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M Colombini

Publications and source records attributed to M Colombini.

At least 73 records · Page 4Linked to original sources
PubMed1990

Group IIIA-metal hydroxides indirectly neutralize the voltage sensor of the voltage-dependent mitochondrial channel, VDAC, by interacting with a dynamic binding site.

The voltage-dependent, anion-selective mitochondrial channel, VDAC, undergoes two different conformational changes from the open to a closed state under positive and negative applied electric fields. Micromolar quantities of aluminum hydroxide and other metal trihydroxides have recently been shown to be able to inhibit this voltage-dependent closure (Dill et al. (1987) J. Membr. Biol. 99, 187-196; Zhang and Colombini (1989) Biochim. Biophys. Acta 991, 68-78). It was suggested that the inhibition results from the neutralization of the positively charged voltage sensors by the metal species. In the present study, the dynamics of the metal-binding site accompanying channel closure was investigated by adding In(OH)3 to only one side of the membrane and examining its effect on the channel's gating processes. Indium added to open channels inhibited channel closure only when the metal-containing side was on the lower potential side of the applied field. If indium was added only to the higher-potential side, the channels closed and tended to remain closed after the field was abolished. The addition of metal hydroxide after closing the channels with a negative potential on the metal side did not result in channel re-opening as would be expected for sensor neutralization. Inhibition occurred immediately, however, if the channels were first allowed to open briefly. The closed-state selectivity seemed to be very similar in the absence or presence of the metal, indicating that the metal-binding sites are not located within the pore of the channel in the closed conformation. The results are consistent with a voltage-dependent translocation across the membrane of each of two metal-binding sites on VDAC. This translocation is tightly coupled with channel opening and closing.

Anions↗
PubMed1990

Selectivity changes in site-directed mutants of the VDAC ion channel: structural implications.

The gene encoding the yeast mitochondrial outer membrane channel VDAC was subjected to site-directed mutagenesis to change amino acids at 29 positions to residues differing in charge from the wild-type sequence. The mutant genes were then expressed in yeast, and the physiological consequences of single and multiple amino acid changes were assessed after isolation and insertion of mutant channels into phospholipid bilayers. Selectivity changes were observed at 14 sites distributed throughout the length of the molecule. These sites are likely to define the position of the protein walls lining the aqueous pore and hence, the transmembrane segments. These results have been used to develop a model of the open state of the channel in which each polypeptide contributes 12 beta strands and one alpha helix to form the aqueous transmembrane pathway.

Amino Acid Sequence↗
PubMed1989

Inhibition by aluminum hydroxide of the voltage-dependent closure of the mitochondrial channel, VDAC.

Micromolar quantities of aluminum have been found (Dill et al. (1987) J. Membrane Biol. 99, 187-196) to reduce the voltage dependence of the mitochondrial outer membrane channel, VDAC, from Neurospora crassa. In the present study, various metallic and organic ions were tested for possible aluminum-like effect, and only the trivalent metals exhibited a similar ability to reduce the channels voltage dependence. However, trivalency alone was not sufficient because lanthanum (III) had no effect. Quantitative analyses with three group IIIA metals (A1, Ga, and In) showed that, of the structural characteristics examined, the ability to form sufficient M(OH)3 at experimental pH was the primary property shared by all the effective metals. While providing new insight into the nature of VDAC's sensor, these results also indicate that aluminum-cell interaction may result from the presence of AI(OH)3 in solution in addition to the widely accepted AI3+-mediated interactions. While the [AI3+] is vanishingly low at neutral pH, the trihydroxide is the major form and should be considered as an important candidate for aluminum-induced cellular effects.

Aluminum Hydroxide↗
PubMed1989

Probing the structure of the mitochondrial channel, VDAC, by site-directed mutagenesis: a progress report.

