Large Aqueous Pores Research Articles

Species-specific gene duplication in Clostridia produces variations of cholesterol-dependent cytolysin with different cytotoxicity

Cholesterol-dependent cytolysin (CDC) is a bacterial toxin that binds to eukaryotic cholesterol-containing membranes, forms oligomeric complexes, and is inserted into the bilayer to create large aqueous pores. Recently, we reported a species-specific duplication of the hemolysin gene in group III Clostridium botulinum. The duplicated genes (bly1 and bly2) encoded two separate CDC proteins (botulinolysins; BLY1 and BLY2). Here, we aimed to investigate whether BLY1 and BLY2 exert differential cytotoxicity. We isolated two bly genes from C. botulinum and evaluated the cytotoxicity of two recombinant BLY proteins (rBLY1 and rBLY2) in HeLa, IEC-6, and NRK cells. rBLYs were cytotoxic to equine erythrocytes. rBLY1 showed higher hemolytic activity than rBLY2. rBLY2 showed no or very weak cytotoxicity to the HeLa, IEC-6, and NRK cells, whereas rBLY1 showed high cytotoxicity to these cells. The comparison of the amino acid sequence of BLYs with those of other CDCs revealed that the already-known amino acid residues involved in cholesterol-containing membrane recognition, oligomerization, and insertion into membranes are well conserved in both BLYs. However, several amino acid substitutions were observed in the conserved regions, particularly in L2 and L3 regions involved in cell binding. These findings suggest that gene duplication in group III C. botulinum evolved distinct functional specializations, and differential cytotoxicity of BLY1 and BLY2 could be due to the amino acid substitution in the conserved regions. However, the structural and functional comparisons of the two BLYs are essential to gain insights into the function of the CDCs.

Cold-set oat protein isolate--gellan gum binary gels with various microstructures: Fabrication, characterization, formation mechanism, and controlled release properties

Cold-set oat protein isolate (OPI) and gellan gum (GG) binary gels with various microstructures was fabricated with different amounts glucono-δ-lactone (GDL) as a carrier to deliver bioactive compounds. OPI and GG formed gel-like mixture at pH 8 mainly via hydrophobic interactions with a binding constant (Ka) of 3.85 × 104 M−1, while the mixture showed highest storage modulus (G′) with 0.1% GG (w/w, based on the dry weight of protein). Novel binary gels fabricated with 7% OPI (w/v) and 0.1% GG (w/w) (OGG) were then developed through addition of various amounts of GDL. For the gels prepared with 1.5% and 4.0% GDL (w/w, based on the dry weight of protein, pH 7.1 and 6.3, respectively), heterogeneous network formed by co-aggregated OPI and GG with a small amount of small GG particles dispersed within large aqueous pores were fabricated mainly through hydrophobic interactions. For OGG-3 (9.0% GDL, w/w) and OGG-4 (20.0% GDL, w/w), the final pH was 5.1 and 4.1 that around or lower than OPI's isoelectric point, hydrogen bonding and electrostatic attractive forces were developed between two polymers since OPI contained neat or positive charges, resulting in a compact coupled OPI-GG network structure. Heterogeneous network with small GG particles dispersed in pores allowed OGG-2 (4.0% GDL, w/w) had comparable good gel hardness to the gel formed with 9.0% GDL (w/w, OGG-3) with coupled microstructure, whereas OGG-3 showed a better ability to restrict the swelling induced release of riboflavin in SGF, and allowed sustainable release of riboflavin in SIF. Therefore, OPI-GG binary gels with various microstructures prepared by simple acidification method at ambient temperature have great potential to be used as delivery systems for heat-sensitive bioactive compounds in food and non-food applications.

Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes κ1 for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients κ1 probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/l) and DMSO (2250 mOsm/l) the filtration coefficient L(p) in the presence of a penetrating cryoprotectant (L(pDMSO) = (4.45 ± 0.04) x 10(-14) m3/Ns) is 3 orders lower compared to the case with electrolyte (L(pNaCl) = (2.25 ± 0.25) x 10(-11) m3/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.

