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1.
Conformational change in the mitochondrial channel, VDAC, detected by electron cryo-microscopy. 总被引:1,自引:0,他引:1
Crystalline arrays of the voltage-dependent channel, VDAC, can be produced by treatment of Neurospora mitochondrial outer membranes with phospholipase A2. The membrane crystals undergo a lateral phase transition (lattice contraction) that can be induced by an amphipathic polyanion, which also reduces the channel's gating potential. Electron cryo-microscopy of frozen-hydrated crystals indicates that the mean projected diameters of the channels do not decrease with lattice contraction. Instead, contraction is associated with the disappearance of lateral protein "arms" that normally extend between the channels. A model is presented that explains the changes in channel packing and gating potential in terms of a conformational change involving the movement of a protein "arm" between the bilayer and the channel. 相似文献
2.
Polyanions decelerate the kinetics of positively charged gramicidin channels as shown by sensitized photoinactivation
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Antonenko YN Borisenko V Melik-Nubarov NS Kotova EA Woolley GA 《Biophysical journal》2002,82(3):1308-1318
The effects of different anionic polymers on the kinetic properties of ionic channels formed by neutral gramicidin A (gA) and its positively charged analogs gramicidin-tris(2-aminoethyl)amine (gram-TAEA) and gramicidin-ethylenediamine (gram-EDA) in a bilayer lipid membrane were studied using a method of sensitized photoinactivation. The addition of Konig's polyanion caused substantial deceleration of the photoinactivation kinetics of gram-TAEA channels, which expose three positive charges to the aqueous phase at both sides of the membrane. In contrast, channels formed of gram-EDA, which exposes one positive charge, and neutral gA channels were insensitive to Konig's polyanion. The effect strongly depended on the nature of the polyanion added, namely: DNA, RNA, polyacrylic acid, and polyglutamic acid were inactive, whereas modified polyacrylic acid induced deceleration of the channel kinetics at high concentrations. In addition, DNA was able to prevent the action of Konig's polyanion. In single-channel experiments, the addition of Konig's polyanion resulted in the appearance of long-lived gram-TAEA channels. The deceleration of the gram-TAEA channel kinetics was ascribed to electrostatic interaction of the polyanion with gram-TAEA that reduces the mobility of gram-TAEA monomers and dimers in the membrane via clustering of channels. 相似文献
3.
The mitochondrial outer membrane channel, VDAC, is regulated by a synthetic polyanion 总被引:9,自引:0,他引:9
A synthetic polyanion has been found to modulate the properties of the mitochondrial outer membrane channel, VDAC. This 10 kDa polyanion, first synthesized and described by Konig and co-workers, is a 1:2:3 copolymer of methacrylate, maleate, and styrene. It had been shown to interfere with the access of metabolites to the mitochondrial inner spaces. Here we show that, at nanomolar levels, the polyanion increases the voltage dependence of VDAC channels over 5-fold. Some channels seem to be totally blocked while others display the higher voltage dependence and are able to close at very low membrane potentials (5 mV). At 27 micrograms/ml polyanion, VDAC channels are closed while inserted into liposomes in the absence of any applied potential. The closed state of VDAC induced by the polyanion has similar properties to the closed state induced by elevated membrane potentials. The physical size of the polyanion-induced closed state (in VDAC-containing liposomes) is about 0.9 nm in radius. How this estimate fits with estimates of the channel's open state and estimated volume changes between the open and closed states, is discussed. 相似文献
4.
Low-dose electron microscopic images have been recorded from membrane crystals of the mitochondrial, voltage-dependent anion-selective channel, embedded in aurothioglucose. There is considerable variation in the high-resolution detail present in correlation averages computed from these images. Correspondence analysis reveals three classes of "control" averages, with main components of variation involving projected size of the pores and density modulations around the pores and in the corners of the unit cells away from the pores. Pretreatments that affect the functional state of the channel also affect the array averages. In particular, there appears to be a general correlation between the expected effector-induced state (i.e., open and closed) and the projected diameter of the channel lumens in the crystalline arrays. 相似文献
5.