The voltage-dependent anion-selective channel (VDAC) of the mitochondrial outer membrane is formed by a small (approximately 30 kDa) polypeptide, but shares with more complex channels the properties of voltage-dependent gating and ion selectivity. Thus, it is a useful model for studying these properties. The molecular biology techniques available in yeast allow us to construct mutant versions of the cloned yeast VDAC gene in vitro, using oligonucleotide-directed mutagenesis, and to express the mutant genes in yeast cells in the absence of wild-type VDAC. We find that one substitution mutation (lys 61 to glu) alters the selectivity of VDAC.

Amino Acid Sequence↗
PubMed1988

The mitochondrial outer membrane channel, VDAC, is modulated by a soluble protein.

The mitochondrial outer membrane channel, VDAC, serves as the primary permeability pathway for metabolite flux between cytoplasmic and mitochondrial compartments. VDAC can occupy several conformational states differing in ion conductivity. Small transmembrane potentials cause transitions from open- to closed-channel conformations. A soluble mitochondrial protein enhances the channel's response to voltage by increasing the rate of channel closing; inducing the occupation of lower conductance states; and decreasing the rate of channel reopening. This protein modulator acts at very low concentrations and its role in the cell may be to regulate the permeability of the mitochondrial outer membrane by inducing channel closure.

Electric Conductivity↗
PubMed1987

The mitochondrial outer membrane channel, VDAC, is regulated by a synthetic polyanion.

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.

Fungal Proteins↗
PubMed1987

Regulation of the mitochondrial outer membrane channel, VDAC.

The channel-forming protein, VDAC, located in the mitochondrial outer membrane, is probably responsible for the high permeability of the outer membrane to small molecules. The ability to regulate this channel in vitro raises the possibility that VDAC may perform a regulatory role in vivo. VDAC exists in multiple, quasi-degenerate conformations with different permeability properties. Therefore a modest input of energy can change VDAC's conformation. The ability to use a membrane potential to convert VDAC from a high (open) to a low (closed) conducting form indicates the presence of a sensor in the protein that allows it to respond to the electric field. Titration and modification experiments point to a polyvalent, positively charged sensor. Soluble, polyvalent anions such as dextran sulfate and Konig's polyanion seem to be able to interact with the sensor to induce channel closure. Thus there are multiple ways of applying a force on the sensor so as to induce a conformational change in VDAC. Perhaps cells use one or more of these methods.

Electric Conductivity↗
PubMed1987

Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast.

The outer mitochondrial membranes of all organisms so far examined contain a protein which forms voltage-dependent anion selective channels (VDAC) when incorporated into planar phospholipid membranes. Previous reports have suggested that the yeast (Saccharomyces cerevisiae) outer mitochondrial membrane component responsible for channel formation is a protein of 29,000 daltons which is also the major component of this membrane. In this report, we describe the purification of this 29,000-dalton protein to virtual homogeneity from yeast outer mitochondrial membranes. The purified protein readily incorporates into planar phospholipid membranes to produce ionic channels. Electrophysiological characterization of these channels has demonstrated they have a size, selectivity and voltage dependence similar to VDAC from other organisms. Biochemically, the purified protein has been characterized by determining its amino acid composition and isoelectric point (pI). In addition, we have shown that the purified protein, when reconstituted into liposomes, can bind hexokinase in a glucose-6-phosphate dependent manner, as has been shown for VDAC purified from other sources. Since physiological characterization suggests that the functional parameters of this protein have been conserved, antibodies specific to yeast VDAC have been used to assess antigenic conservation among mitochondrial proteins from a wide number of species. These experiments have shown that yeast VDAC antibodies will recognize single mitochondrial proteins from Drosophila, Dictyostelium and Neurospora of the appropriate molecular weight to be VDAC from these organisms. No reaction was seen to any mitochondrial protein from rat liver, rainbow trout, Paramecium, or mung bean. In addition, yeast VDAC antibodies will recognize a 50-kDa mol wt protein present in tobacco chloroplasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1987

Elimination and restoration of voltage dependence in the mitochondrial channel, VDAC, by graded modification with succinic anhydride.