The N-Terminal Half of the Connexin Protein Contains the Core Elements of the Pore and Voltage Gates

Connexins form channels with large aqueous pores that mediate fluxes of inorganic ions and biological signaling molecules. Studies aimed at identifying the connexin pore now include a crystal structure that provides details of putative pore-lining residues that need to be verified using independent biophysical approaches. Here we extended our initial cysteine-scanning studies of the TM1/E1 region of Cx46 hemichannels to include TM2 and TM3 transmembrane segments. No evidence of reactivity was observed in either TM2 or TM3 probed with small or large thiol-modifying reagents. Several identified pore residues in E1 of Cx46 have been verified in different Cx isoforms. Use of variety of thiol reagents indicates that the connexin hemichannel pore is large and flexible enough, at least in the extracellular part of the pore funnel, to accommodate uncommonly large side chains. We also find that that gating characteristics are largely determined by the same domains that constitute the pore. These data indicate that biophysical and structural studies are converging towards a view that the N-terminal half of the Cx protein contains the principal components of the pore and gating elements, with NT, TM1 and E1 forming the pore funnel.

Loop Gating of Connexin Hemichannels Involves Movement of Pore-lining Residues in the First Extracellular Loop Domain

Unapposed connexin hemichannels exhibit robust closure in response to membrane hyperpolarization and extracellular calcium. This form of gating, termed "loop gating," is largely responsible for regulating hemichannel opening, thereby preventing cell damage through excessive flux of ions and metabolites. The molecular components and structural rearrangements underlying loop gating remain unknown. Here, using cysteine mutagenesis in Cx50, we demonstrate that residues at the TM1/E1 border undergo movement during loop gating. Replacement of Phe(43) in Cx50 with a cysteine resulted in small or no appreciable membrane currents. Bath application of dithiothreitol or TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine), reagents that exhibit strong transition metal chelating activity, led to robust currents indicating that the F43C substitution impaired hemichannel function, producing "lock-up" in a closed or poorly functional state due to formation of metal bridges. In support, Cd(2+) at submicromolar concentrations (50-100 nm) enhanced lock-up of F43C hemichannels. Moreover, lock-up occurred under conditions that favored closure, indicating that the sulfhydryl groups come close enough to each other or to other residues to coordinate metal ions with high affinity. In addition to F43C, metal binding was also found for G46C, and to a lesser extent, D51C substitutions, positions found to be pore-lining in the open state using the substituted-cysteine accessibility method, but not for A40C and A41C substitutions, which were not found to reside in the open pore. These results indicate that metal ions access the cysteine side chains through the open pore and that closure of the loop gate involves movement of the TM1/E1 region that results in local narrowing of the large aqueous connexin pore.

Evaluation of Hydrophilic Permeant Transport Parameters in the Localized and Non-Localized Transport Regions of Skin Treated Simultaneously With Low-Frequency Ultrasound and Sodium Lauryl Sulfate

The porosity (epsilon), the tortuosity (tau), and the hindrance factor (H) of the aqueous pore channels located in the localized transport regions (LTRs) and the non-LTRs formed in skin treated simultaneously with low-frequency ultrasound (US) and the surfactant sodium lauryl sulfate (SLS), were evaluated for the delivery of four hydrophilic permeants (urea, mannitol, raffinose, and inulin) by analyzing dual-radiolabeled diffusion masking experiments for three different idealized cases of the aqueous pore pathway hypothesis. When epsilon and tau were assumed to be independent of the permeant radius, H was found to be statistically larger in the LTRs than in the non-LTRs. When a distribution of pore radii was assumed to exist in the skin, no statistical differences in epsilon, tau, and H were observed due to the large variation in the pore radii distribution shape parameter (3 A to infinity). When infinitely large aqueous pores were assumed to exist in the skin, epsilon was found to be 3-8-fold greater in the LTRs than in the non-LTRs, while little difference was observed in the LTRs and in the non-LTRs for tau. This last result suggests that the efficacy of US/SLS treatment may be enhanced by increasing the porosity of the non-LTRs.

The Role of the GX9GX3G Motif in the Gating of High Voltage-activated Ca2+ Channels

The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) Ca(V) channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca(V)1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly(432) and Gly(436) in Ca(V)1.2) to form a triglycine motif unique to HVA Ca(V) channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca(2+) channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca(V)1.2, alpha-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with Ca(V)1.2 wild type > G770A > G422A congruent with G436A >> G432A (from the fastest to the slowest). Mutations at position Gly(432) produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly(436) to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX(3)G residues blunted Ca(2+)-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca(V)2.3, mutation of the distal glycine Gly(352) impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX(3)G motif in the voltage-dependent activation and inactivation gating of HVA Ca(V) channels with the distal glycine residue being mostly involved in the inactivation gating.

Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit β-strand alignment

Cholesterol-dependent cytolysins are bacterial protein toxins that bind to cholesterol-containing membranes, form oligomeric complexes and insert into the bilayer to create large aqueous pores. Membrane-dependent structural rearrangements required to initiate the oligomerization of perfringolysin O monomers have been identified, as have the monomer-monomer interaction surfaces, using site-specific mutagenesis, disulfide trapping and multiple fluorescence techniques. Upon binding to the membrane, a structural element in perfringolysin O moves to expose the edge of a previously hidden beta-strand that forms the monomer-monomer interface and is required for oligomer assembly. The beta-strands that form the interface each contain a single aromatic residue, and these aromatics appear to stack, thereby aligning the transmembrane beta-hairpins of adjacent monomers in the proper register for insertion. Collectively, these data reveal a novel membrane binding-dependent mechanism for regulating cytolysin monomer-monomer association and pore formation.

Transplant: immunology and treatment of rejection

Transplant: immunology and treatment of rejection

Mechanosensitive channels in bacteria as membrane tension reporters.

The purpose of this short review is to discuss recent data on the molecular structure and mechanism of gating of MscL, a mechanosensitive channel of large conductance from Escherichia coli. MscL is the first isolated molecule shown to convert mechanical stress of the membrane into a simple response, the opening of a large aqueous pore. The functional complex appears to be a stable homo-pentamer of 15-kDa subunits, the gating transitions in which are driven by stretch forces conveyed through the lipid bilayer. We have measured the open probability of MscL and the kinetics of transitions as a function of membrane tension. The parameters extracted from the single-channel current recordings and dose-response curves such as the energy difference between the closed, open, and intermediate conducting states, and the transition-related changes in protein dimensions suggest a large conformational rearrangement of the channel complex. The estimations show that in native conditions MscL openings could be driven primarily by forces of osmotic nature. The thermodynamic and spatial parameters reasonably correlate with the available data on the structure of a single MscL subunit and multimeric organization of the complex. Combined with the functional analysis of mutations, these data give grounds to hypotheses on the nature of the channel mechanosensitivity.

Cloning and functional characterisation of the mouse P2X 7 receptor

We have isolated a 1785-bp complementary DNA (cDNA) encoding the murine P2X 7 receptor subunit from NTW8 mouse microglial cells. The encoded protein has 80% and 85% homology to the human and rat P2X 7 subunits, respectively. Functional properties of the heterologously expressed murine P2X 7 homomeric receptor broadly resembled those of the P2X 7 receptor in the native cell line. However, marked phenotypic differences were observed between the mouse receptor, and the other P2X 7 receptor orthologues isolated with respect to agonist and antagonist potencies, and the kinetics of formation of the large aqueous pore.

VDAC channels mediate and gate the flow of ATP: implications for the regulation of mitochondrial function

The mitochondrial channel, VDAC, forms large (3 nm in diameter) aqueous pores through membranes. We measured ATP flow (using the luciferin/luciferase method) through these channels after reconstitution into planar phospholipid membranes. In the open state of VDAC, as many as 2 x 10(6) ATP molecules can flow through one channel per second. The half-maximum rate occurs at approximately 75 mM ATP. The permeability of a single channel for ATP is 1.1 x 10(-14) cm3/s (about 1 cm/s after correcting for cross-sectional area), which is 100 times less than the permeability for chloride and 10 times less than that for succinate. Channel closure results in a 50% reduction in conductance, showing that monovalent ions are still quite permeable, yet ATP flux is almost totally blocked. This is consistent with an electrostatic barrier that results in inversion of the selectivity of the channel and could be an example of how large channels selectively control the flow of charged metabolites. Thus VDAC is ideally suited to controlling the flow of ATP between the cytosol and the mitochondrial spaces.

Oriented channel insertion reveals the motion of a transmembrane beta strand during voltage gating of VDAC.

Yeast VDAC channels (isolated from the mitochondrial outer membrane) form large aqueous pores whose walls are believed to consist of 1 alpha helix and 12 beta strands. Each channel has two voltage-gating processes: one closes the channels at positive potentials, the other at negative. When VDAC is reconstituted into phospholipid (soybean) membranes, the two gating processes have virtually the same steepness of voltage dependence and the same midpoint voltage. Substituting lysine for glutamate at either end of one putative beta strand (E145K or E152K) made the channels behave asymmetrically, increasing the voltage dependence of one gating process but not the other. The asymmetry was the same whether 1 or 100 channels were in the membrane, indicating oriented channel insertion. However, the direction of insertion varied from membrane to membrane, indicating that the insertion of the first channel was random and subsequent insertions were directed by the previously inserted channel(s). This raises the prospect of an auto-directed insertion with possible implications to protein targeting in cells. Each of the mutations affected a different gating process because the double mutant increased voltage dependence of both processes. Thus this strand may slide through the membrane in one direction or the other depending on the gating process. We propose that the model of folding for VDAC be altered to move this strand into the sensor region of the protein where it may act as a tether and guide/restrict the motion of the sensor.