Carmen A. Mannella 《Journal of bioenergetics and biomembranes》1987,19(4):329-340
The channel protein in the outer membrane ofNeurospora crassa mitochondria, VDAC, forms extended planar crystals on the membrane. The arrays, which are induced by phospholipase A2, are polymorphic, varying from parallelogram (P) to near-rectangular (R) geometry with increased phospholipase treatment. Computer-based analysis of projection images of negatively stained VDAC arrays indicates that the protein forms a transmembrane channel in the P array. Comparison of average images of arrays embedded in different negative stains suggests that the bore of the channel is 2–2.5 nm. The locations of functionally important lysine clusters on VDAC are inferred from the effects of succinylation on projection images of arrays negatively stained with phosphotungstate. Projection images of unstained frozen-hydrated arrays indicate the general shape of the channel and suggest each channel is formed by one 31-kDa VDAC polypeptide. 相似文献
6.
Krasnikov BF Melik-Nubarov NS Zorova LD Kuzminova AE Isaev NK Cooper AJ Zorov DB 《American journal of physiology. Cell physiology》2011,300(5):C1193-C1203
A synthetic polyanion composed of styrene, maleic anhydride, and methacrylic acid (molar ratio 56:37:7) significantly inhibited the respiration of isolated rat liver mitochondria in a time-dependent fashion that correlated with 1) collapse of the mitochondrial membrane potential and 2) high amplitude mitochondrial swelling. The process is apparently Ca(2+) dependent. Since it is blocked by cyclosporin A, the process is ascribed to induction of the mitochondrial permeability transition. In mitoplasts, i.e., mitochondria lacking their outer membranes, the polyanion rapidly blocked respiration. After incubation of rat liver mitochondria with the polyanion, cytochrome c was released into the incubation medium. In solution, the polyanion modified by conjugation with fluorescein formed a complex with cytochrome c. Addition of the polyanion to cytochrome c-loaded phosphatidylcholine/cardiolipin liposomes induced the release of the protein from liposomal membrane evidently due to coordinated interplay of Coulomb and hydrophobic interactions of the polymer with cytochrome c. We conclude that binding of the polyanion to cytochrome c renders it inactive in the respiratory chain due to exclusion from its native binding sites. Apparently, the polyanion interacts with cytochrome c in mitochondria and releases it to the medium through breakage of the outer membrane as a result of severe swelling. Similar properties were demonstrated for the natural polyanion, tobacco mosaic virus RNA. An electron microscopy study confirmed that both polyanions caused mitochondrial swelling. Exposure of cerebellar astroglial cells in culture to the synthetic polyanion resulted in cell death, which was associated with nuclear fragmentation. 相似文献
7.
Three kinds of polyion complex membranes were prepared on a glassy carbon electrode: polycation (poly-L-lysine)-rich membrane, polyanion (DNA)-rich membrane, and equivalent membrane. The permeation of electroactive species (e.g., hydrogen peroxide, L-ascorbate, urate, dopamine) through the membrane was measured by the oxidation current of species at base electrode. Permeation of the anionic species can be depressed through the anion-rich membrane, and permeation of the cation can also be regulated through the cation-rich membrane. It is obvious that the charge exclusion can be controlled by changing the component ratio of polycation and polyanion during preparation. 相似文献
8.
Structure of soluble and membrane-bound human annexin V. 总被引:5,自引:0,他引:5
Annexins are a family of water-soluble proteins that bind to membranes in a calcium-dependent manner. Some members have been shown to exhibit voltage-dependent calcium channel activity, a property characteristic of integral membrane proteins. The structures of human annexin V in crystals obtained from aqueous solution and in two-dimensional crystals when bound to phospholipid layers have been determined by X-ray and electron crystallography, respectively. They are compared here. Both structures show close correspondence, suggesting that annexins attach to phospholipid membranes without substantial structural change. These observations, together with biochemical data, lead to the conclusion that annexin V interacts with phospholipid membranes with its convex face. We propose that binding is mediated by direct interaction between the phosphoryl headgroups and the calcium bound to polypeptide loops protruding from the convex face. The membrane area covered by annexin may thus become disordered and permeable allowing calcium flux through the membrane and the central channel-like structure found in annexin molecules. 相似文献
9.
The ryanodine receptor (RyR) is the largest known membrane protein with a total molecular mass of 2.3 x 10(3) kDa. Well ordered, two-dimensional (2D) crystals are an essential prerequisite to enable RyR structure determination by electron crystallography. Conventionally, the 2D crystallization of membrane proteins is based on a 'trial-and-error' strategy, which is both time-consuming and chance-directed. By adopting a new strategy that utilizes protein sequence information and predicted transmembrane topology, we successfully crystallized the RyR on positively charged lipid membranes. Image processing of negatively stained crystals reveals that they are well ordered, with diffraction spots of IQ < or = 4 extending to approximately 20 angstroms, the resolution attainable in negative stain. The RyR crystals obtained on the charged lipid membrane have characteristics consistent with 2D arrays that have been observed in native sarcoplasmic reticulum of muscle tissues. These crystals provide ideal materials to enable structural analysis of RyR by high-resolution electron crystallography. Moreover, the reconstituted native-like 2D array provides an ideal model system to gain structural insights into the mechanism of RyR-mediated Ca2+ signaling processes, in which the intrinsic ability of RyR oligomers to organize into a 2D array plays a crucial role. 相似文献
10.