The major permeability pathways of the outer mitochondrial membrane are the voltage-gated channels called VDAC. It is known that the conductance of these channels decreases as the transmembrane voltage is increased in the positive or negative direction. These channels are known to display a preference for anions over cations of similar size and valence. It was proposed (Doring & Colombini, 1985b) that a set of positive charges lining the channel may be responsible for both voltage dependence and selectivity. A prediction of this proposal is that progressive replacement of the positive charges with negative charges should at first diminish, and then restore, voltage dependence. At the same time, the channel's preference for anions over cations should diminish then reverse. Succinic anhydride was used to perform these experiments as it replaces positively charged amino groups with negatively charged carboxyl groups. When channels, which had been inserted into phospholipid membranes, were treated with moderate amounts of the anhydride, they lost their voltage dependence and preference for anions. With further succinylation, voltage dependence was regenerated while the channels became cation selective. The voltage needed to close one-half of the channels increased in those treatments in which voltage dependence was diminished. As voltage dependence was restored, the voltage needed to close half of the channels decreased. The energy difference between the open and closed state in the absence of an applied field changed little with succinylation, indicating that the procedure did not cause large changes in VDAC's structure but specifically altered those charges responsible for voltage gating and selectivity.

Intracellular Membranes↗
PubMed1987

Voltage gating in VDAC is markedly inhibited by micromolar quantities of aluminum.

The mitochondrial outer membrane contains voltage-gated channels called VDAC that are responsible for the flux of metabolic substrates and metal ions across this membrane. The addition of micromolar quantities of aluminum chloride to phospholipid membranes containing VDAC channels greatly inhibits the voltage dependence of the channels' permeability. The channels remain in their high conducting (open) state even at high membrane potentials. An analysis of the change in the voltage-dependence parameters revealed that the steepness of the voltage dependence decreased while the voltage needed to close half the channels increased. The energy difference between the open and closed states in the absence of an applied potential did not change. Therefore, the results are consistent with aluminum neutralizing the voltage sensor of the channel. pH shift experiments showed that positively charged aluminum species in solution were not involved. The active form was identified as being either (or both) the aluminum hydroxide or the tetrahydroxoaluminate form. Both of these could reasonably be expected to neutralize a positively charged voltage sensor. Aluminum had no detectable effect on either single-channel conductance or selectivity, indicating that the sensor is probably not located in the channel proper and is distinct from the selectivity filter.

Aluminum↗
PubMed1987

Ultrasteep voltage dependence in a membrane channel.

A mechanism for regulating voltage-gated channels is presented. The treatment amplifies the effect of the applied membrane potential resulting in a dramatic increase in the channel's voltage dependence. Addition of a large polyvalent anion to the medium bathing a phospholipid bilayer containing the voltage-dependent channel from the mitochondrial outer membrane, VDAC, induced up to a 12-fold increase in the channel's voltage sensitivity. The highest polyvalent anion concentration tested resulted in an e-fold conductance change for a 0.36-mV change in membrane potential. On the low end, a concentration of 2 microM resulted in a 50% increase in VDAC voltage dependence. A mechanism based on polyvalent anion accumulation in the access resistance region at the mouth of the pore is consistent with all findings. Perhaps the voltage dependence of voltage-gated channels is amplified in vivo by polyvalent ions. If so, the control of excitable phenomena may be under much finer regulation than that provided by membrane potential alone.

Anions↗
PubMed1986

Hexokinase receptor complex in hepatoma mitochondria: evidence from N,N'-dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC.

