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M Colombini

Publications and source records attributed to M Colombini.

At least 55 records · Page 3Linked to original sources
PubMed1994

Well being in patients on CAPD and hemodialysis.

In the present multicenter study, 120 pts who had been treated by both hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) for at least 6 months each, were invited to answer questions on 34 matters, to compare symptoms and their well-being while on the two treatments. Patients were invited to choose HD or CAPD and indicate the reasons for their choice. For 28 patients the first treatment was HD and for 92 CAPD. The mean time between the change of therapy and the study was 46 +/- 35 months. Their final choices were found to be strictly related to the present treatment (p < 0.001). The reasons for choice of CAPD were: more free time (21%), more freedom (67%), better well-being (44%), less worry (5%); for HD they were: more free time (53%), better well-being (39%), less worry (13%), no need for a peritoneal catheter and fewer clinical complications (19%). The catheter was considered more cumbersome than the A.V. fistula, the time involved was considered to be shorter on HD by 52 patients and on CAPD by 39, thirst and cramps were considered to be more frequent and severe on CAPD by half of the patients. The prevalence and severity of problems and symptoms and choice of treatment were not related to sex, job, education or age.

Adult↗
PubMed1993

Mitochondrial voltage-dependent anion channel. Immunochemical and immunohistochemical characterization in rat brain.

The purified mitochondrial benzodiazepine receptor (mBzR) is a complex comprising the voltage-dependent anion channel (VDAC), adenine nucleotide carrier, and an 18-kDa protein that binds isoquinoline carboxamide ligands (McEnery, M. W., Snowman, A. M., Trifiletti, R. R., and Snyder, S. H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3170-3174). An antiserum raised against the mBzR complex reacts selectively with VDAC and is used, along with purification, electrophysiological and immunohistochemical techniques, to characterize the properties and distribution of rat brain VDAC. Although purified VDAC displays biochemical and electrical conductance properties similar to VDAC from other sources, the immunohistochemical distribution of VDAC in rat brain is heterogeneous with pronounced regional variations; the pontine nuclei, the supraoptic nucleus, Purkinje cells of the cerebellum, and the caudate putamen evidence the highest density. The distribution of VDAC is inclusive of the more discretely localized 18-kDa mBzR protein, suggesting that only a portion of the total VDAC participates in the mBzR. The histochemical localizations of the mitochondrial marker enzymes glutamate dehydrogenase and cytochrome c oxidase also indicate marked regional variability in both mitochondrial content and composition. The discrete expression of VDAC reflects a striking heterogeneity of rat brain mitochondria and underlying differences in the utilization of mitochondrial outer membrane ion channels.

Animals↗
PubMed1993

The outer mitochondrial membrane channel, VDAC, is modulated by a protein localized in the intermembrane space.

The mitochondrial outer membrane channel, VDAC, provides a pathway for the flux of metabolites between the cytoplasm and mitochondrion. VDAC is voltage-dependent and occupies states of differing conductivity and ion selectivity that are dependent on transmembrane potential. A protein, derived from preparations of mitochondria, has been shown to increase the voltage dependence of VDAC and is called the VDAC modulator. Both VDAC and the VDAC modulator have been extensively characterized by reconstitution into planar lipid bilayers. In order for the VDAC modulator to have physiological significance it must have physical access to VDAC in the cell. This constraint dictates that the modulator be an extrinsic outer mitochondrial membrane protein, occupy the mitochondrial intermembrane space, or be a cytoplasmic constituent. To address the question of subcellular localization, purified mitochondria were selectively lysed with digitonin or treated with trypsin while resuspended in hypo-osmotic or iso-osmotic medium. Marker enzymes and modulator activity were monitored during the various treatments. Results indicate that the integrity of the outer membrane was necessary to prevent modulator release or protection from trypsin digestion. Outer membrane lysis, under conditions where the inner membrane remained intact, resulted in modulator release or inactivation by trypsin. These results suggest an intermembrane space location for the VDAC modulator in the mitochondrion.

Digitonin↗
PubMed1993

Mapping of residues forming the voltage sensor of the voltage-dependent anion-selective channel.

Voltage-gated ion-channel proteins contain "voltage-sensing" domains that drive the conformational transitions between open and closed states in response to changes in transmembrane voltage. We have used site-directed mutagenesis to identify residues affecting the voltage sensitivity of a mitochondrial channel, the voltage-dependent anion-selective channel (VDAC). Although charge changes at many sites had no effect, at other sites substitutions that increased positive charge also increased the steepness of voltage dependence and substitutions that decreased positive charge decreased voltage dependence by an appropriate amount. In contrast to the plasma membrane K+ and Na+ channels, these residues are distributed over large parts of the VDAC protein. These results have been used to define the conformational transitions that accompany voltage gating of an ion channel. This gating mechanism requires the movement of large portions of the VDAC protein through the membrane.

