upon a contact with a glass micropipette. Low pH of solution inside the pipette triggered fusion that could be registered by a change in the conductance and integral fluorescence of the BLM patch. It has been shown that the fusion initiation is followed by an increase of fluorescence signal due to the probe redistribution from the virus membrane to the BLM fragment. The increase in fluorescence was accompanied by changes in conductance. Usually, from two to five periods of the channel activity were observed, each of which probably corresponded to fusion of a single virion. It has been found that electric activity was completely inhibited by amantadine known as a blocking agent of M2 channels. This allows one to suggest that the observed changes in conductance are connected with the activity of M2 channels in the virus membrane, whose electric accessibility was the result of fusion of single virions with BLM.     

Effect of polymyxin B on the conductivity of negatively charged bilayer lipid membranes

Effect of polymyxin B on the planar bilayer lipid membranes (BLM) formed from synthetic phosphatidic acid has been studied. The addition of cholesterol to phospholipid in molar ratio 1 : 2 was followed by an increase of BLM conductance from 2 x 10(-8) to 3 x 10(-7) Ohm-1 cm-2. It was suggested that the observed increase of conductance was due to the fluidity of the membrane matrix in the presence of cholesterol. It was shown that 10(-6)--10(-5) M polymyxin slightly affected the conductance of BLM from phosphatidic acid. It was found that polymyxin increased conductance of negatively charged BLM modified by palmitic acid from 10(-8) to 10(-6) Ohm-1 cm-2.     

Interaction between filamentous actin and lipid bilayer causes the increase of syringomycin E channel-forming activity

The effect of filamentous (F) actin on the channel-forming activity of syringomycin E (SRE) in negatively charged and uncharged bilayer lipid membranes (BLM) was studied. F-actin did not affect the membrane conductance in the absence of SRE. No changes in SRE-induced membrane conductance were observed when the above agents were added to the same side of BLM. However, the opposite side addition of F-actin and SRE provokes a multiple increase in membrane conductance. The similar voltage dependence of membrane conductance, equal values of single channel conductance and the effective gating charge of the channels upon F-actin action suggests that the actin-dependent increase in BLM conductance may result from an increase in the number of opened SRE-channels. BLM conductance kinetics depends on the sequence of SRE and F-actin addition, suggesting that actin-dependent rise of conductance may be induced by BLM structural changes that follow F-actin adsorption. F-actin exerted similar effect on membrane conductance of both negatively charged and uncharged bilayers, as well as on conductance of BLM with high ionic strength bathing solution, suggesting the major role for hydrophobic interactions in F-actin adsorption on lipid bilayer.     

Presenilin/γ-secretase regulates neurexin processing at synapses

Neurexins are a large family of neuronal plasma membrane proteins, which function as trans-synaptic receptors during synaptic differentiation. The binding of presynaptic neurexins to postsynaptic partners, such as neuroligins, has been proposed to participate in a signaling pathway that regulates synapse formation/stabilization. The identification of mutations in neurexin genes associated with autism and mental retardation suggests that dysfunction of neurexins may underlie synaptic defects associated with brain disorders. However, the mechanisms that regulate neurexin function at synapses are still unclear. Here, we show that neurexins are proteolytically processed by presenilins (PS), the catalytic components of the γ-secretase complex that mediates the intramembraneous cleavage of several type I membrane proteins. Inhibition of PS/γ-secretase by using pharmacological and genetic approaches induces a drastic accumulation of neurexin C-terminal fragments (CTFs) in cultured rat hippocampal neurons and mouse brain. Neurexin-CTFs accumulate mainly at the presynaptic terminals of PS conditional double knockout (PS cDKO) mice lacking both PS genes in glutamatergic neurons of the forebrain. The fact that loss of PS function enhances neurexin accumulation at glutamatergic terminals mediated by neuroligin-1 suggests that PS regulate the processing of neurexins at glutamatergic synapses. Interestingly, presenilin 1 (PS1) is recruited to glutamatergic terminals mediated by neuroligin-1, thus concentrating PS1 at terminals containing β-neurexins. Furthermore, familial Alzheimer's disease (FAD)-linked PS1 mutations differentially affect β-neurexin-1 processing. Expression of PS1 M146L and PS1 H163R mutants in PS-/- cells rescues the processing of β-neurexin-1, whereas PS1 C410Y and PS1 ΔE9 fail to rescue the processing defect. These results suggest that PS regulate the synaptic function and processing of neurexins at glutamatergic synapses, and that impaired neurexin processing by PS may play a role in FAD.     

Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd²⁺, La³⁺ or Tb³⁺ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.     

Interaction of all-<Emphasis Type="Italic">trans</Emphasis>-Retinal with bilayer lipid membranes

The interaction of all-trans-retinal (hereinafter referred to as retinal) with planar bilayer lipid membranes has been studied. Addition of retinal into aqueous solutions on both sides of the membrane formed from diphytanoilphosphatidylcholine (DPhPC) or its mixture with diphytanoilphosphatidylethanolamine (DPhPC/DPhPE in w/w proportion of 3: 5) led to a change of conductance induced by ionophores nonactin (increase of conductance) or pentachlorophenol (decrease). Increase of nonactin-induced conductance was dependent on the membrane lipid composition and was two times higher in the case of DPhPC/DPhPE mixture. The change of conductance caused by ionophores of different signs (plus or minus) had different direction suggesting the influence of the retinal on the dipole potential upon its incorporation into BLM. The boundary potentials difference measured by the intramembrane field compensation method (IFC) after the retinal addition on one side of the membrane did not exceed 2.5 mV suggesting that its distribution in the bilayer is almost symmetrical. The illumination of the retinal-containing BLM caused a decrease in its lifetime when the membranes were formed from unsaturated lipids. Retinal incorporated into BLM led also to photoinactivation of the gramicidin channels. The process was completely inhibited by a singlet oxygen quencher (sodium azide). These results indicate that retinal accumulated in the membrane can affect both membrane proteins and the unsaturated lipids by their oxidation by the singlet oxygen.     

Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.     

Ion permeability of bilayer membranes formed from synthetic phospholipids in the phase transition region

Ion permeability of black lipid membranes formed from synthetic phospholipids has been studied. The resistance of BLM formed from phosphatidylcholine, tiophosphatidylcholine, threealkylphosphate and threealkyltiophosphate was 10(7)--10(8) Ohm.cm2. It was shown that the membrane potential of the 10--30 mV arised in KCl gradient indicating the preference cation conductance in synthetic lipid membranes. A sharp decrease of the membrane conductance near to the phase transition temperature was discovered. The change of conductance by phase transition temperature was sensitive to chemical nature of the polar head of phospholipids used.     

Channel properties of the purified glutamate receptor from rat brain reconstituted in planar lipid bilayer membrane

The properties of the purified rat brain glutamate receptor (GluR), reconstituted in planar lipid bilayer (BLM) were characterised. The single channel currents activated by glutamate and aspartate were similar. The different kinetics of current fluctuation were observed. Paroxysms of channel activity seems to be resulted from the transit of GluR through its active conformation from which it can open several times before desensitising. The effect of concanovaline A (Con A) as an agent blocking desensitisation of glutamatergic synapses was investigated. It was shown that Con A evokes high levels of conductivity and prolonged opening events of channels. Another agent, which stabilises glutamate activated conductivity, dithiothreitol (DTT), evokes "chronic" channel activity. This study demonstrates that purified GluR reconstituted in planar lipid bilayers exhibits the ion-conductivity properties that are associated with the postsynaptic membrane.     

多重扫描伏安法在平面双分子脂膜(BLM)研究中的应用

本文阐明了伏安技术对重组膜的各种电性质测量原理和方法,并用多重扫描技术着重研究了膜电导的可逆跃迁变化,以及扫描速度和电压的影响,结果指出,伏安法可能发展成一种研究重组膜离子通道现象的新手段.     