In rapidly growing, highly glycolytic hepatoma cells as much as 65% of the total cell hexokinase is bound to the outer mitochondrial membrane [Parry, D.M., & Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912]. In this paper, we describe the purification to apparent homogeneity of a mitochondrial pore-forming protein from the highly glycolytic AS-30D rat hepatoma cell line. The purified protein shows a single 35 000-dalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an amino acid composition slightly more hydrophobic than that of the rat liver pore protein (also known as VDAC or mitochondrial porin), and a channel-forming activity of 136 channels min-1 (microgram of protein)-1. In addition to displaying the properties characteristic of VDAC (single-channel conductance, voltage dependence, and preference for anions), we observe that the AS-30D VDAC protein is one of only three mitochondrial proteins that bind [14C]dicyclohexylcarbodiimide (DCCD) at relatively low dosages (2 nmol of DCCD/mg of mitochondrial protein). Significantly, treatment of intact mitochondria isolated from either rat liver or the AS-30D hepatoma with DCCD results in an almost complete inhibition of their ability to binding hexokinase. Fifty percent inhibition of binding occurs at less than 2 nmol of DCCD/mg of mitochondrial protein. In contrast to DCCD, water-soluble carbodiimides are without effect on hexokinase binding. These results suggest that the pore-forming protein of tumor mitochondria forms at least part of the hexokinase receptor complex. In addition, they indicate that a carboxyl residue located within a hydrophobic region of the receptor complex may play a critical role in hexokinase binding.

Amino Acids↗
PubMed1985

Voltage dependence and ion selectivity of the mitochondrial channel, VDAC, are modified by succinic anhydride.

The mitochondrial protein VDAC forms voltage-dependent anion-selective channels in planar phospholipid membranes. When succinic anhydride was added to these membranes, it virtually eliminated VDAC's voltage-dependence and changed its selectivity from anion to cation. These results were obtained without large changes in open-channel conductance or in energy difference between the open and closed states in the absence of a field. Since succinic anhydride converts amino groups into carboxyl groups, we propose that amino groups are responsible for VDAC's voltage-dependence and anion selectivity. Perhaps the same charges are responsible for both functions.

Animals↗
PubMed1985

The mitochondrial voltage-dependent channel, VDAC, is modified asymmetrically by succinic anhydride.

In the accompanying paper, succinic anhydride was shown to react with the outer mitochondrial membrane channel-forming protein, VDAC, resulting in the loss of its voltage dependence. In this paper, the anhydride was added to VDAC held in a particular conformational state by means of an applied electric field. VDAC was inserted into the membranes from the cis side and the anhydride was added either to the cis or trans side. Channels modified in the open state behaved similarly whether anhydride was added to the cis or trans side. Modifications of VDAC in either of the two closed states did not. Modifications resulting in the loss of voltage-dependence occurred primarily when anhydride was added to the negative side of the membrane irrespective of which closed state the VDAC was in indicating that the accessibility of the gating charges alternated between the cis and trans sides as the channel's conformation was changed from one closed state to the other. Despite the pronounced asymmetry, in general the resulting channels behaved in the same way in response to either positive or negative fields. A model consistent with the results is presented which proposes that the same gating charges are responsible for channel closure at both positive and negative fields.

Animals↗
PubMed1985

Evidence for titratable gating charges controlling the voltage dependence of the outer mitochondrial membrane channel, VDAC.

A voltage-dependent anion-selective channel, VDAC, is found in outer mitochondrial membranes. VDAC's conductance is known to decrease as the transmembrane voltage is increased in either the positive or negative direction. Charged groups on the channel may be responsible for this voltage dependence by allowing the channel to respond to an applied electric field. If so, then neutralization of these charges would eliminate the voltage dependence. Channels in planar lipid bilayers which behaved normally at pH 6 lost much of their voltage dependence at high pH. Raising the pH reduced the steepness of the voltage dependence and raised the voltage needed to close half the channels. In contrast, the energy difference between the open and closed state in the absence of a field was changed very little by the elevated pH. The groups being titrated had an apparent pK of 10.6. From the pK and chemical modification, lysine epsilon amino groups are the most likely candidates responsible for VDAC's ability to respond to an applied electric field.

Animals↗