Amino Acid Sequence↗
PubMed1993

Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane is a small abundant protein found in all eukaryotic kingdoms which forms a voltage-gated pore when incorporated into planar lipid bilayers. VDAC is also the site of binding of the metabolic enzymes hexokinase and glycerol kinase to the mitochondrion in what may be a significant metabolic regulatory interaction. Recently, there has been speculation that there may be multiple forms of VDAC in mammals which differ in their localization in the outer mitochondrial membrane and in their physiological function. In this report, we describe the identification and characterization of two human cDNAs encoding VDAC homologs (HVDAC1 and HVDAC2). To confirm VDAC function, each human protein has been expressed in yeast lacking the endogenous VDAC gene. Human proteins isolated from yeast mitochondria formed channels with the characteristics expected of VDAC when incorporated into planar lipid bilayers. In addition, expression of the human proteins in such strains can complement phenotypic defects associated with elimination of the endogenous yeast VDAC gene. Since VDAC is the site of binding of hexokinase to the outer mitochondrial membrane, the binding capacity of each VDAC isoform expressed in yeast mitochondria was assessed. When compared with the binding of hexokinase to mitochondria lacking VDAC, the results show that mitochondria expressing HVDAC1 are capable of specifically binding hexokinase, whereas mitochondria expressing HVDAC2 only bind hexokinase at background levels. The expression of each human cDNA has been assessed by Northern blot and polymerase chain reaction techniques. With one exception, each is expressed in all human cell lines and tissues examined.

Amino Acid Sequence↗
PubMed1993

Zero-current potentials in a large membrane channel: a simple theory accounts for complex behavior.

Flow of ions through large channels is complex because both cations and anions can penetrate and multiple ions can be in the channel at the same time. A modification of the fixed-charge membrane theory of Teorell was reported (Peng, S., E. Blachly-Dyson, M. Forte, and M. Colombini. 1992. Biophys. J. 62:123-135) in which the channel is divided into two compartments: a relatively charged cylindrical shell of solution adjacent to the wall of the pore and a relatively neutral central cylinder of solution. The zero-current (reversal) potential results in current flow in opposite directions in these two compartments. This description accounted rather well for the observed reversal potential changes following site-directed mutations. Here we report the results of systematic tests of this simple theory with the mitochondrial channel, VDAC (isolated from Neurospora crassa), reconstituted into planar phospholipid membranes. The variation of the observed reversal potential with transmembrane activity ratio, ionic strength, ion mobility ratio, and net charge on the wall of the pore are accounted for reasonably well. The Goldman-Hodgkin-Katz theory fails to account for the observations.

Biophysical Phenomena↗
PubMed1992

Isolation and cloning of a voltage-dependent anion channel-like Mr 36,000 polypeptide from mammalian brain.

A polypeptide of M(r) 36,000 (36 kDa) was isolated from detergent-solubilized membrane fractions of mammalian brain on a benzodiazepine affinity column utilized for the purification of the gamma-aminobutyric acid/benzodiazepine receptor protein, followed by preparative gel electrophoresis. Partial protein sequence for two fragments of the 36-kDa polypeptide allowed the isolation of cDNA clones from a rat hippocampal library. An open reading frame coding a sequence of 295 amino acid residues containing the two probe peptide sequences with minor differences, and a putative N-terminal signal peptide of 25 residues was found. Hydropathy index revealed no regions of alpha-helix suitable for membrane spanning, but several areas of alternating hydrophilic and hydrophobic residues consistent with beta-strands. The sequence of this brain protein was 24% identical to that of a yeast mitochondrial protein, the voltage-dependent anion channel (VDAC), and over 70% identical with the VDAC from human B lymphocytes. The gamma-aminobutyric acid type A (GABAA) receptor/36-kDa preparation purified on benzodiazepine affinity column has channel-forming activity in lipid bilayer membranes that is virtually identical to VDAC isolated from mitochondria of various sources, indicating that the 36-kDa protein is a new member of the VDAC family of proteins. An antiserum raised against the purified 36-kDa polypeptide was able to precipitate [3H]muscimol binding activity, indicating a tight association with the GABAA receptor protein in vitro and copurification on the benzodiazepine affinity column due to this association. Further studies are needed to determine whether such an association occurs in vivo.

Amino Acid Sequence↗
PubMed1992

Regulation of mitochondrial respiration by controlling the permeability of the outer membrane through the mitochondrial channel, VDAC.