Delivering the goods: Activity-induced exocytosis and AMPA receptor insertion within a post-synaptic membrane compartment depends on syntaxin 4

The activity-dependent strengthening of neural transmission at individual synapses has long been postulated to underlie learning and memory in the brain, and current wisdom strongly suggests that molecular modifications within both the pre- and post-synaptic nerve terminals contribute to this strengthening process (i.e. long-term potentiation or LTP). At excitatory, glutamatergic synapses, the dynamic insertion and retrieval of ionotropic glutamate receptors into and from the post-synaptic plasma membrane have been implicated in synaptic plasticity, however, the site(s) for these trafficking events and the molecules involved have not be clearly elucidated. Biochemical studies have identified SNARE proteins as critical mediators of membrane fusion events in many cell types, including neurons, and several bacterial toxins are known to interfere with neurotransmission by disrupting the function of membrane-bound SNARE proteins, such as VAMP and syntaxin. Using high resolution imaging techniques, the authors have characterized the molecular processes underlying activity-driven membrane fusion events within dendritic spines, tiny membrane protrusions only 1-2 microns in size. Their data demonstrate that syntaxin 4 functions as a key SNARE protein for the exocytic insertion of glutamate receptors and the membrane trafficking events contributing to synaptic plasticity.     

Axonal speeding: shaping synaptic potentials in small neurons by the axonal membrane compartment

The role of the axonal membrane compartment in synaptic integration is usually neglected. We show here that in interneurons of the cerebellar molecular layer, where dendrites are so short that the somatodendritic domain can be considered isopotential, the axonal membrane contributes a significant part of the cell input capacitance. We examine the impact of axonal membrane on synaptic integration by cutting the axon with two-photon illumination. We find that the axonal compartment acts as a sink for signals generated at fast conductance synapses, thus increasing the initial decay rate of corresponding synaptic potentials over the value predicted from the resistance-capacitance (RC) product of the cell membrane; signals generated at slower synapses are much less affected. This mechanism sharpens the spike firing precision of fast glutamatergic inputs without resorting to multisynaptic pathways.     

Piezoeffect, baseline conductivity and filtration coefficients of lipid bilayer membrane

A piezoeffect in bilayer lipid membranes (BLM), i.e. a generation of alternate electrical current y through a membrane under alternate pressure gradient in the absence of constant transmembrane potential is investigated. It is shown that y is not connected with the membrane deformations, it is generated with electroosmotic flow of K+ ions, its amplitude increases with a rise of BLM phonic conductance and is probably connected with defects in BLM structure. The value of the filtration coefficient Lp is estimated.     

Study of conductance changes of bilayer lipid membrane induced by electric field

Changes in the bilayer lipid membrane (BLM) conductance induced by electric field were studied. BLMs were formed from diphytanoylphosphocholine (DPhPC) solution in squalene. Certain time after a constant voltage (200-500 mV) was applied to the BLM in the voltage-clamp mode, the BLM conductance started to grow up to approximately 10 nS until the BLM ruptured. The conductance often changed abruptly (with the front duration of less than 33 micros) and then stabilized for a relatively long time (up to 10; 300 ms on average) thus resembling the ion channel activity. The mean amplitude of conductance steps was 650 pS. However, in some cases a slow conductance drift was recorded. When N-methyl-D-glucamine/glutamate ions were used instead of KCl, the conductance changes became 5 times smaller. We suggest that formation in the BLM of single pores approximately 1 nm in diameter should result in the observed changes in BLM conductance. The BLM conductance growth was due to consecutive opening of several such pores. When the electric field amplitude was abruptly decreased (down to 50-100 mV), the conductance dropped rapidly to the background value. When we increased the voltage again, the BLM conductance right after the increase depended on the time BLM spent under "weak" electric field. If this time exceeded 500 ms, the conductance was at the background level, but when the time was diminished, the conductance reached the value recorded before the voltage decrease. These data imply that the closure of the pores should lead to the formation in BLM of small defects (prepores) that can be easily transformed into pores when the voltage is increased. The lifetimes of such prepores did not exceed 500 ms.     

Modeling of damage to cell membranes during photodynamic therapy: photosensitization of planar lipid bilayers by hematoporphyrin dimethyl ether

The variations of electrical conductance of planar bilayer lipid membranes (BLM) sensitized by hematoporphyrin dimethyl ether under visible light illumination were studied. The conductance of BLM does not change for some period after switching on the light, then an increase in the conductance starts and the membrane breaks. This "induction" time does not depend on addition of azide or ferricyanide to the solution, on addition of BHT to the lipid and on substitution of air for argon in the cell. The induction time for any new BLM, formed in the same cell immediately after the previous membrane was broken, is shorter. The variation of BLM boundary potentials during induction time was not observed. The results obtained suggest that the photodamage of BLM sensitized by HPD leads to accumulation of uncharged reaction products and oxygen does not take part in this process.     