An ADP‐sensitive cyclosporin‐A‐binding protein in rat liver mitochondria

Mitochondria contain a structure which forms a large aqueous pore in the inner membrane after Ca2+ overload in the presence of Pi. In the present study, pore activation in liver mitochondria was monitored using the collapse of the inner membrane potential (delta psi). Ca(2+)-induced pore opening (delta psi collapse) was prevented by the immunosuppressant cyclosporin A, but cyclosporin A did not reverse pore opening (i.e. allow delta psi regeneration) unless ADP was also added. At concentrations that produced substantial pore blockade, [3H]cyclosporin partitioned more or less equally between membrane and soluble fractions, but the distribution was shifted slightly to the membranes in the presence of ADP. ADP also increased the binding of [3H]cyclosporin A to membranes washed free of soluble components. The indication that cyclosporin A inhibition of the pore is mediated by an ADP-sensitive membrane component was examined using a tritiated photoactivable derivative of cyclosporin A. ADP selectively increased covalent binding of this derivative to a membrane component. This component eluted from molecular-sizing columns as a 13-17-kDa-protein in the presence of 0.5% Chaps as detergent and migrated as a 10-kDa (approximately) protein in SDS/PAGE. These findings provide the first evidence that a protein of approximately 10 kDa may be part of the cyclosporin-A receptor of the Ca(2+)-activated pore. The possible implications of these findings are discussed.

ATP-dependent ionic permeability on nuclear envelope in in situ nuclei of Xenopus oocytes.

The nuclear envelope represents a structural and functional barrier between cytoplasm and nucleoplasm. Small molecules and solutes passively cross the nuclear envelope, whereas the transport of large proteins and RNA requires metabolic energy. Using in situ Xenopus oocyte nuclei, we characterized ATP-dependent ionic permeabilities on the external surface of the envelope. The presence, but not necessarily the hydrolysis, of ATP is crucial to maintaining the channels in an open state. Localization of the ionic channels is still unclear. From morphologic and current kinetics data, we suggest a relation between the ionic channels and the nuclear pores. We try, in this way, to explain the apparent contradiction between the presence of ion-selective channels in parallel with large aqueous pores on the nuclear envelope. Under this hypothesis, variations in the metabolic energy content of the cytoplasm would induce nucleocytoplasmic passive exchanges. The distribution and movement of charged particles across the nuclear envelope may influence many cytoplasmic functions. Regulation of the current by ATP could play an important role in hormonal stimulation, divalent ion permeation into the nucleus, and cell cycle mechanisms.

Channels in mitochondrial membranes: knowns, unknowns, and prospects for the future.

Rapid diffusion of hydrophilic molecules across the outer membrane of mitochondria has been related to the presence of a protein of 29 to 37 kDa, called voltage-dependent anion channel (VDAC), able to generate large aqueous pores when integrated in planar lipid bilayers. Functional properties of VDAC from different origins appear highly conserved in artificial membranes: at low transmembrane potentials, the channel is in a highly conducting state, but a raise of the potential (both positive and negative) reduces drastically the current and changes the ionic selectivity from slightly anionic to cationic. It has thus been suggested that VDAC is not a mere molecular sieve but that it may control mitochondrial physiology by restricting the access of metabolites of different valence in response to voltage and/or by interacting with a soluble protein of the intermembrane space. The latest application of the patch clamp and tip-dip techniques, however, has indicated both a different electric behavior of the outer membrane and that other proteins may play a role in the permeation of molecules. Biochemical studies, use of site-directed mutants, and electron microscopy of two-dimensional crystal arrays of VDAC have contributed to propose a monomeric beta barrel as the structural model of the channel. An important insight into the physiology of the inner membrane of mammalian mitochondria has come from the direct observation of the membrane with the patch clamp. A slightly anionic, voltage-dependent conductance of 107 pS and one of 9.7 pS, K(+)-selective and ATP-sensitive, are the best characterized at the single channel level. Under certain conditions, however, the inner membrane can also show unselective nS peak transitions, possibly arising from a cooperative assembly of multiple substrates.