Mitochondrial functions depend not only on the properties of the particular enzyme systems, but also on the continual flux of metabolites between the cytoplasm and mitochondrial spaces. We report the results of experiments that strongly indicate that a soluble mitochondrial protein can regulate mitochondrial respiration by reducing the permeability of the outer membrane. This protein is known as the VDAC modulator because it induces the outer mitochondrial membrane channel, VDAC, to close. When added to intact mitochondria, the modulator reduces the ADP-stimulated respiration. This inhibition can be prevented by damaging the outer membrane prior to modulator addition. Another mitochondrial activity, adenylate kinase, is reduced by 40% by the addition of the VDAC modulator to intact mitochondria. Again, damaging the outer membrane removed the modulator effect. Dextran sulfate, an artificial polyanion that acts on VDAC channels in a similar way to the VDAC modulator, has the same effects on intact mitochondria. The findings correlate well with observations of the actions of the VDAC modulator on reconstituted VDAC channels, in which the modulator induces the channel to enter a very low conductive state. The ability of a mitochondrial protein to regulate mitochondrial activities by reducing the permeability of the outer membrane further fuels the hypothesis that this membrane participates in the overall regulation of mitochondrial functions.

Adenylate Kinase↗
PubMed1992

Toward the molecular structure of the mitochondrial channel, VDAC.

A summary is presented of the most recent information about the structure and mechanism of closure of the mitochondrial channel, VDAC. Considerable information has come from studies involving electron microscopy of two-dimensional crystals and from electrophysiological studies of wild-type channels and site-directed mutants. Available evidence points to a beta-barrel as the basic structural model for VDAC. Two models for voltage- or effector- induced closure have been proposed, the first involving removal of strands from the wall of the pore, the second invoking movement of protein domains into the lumen. Experimental strategies to resolve the actual mechanism are presented.

Amino Acid Sequence↗
PubMed1992

Determination of the number of polypeptide subunits in a functional VDAC channel from Saccharomyces cerevisiae.

Genes encoding VDAC proteins containing specific site-directed amino acid alterations were introduced into wild-type Saccharomyces cerevisiae. The mutant VDAC proteins form channels with ion selectivities very different from that of the wild-type channel. Therefore, the resulting yeast strains express two different genes capable of coding for functional, yet distinct, VDAC channels. If VDAC were an oligomeric channel, analysis of VDAC from these strains should have revealed not only the presence of channels with wild-type or mutant selectivity but also channels with intermediate selectivities. While channels with wild-type and mutant selectivities were observed with approximately equal frequency, no channels with intermediate selectivity were observed. Sufficient observations were performed with two different mutant genes K61E.K65E and K19E.K61E) that the likelihood of having missed hybrid channels was less than 1 in 10(7). These findings favor the hypothesis that each functional VDAC channel is composed of a single 30-kDa polypeptide chain.

Fungal Proteins↗
PubMed1992

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel, VDAC.

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found in Neurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 micrograms/ml) increases the voltage dependence of VDAC channels over 2-3-fold. At higher protein concentrations (50-100 micrograms/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifier in vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.

Electrophysiology↗
PubMed1992

Large scale rearrangement of protein domains is associated with voltage gating of the VDAC channel.

The VDAC channel of the mitochondrial outer membrane is voltage-gated like the larger, more complex voltage-gated channels of the plasma membrane. However, VDAC is a low molecular weight (30 kDa), abundant protein, which is readily purified and reconstituted, making it an ideal system for analyzing the molecular basis for ion selectivity and voltage-gating. We have probed the VDAC channel by subjecting the cloned yeast (S. cerevisiae) VDAC gene to site-directed mutagenesis and introducing the resulting mutant channels into planar bilayers to detect the effects of specific sequence changes on channel properties. This approach has allowed us to formulate and test a model of the open state structure of the VDAC channel. Now we have applied the same approach to analyzing the structure of the channel's low-conducting "closed state" (essentially closed to important metabolites). We have identified protein domains forming the wall of the closed conformation and domains that seem to be removed from the wall of the pore during channel closure. The latter can explain the reduction in pore diameter and volume and the dramatically altered channel selectivity resulting from the channel closure. This process would make a natural coupling between motion of the sensor and channel gating.

Amino Acid Sequence↗
PubMed1992

Effect of simvastatin in CAPD patients with hypercholesterolemia.

The effect of simvastatin on serum total and HDL cholesterol and total triglyceride levels in 20 hypercholesterolemic patients on CAPD treatment was studied. The drug was given at the initial dose of 10 mg/day which was doubled up to 40 mg/day. Two non-compliant patients stopped the drug in the first week of treatment. One patient had vomiting and stopped simvastatin. One patients reduced the dose from 20 to 10 mg/day because of increase in CPK level. The study was completed in 16 patients. Serum cholesterol decreased from 318 +/- 39 to 208 +/- 34 mg/dl (p < 0.001), triglyceride from 317 +/- 129 to 278 +/- 160 mg/dl and HDL cholesterol from 43 +/- 13 to 35 +/- 11 mg/dl. The effective does was 10 mg/day in 4 cases, 20 mg/dl in 7 and 40 mg/dl in 5. In CAPD patients, simvastatin is safe and effective in lowering serum cholesterol. The clinical significance of the decrease in HDL cholesterol and its possible effect on clinical outcome are still unknown.