11.
Numerous protein plaques cover the apical surface of mammalian urinary bladder epithelial cells. These plaques contain four integral membrane proteins, called uroplakins, which form a well-ordered array of hexameric complexes. The 3D structure of these naturally occurring 2D crystals was studied by cryo-electron-crystallographic methods using a slow-scan charged-coupled device (CCD) camera to record the electron micrographs. A 1.2 nm projection map calculated from untilted crystals shows that each hexamer comprises a ring of six inner and six outer domains at a radius of 5.7 nm and 9.2 nm respectively. The 3D structure shows that the mass is distributed strongly asymmetrically with respect to the membrane, with most of the mass protruding from the luminal face. Both domains in the asymmetric unit traverse the membrane and protrude from the membrane on the cytoplasmic side. On the luminal side, the two domains are bridged forming a stretched arc. The total thickness of the complex is about 13.2 nm. A model of the urothelial plaque reveals that contacts between the hexamers are much less extended than within the hexamers. 相似文献
12.
《Cell calcium》2019
Essential for physiology, transient receptor potential (TRP) channels constitute a large and diverse family of cation channels functioning as cellular sensors responding to a vast array of physical and chemical stimuli. Detailed understanding of the inner workings of TRP channels has been hampered by a lack of atomic structures, though structural biology of TRP channels has been an enthusiastic endeavor since their molecular identification two decades ago. These multi-domain integral membrane proteins, exhibiting complex polymodal gating behavior, have been a challenge for traditional X-ray crystallography, which requires formation of well-ordered protein crystals. X-ray structures remain limited to a few TRP channel proteins to date. Fortunately, recent breakthroughs in single-particle cryo-electron microscopy (cryo-EM) have enabled rapid growth of the number of TRP channel structures, providing tremendous insights into channel gating and regulation mechanisms and serving as foundations for further mechanistic investigations. This brief review focuses on recent exciting developments in structural biology of a subset of TRP channels, the calcium-permeable, non-selective and thermosensitive vanilloid subfamily of TRP channels (TRPV1-4), and the permeation and gating mechanisms revealed by structures. 相似文献
13.
It is notoriously difficult to produce crystals of membrane proteins that diffract to sufficient resolution for structural studies by X-ray crystallography. Crystals of a prokaryotic CLC chloride channel that were initially unacceptable for structural analysis improved in both quality and diffraction limit by a process of dehydration. The loss of water decreased the dimensions of the unit cell axes by up to 25 A, improved the diffraction limit from 8.0 to 4.0 A, and decreased the mosaicity to values of approximately 1 degrees. Dehydration of integral membrane protein crystals should be one of the procedures included in the initial screening for appropriate crystals and as a method of improving the diffraction limits of existing crystals. 相似文献
14.
A novel crystallization method for visualizing the membrane localization of potassium channels.
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The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel. 相似文献
15.
The effect of polyanion, poly(vinylsulfate), used as a model of negatively charged surface, on ferric cytochrome c (ferricyt c) structure in acidic pH has been studied by absorbance spectroscopy, circular dichroism (CD), tryptophan (Trp) fluorescence and microcalorimetry. The polyanion induced only small changes in the native structure of the protein at neutral pH, but it profoundly shifted the acid induced high spin state of the heme in the active center of cyt c to a more neutral pH region. Cooperativity of the acidic transition of ferricyt c in the presence of the polyanion was disturbed, in comparison with uncomplexed protein, as followed from different apparent pK(a) values observed in a distinct regions of the ferricyt c electronic absorbance spectrum (4.55+/-0.08 in the 620 nm band region and 5.47+/-0.15 in the Soret region). The ferricyt c structure in the complex with the polyanion at acidic pH (below pH 5.0) has properties of a molten globule-like state. Its tertiary structure is strongly disturbed according to CD and microcalorimetry measurements; however, its secondary structure, from CD, is still native-like and ferricyt c is in a compact state as evidenced by quenched Trp fluorescence. These findings are discussed in the context of the molten globule state of proteins induced on a negatively charged membrane surface under physiological conditions. 相似文献
16.