The exocytotic fusion pore modeled as a lipidic pore.

Freeze-fracture electron micrographs from degranulating cells show that the lumen of the secretory granule is connected to the extracellular compartment via large (20 to 150 nm diameter) aqueous pores. These exocytotic fusion pores appear to be made up of a highly curved bilayer that spans the plasma and granule membranes. Conductance measurements, using the patch-clamp technique, have been used to study the fusion pore from the instant it conducts ions. These measurements reveal the presence of early fusion pores that are much smaller than those observed in electron micrographs. Early fusion pores open abruptly, fluctuate, and then either expand irreversibly or close. The molecular structure of these early fusion pores is unknown. In the simplest extremes, these early fusion pores could be either ion channel like protein pores or lipidic pores. Here, we explored the latter possibility, namely that of the early exocytotic fusion pore modeled as a lipid-lined pore whose free energy was composed of curvature elastic energy and work done by tension. Like early exocytotic fusion pores, we found that these lipidic pores could open abruptly, fluctuate, and expand irreversibly. Closure of these lipidic pores could be caused by slight changes in lipid composition. Conductance distributions for stable lipidic pores matched those of exocytotic fusion pores. These findings demonstrate that lipidic pores can exhibit the properties of exocytotic fusion pores, thus providing an alternate framework with which to understand and interpret exocytotic fusion pore data.

Synthesis and characterization of thermally reversible macroporous poly(N‐isopropylacrylamide) hydrogels

AbstractMacroporous hydrogels are characterized by large pore sizes, high pore volumes, and high specific surface area. Besides these characteristics, macroporous hydrogels based on thermally reversible polymers respond to temperature changes much faster than hydrogels prepared by a conventional method. Crosslinked poly(N‐isopropylacrylamide) (polyNIPAAm) forms a thermally reversible hydrogel which shows a lower critical solution temperature (LCST) ca. 33°C in aqueous solutions. We have synthesized thermally reversible polyNIPAAm hydrogels having macroporous structures by a new method. These macroporous hydrogels have large pore volumes, large average pore sizes, and faster macromolecule permeation rates in comparison to conventional polyNIPAAm hydrogels synthesized by a conventional method. Compared with conventional polyNIPAAm hydrogels, the macroporous polyNIPAAm hydrogels have higher swelling ratios at temperatures below the LCST and exhibit faster deswelling and reswelling rates. The deswelling rates are especially rapid. These thermally reversible macroporous hydrogels may be very useful in controlled active agent delivery and toxin removal, as well as dewatering of solutions. Peptides or proteins may behave as if they were in bulk solution within the large aqueous pores, and this may reduce their inactivation when such gels are used for their storage and later release. The gels may also be useful in microrobotic devices due to their fast response to temperature. © 1992 John Wiley & Sons, Inc.

Controlled release of amylase from a thermal and pH-sensitive, macroporous hydrogel

A heterogeneous hydrogel containing silicone rubber domains has been synthesized from a thermal and pH sensitive polymer containing N-isopropyl acrylamide (NIPAAm), acrylic acid (AAc), a divinyl silicone rubber plus a small amount of crosslinker. These gels are unswollen at 37°C and pH 1.4 and swell at pH 7.4 where they form large aqueous pores. Therefore, they could be useful for pH-controlled enteric delivery of proteins. Amylase has been loaded and released from such gels under conditions simulating gastrointestinal transport in the study reported here.

23] Movement from trans-Golgi network to cell surface in semiintact cells

Publisher Summary This chapter describe a method that utilizes semiintact Chinese hamster ovary (CHO) cells to reconstitute vesicular traffic from the trans-Golgi region to the plasma membrane. The assay utilizes a bacterial cytolysin, streptolysin O (SL-O), to introduce large aqueous pores selectively into the plasma membrane. The general application of SL-O to permeabilize cells is reviewed. As this method produces minimal damage to the cell surface, it is suitable for studying various transport pathways between the trans-Golgi and the plasma membrane. This approach is successfully utilized by the group to study intracellular transport in CHO cells. The trans-Golgi is a key compartment involved in tracking among the cellular organelles. Newly synthesized proteins to be released into the extracellular space, incorporated into the plasma membrane, or localized to lysosomes all traverse the same compartments until they reach the trans-Golgi. At the trans-Golgi network, these proteins are sorted from one another and packaged into unique classes of vesicles that are targeted to distinct post-Golgi destinations.