Anticholesteremic Agents↗
PubMed1991

Patch clamping VDAC in liposomes containing whole mitochondrial membranes.

Whole mitochondrial membranes isolated from Neurospora crassa were reconstituted into liposomes and patch clamped. Clear activity characteristic of the mitochondrial channel VDAC was found, namely: open state conductance of 650 pS (in 150 mM KCl, 1 mM CaCl2, 20 mM HEPES, pH 7.2), voltage-dependent closure at both positive and negative potentials, change in conductance upon channel closure of about 450 pS in response to negative and positive potentials, and increased voltage dependence in the presence of König's polyanion. This is the first clear demonstration of VDAC single channels using the patch-clamp technique, even though others used this method before to study whole mitochondrial membranes and liposomes containing mitochondrial proteins. We also found one other channel with a conductance change of about 120 pS.

Electric Conductivity↗
PubMed1991

Surface topography and molecular stoichiometry of the mitochondrial channel, VDAC, in crystalline arrays.

The mitochondrial outer membrane contains a protein, called VDAC, that forms large aqueous pores. In Neurospora crassa outer membranes, VDAC forms two-dimensional crystalline arrays whose size and frequency can be greatly augmented by lipase treatment of these membranes (C. Mannella, Science 224, 165, 1984). Fourier filtration and surface reconstruction of freeze-dried/shadowed (45 degrees) arrays produced detailed images of two populations of crystals, whose lattices are mirror images of each other. Most likely, this technique has revealed both surfaces of the same two-dimensional crystal with lattice parameters: a = 12.3 +/- 0.1 nm, b = 11.2 +/- 0.1 nm, and theta = 109 +/- 1 degree. Three-dimensional reconstructions of the surface reliefs on both sides of the crystal show them to be very similar. The majority of the protein forming the channel appears to be at or below the level of the membrane. To address the issue of the number of 30-kDa polypeptides that form a VDAC channel, measurements of mass per unit area were carried out by analyzing scanning transmission electron micrographs of unstained, freeze-dried arrays. The crystal form used for mass analysis contained the same motif of six stain-accumulating centers per unit cell, with p2 symmetry as in the oblique configuration, but it had a different orientation relative to the lattice lines. These data yielded a surface density of 1.9 +/- 0.2 kDa/nm2, indicating that there is a one-to-one ratio between VDAC polypeptides and the channels visualized in filtered electron micrographs, and that VDAC membrane crystals contain 68% protein and 32% lipid by mass.

Crystallization↗
PubMed1991

Voltage gating of the mitochondrial outer membrane channel VDAC is regulated by a very conserved protein.

Soluble protein preparations obtained from the mitochondrial fractions of three very different organisms, Neurospora crassa, rat, and potato, were discovered to greatly enhance the voltage sensitivity of the mitochondrial outer membrane channel, VDAC. The active ingredient, referred to as the VDAC modulator, increased the rate of voltage-dependent channel closure by approximately 10-fold. The modulator from one species increased the closing rate of VDAC channels from all three species. The activity is pronase sensitive and not mimicked by another negatively charged protein, BSA. The highly conserved property of this modulator suggests an important physiological role in regulating mitochondrial function.

Animals↗
PubMed1991

Gemfibrozil in CAPD patients.

Gemfibrozil was given at a daily oral dose of 600 mg to 28 CAPD patients with serum triglyceride levels higher than 200 mg/dl, after 2 months of low fat low calorie diet. Eleven patients dropped out from the study due to spontaneous withdrawal of the drug or to intercurrent hospitalization. The 17 patients who completed the study showed a significant decrease in serum total (434.6 +/- 207.9 to 237.5 +/- 81.6 mg/dl, p 0.003), VLDL (268.8 +/- 17.8 to 109.2 +/- 66.9 mg/dl, p less than 0.005) and LDL (152.9 +/- 34.6 to 119.2 +/- 30.7 mg/dl, p = 0.004) triglyceride. Serum VLDL cholesterol increased from 108.7 +/- 54.1 to 59.9 +/- 29.0 mg/dl, p = 0.003. Serum HDL cholesterol decreased from 25.4 +/- 4.8 to 35.3 +/- 6.3 mg/dl (p less than 0.001). Apo A1 increased from 117.8 +/- 16.7 to 130.8 +/- 16.7 mg/dl (p less than 0.001) and Apo B increased from 173 +/- 26.7 to 137.8 +/- 21.9 mg/dl (p less than 0.001). No clinical or laboratory side effect was observed in the patients who completed the study. Therefore dropouts were due to the poor patients' compliance rather than to the severity of side effects.

Aged↗