Two-dimensional structure of the membrane domain of human band 3, the anion transport protein of the erythrocyte membrane. 总被引:6,自引:1,他引:5
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The membrane domain of human erythrocyte Band 3 protein (M(r) 52,000) was reconstituted with lipids into two-dimensional crystals in the form of sheets or tubes. Crystalline sheets were monolayers with six-fold symmetry (layer group p6, a = b = 170 A, gamma = 60 degrees), whereas the symmetry of the tubular crystals was p2 (a = 104 A, b = 63 A, gamma = 104 degrees). Electron image analysis of negatively stained specimens yielded projection maps of the protein at 20 A resolution. Maps derived from both crystal forms show that the membrane domain is a dimer of two monomers related by two-fold symmetry, with each monomer consisting of three subdomains. In the dimer, two subdomains of each monomer form a roughly rectangular core (40 x 50 A in projection), surrounding a central depression. The third subdomain of the monomer measures approximately 15 x 25 A in projection and appears to be connected to the other two by a flexible link. We propose that the central depression may represent the channel for anion transport while the third subdomain appears not to be directly involved in channel formation. 相似文献
17.
T. König I. Stipani I. Horvàth F. Palmieri 《Journal of bioenergetics and biomembranes》1982,14(5-6):297-305
A synthetic polyanion (a copolymer of methacrylate, maleate, and styrene in 1:2:3 proportion with an average molecular weight of 10,000 dalton) inhibits the tricarboxylate, oxoglutarate, dicarboxylate, and adenine nucleotide translocators of rat liver mitochondria. The activity versus inhibitor concentration curves are sigmoidal. The inhibition of the oxoglutarate and tricarboxylate translocators by the polyanion is competitive, while that of the adenine nucleotide translocator is of mixed-type. TheK
1 values of the polyanion are the following: for oxoglutarate translocator 4.0 µM, tricarboxylate translocator 1.2 µM, and adenine nucleotide translocator 1.3 µM with ADP and 0.8 µM with ATP. It is suggested that the polyanion acts primarily by increasing the negative charge of the inner membrane at the outer surface, and the sensitivity of the translocators toward the polyanion depends on the number of negative charges of their substrates. 相似文献
18.
Channelrhodopsin-2 (ChR2) is the prototype of a new class of light-gated ion channels that is finding widespread applications in optogenetics and biomedical research. We present a 6-Å projection map of ChR2, obtained by cryo-electron microscopy of two-dimensional crystals grown from pure, heterologously expressed protein. The map shows that ChR2 is the same dimer with non-crystallographic 2-fold symmetry in three different membrane crystals. This is consistent with biochemical analysis, which shows a stable dimer in detergent solution. Comparison to the projection map to bacteriorhodopsin indicates a similar structure of seven transmembrane alpha helices. Based on the projection map and sequence alignments, we built a homology model of ChR2 that potentially accounts for light-induced channel gating. Although a monomeric channel is not ruled out, comparison to other membrane channels and transporters suggests that the ChR2 channel is located at the dimer interface on the 2-fold axis, lined by transmembrane helices 3 and 4. 相似文献
19.
The leucocytosis which can be evoked by the polyanions dextran sulphate (DS), polymethacrylic acid (PMAA) and the copolymer of PMAA and styrene (PMAA--STYR) was studied in mice. After intravenous administration of these polyanions peak numbers of leucocytes were found in the peripheral blood 3 hr after injection. All three types of polyanions increased the number of lymphocytes, granulocytes and monocytes. Dose--response studies revealed that the nature of the polyanion determined the degree of leucocyte mobilization. The most potent mobilizer was found to be DS. This polyanion could evoke a six-fold increase of the number of peripheral blood leucocytes. By means of the membrane fluorescence technique it could be demonstrated that optimal doses of DS, PMAA and PMAA--STYR mobilized both B and T lymphocytes. The ratio between the number of B and T cells mobilized was greater for DS than for the other two polyanions. Intravenous injection of DS, PMAA and PMAA--STYR also increased the number of circulating haemopoietic stem cells (CFU-S). The most potent stem cell mobilizer appeared to be PMAA--STYR. This polyanion evoked a twenty-five-fold increase in the number of CFU-S. 相似文献
20.
Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC. 相似文献