SHANK1 and autism spectrum disorders

Autism spectrum disorders(ASD) are highly heterogeneous pediatric developmental disorders with estimated heritability more than 70%. Although the genetic factors in ASD are mainly unknown, a large number of gene mutations have been found, especially in genes involved in neurogenesis. The Neurexin-Neuroligin-Shank(NRXN-NLGN-SHANK) pathway plays a key role in the formation, maturation and maintenance of synapses, consistent with the hypothesis of neurodevelopmental abnormality in ASD. Presynaptic NRXNs interact with postsynaptic NLGNs in excitatory glutamatergic synapses. SHANK proteins function as core components of the postsynaptic density(PSD) by interacting with multiple proteins. Recently, deletions and point mutations of the SHANK1 gene have been detected in ASD individuals, indicating the involvement of SHANK1 in ASD. This review focuses on the function of SHANK1 protein, Shank1 mouse models, and the molecular genetics of the SHANK1 gene in human ASD.     

Electric field increases the phase transition temperature in the bilayer membrane of phosphatidic acid

The effect of the electric field on the phase transition temperature (Tc) of acidic 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) and 1,2-dipalmitoyl-sn-glycero-3-thionphosphate (thion-DPPA) and zwitterion, i.e. 1,2-dipalmitoyl-rac-3-phosphocholine and 1,2-distearoyl-rac-glycero-3-phosphocholine (DPPC and DSPC), lipids has been investigated. The phase transition was detected using the jump-like increase effect in the conductance of the planar bilayer membrane. A voltage increase to 150 mV has been shown to increase the phase transition temperature in a bilayer lipid membrane (BLM) of phosphatidic acids (DPPA and thion-DPPA) by 8-12 degrees C while the transition temperature in the bilayer of zwitterion lipids (DPPC and DSPC) increases insignificantly. The increasing of Tt in BLM of acidic lipids is attributed to the voltage-induced changes in the molecule packing density.     

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.     

Identification and characterization of SV31, a novel synaptic vesicle membrane protein and potential transporter

Synaptic vesicle proteins govern all relevant functions of the synaptic vesicle life cycle, including vesicle biogenesis, vesicle transport, uptake and storage of neurotransmitters, and regulated endocytosis and exocytosis. In spite of impressive progress made in the past years, not all known vesicular functions can be assigned to defined protein components, suggesting that the repertoire of synaptic vesicle proteins is still incomplete. We have identified and characterized a novel synaptic vesicle membrane protein of 31 kDa with six putative transmembrane helices that, according to its membrane topology and phylogenetic relation, may function as a vesicular transporter. The vesicular allocation is demonstrated by subcellular fractionation, heterologous expression, immunocytochemical analysis of brain sections and immunoelectron microscopy. The protein is expressed in select brain regions and contained in subpopulations of nerve terminals that immunostain for the vesicular glutamate transporter 1 and the vesicular GABA transporter VGaT (vesicular amino acid transporter) and may attribute specific and as yet undiscovered functions to subsets of glutamatergic and GABAergic synapses.     

A Septin-Dependent Diffusion Barrier at Dendritic Spine Necks

Excitatory glutamatergic synapses at dendritic spines exchange and modulate their receptor content via lateral membrane diffusion. Several studies have shown that the thin spine neck impedes the access of membrane and solute molecules to the spine head. However, it is unclear whether the spine neck geometry alone restricts access to dendritic spines or if a physical barrier to the diffusion of molecules exists. Here, we investigated whether a complex of septin cytoskeletal GTPases localized at the base of the spine neck regulates diffusion across the spine neck. We found that, during development, a marker of the septin complex, Septin7 (Sept7), becomes localized to the spine neck where it forms a stable structure underneath the plasma membrane. We show that diffusion of receptors and bulk membrane, but not cytoplasmic proteins, is slower in spines bearing Sept7 at their neck. Finally, when Sept7 expression was suppressed by RNA interference, membrane molecules explored larger membrane areas. Our findings indicate that Sept7 regulates membrane protein access to spines.