The emerging picture of mitochondrial membrane channels.
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Intracellular Membranes↗
Biomedical subjects
C A Mannella
Publications and source records attributed to C A Mannella.
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↗
Binding of a synthetic targeting peptide to a mitochondrial channel protein.
Membrane crystals of the mitochondrial outer membrane channel VDAC (porin) from Neurospora crassa were incubated with a 20-amino-acid synthetic peptide corresponding to the N-terminal targeting region of subunit IV of cytochrome oxidase. The peptide caused disordering and contraction of the crystal lattice of the membrane arrays. Also, new stain-excluding features were observed on the peptide-treated arrays which most likely correspond to sites at which the peptide accumulates. The stain exclusion zones associated with binding of the targeting peptide (and with binding of apocytochrome c in an earlier study) have been localized on a two-dimensional density map of frozen-hydrated, crystalline VDAC previously obtained by cryo-electron microscopy. The results indicate that both the peptide and cytochrome c bind to protein "arms" which extend laterally between the channel lumens. The finding that imported polypeptides bind to a specific region of the VDAC protein implicates this channel in the process by which precursor proteins are recognized at and translocated across the mitochondrial outer membrane.
Amino Acid Sequence↗
The 'ins' and 'outs' of mitochondrial membrane channels.
The outer membrane of the mitochondrion contains thousands of copies of a pore-forming protein called VDAC or porin. Considerable progress has been made towards elucidating the molecular structure of this channel. Moreover, mounting evidence that the permeability of VDAC may be regulated is challenging the textbook notion of the outer membrane as a simple sieve. Numerous other channel activities have been detected by electrophysiol approaches in both the outer and inner mitochondrial membranes. The inner-membrane channels do not appear to be open under normal physiological conditions and so should not dissipate energy-transducing ion gradients. The biological functions of the different classes of mitochondrial channels are uncertain, but several possibilities (including protein translocation) are being explored.
Bacterial Outer Membrane Proteins↗
Classification of projection images of crystalline arrays of the mitochondrial, voltage-dependent anion-selective channel embedded in aurothioglucose.
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.
Aurothioglucose↗
Structural analysis of mitochondrial pores.
Structural information about the channel in the mitochondrial outer membrane, derived from sequence analysis and electron microscopy of two-dimensional crystals, is summarized. A model for the channel is presented, consisting of a cylindrical beta-barrel that is formed by one or two 30-kDa polypeptides, with an alpha-carbon backbone diameter of 3.8 nm. The radial distributions of basic amino acids and lipid-contact regions on the projected cylinder are mapped relative to interchannel bonding sites inferred from channel packing in the arrays. Speculation on the kinds of conformational changes that the channel might undergo is also presented.
Amino Acid Sequence↗
Interaction between the VDAC channel and a polyanionic effector. An electron microscopic study.
The conductance of the voltage-dependent mitochondrial outer membrane channel is modulated by a synthetic anionic polymer. When added to suspensions of membrane crystals of the channel, the polyanion caused disordering of the usual parallelogram array and increased occurrence of a contracted form of the array. Correlation averages obtained from electron microscopic images of the channel crystals indicated a narrowing of the projected channel lumen in the presence of the polyanion and the appearance of new, narrow zones of stain exclusion on the outside of the channel. These effects are interpreted in terms of possible conformational changes induced in the channel by binding of the polyanion.
Electric Conductivity↗
Permeability of the mitochondrial outer membrane to organic cations.
Inhibition of mitochondrial respiration by hydrophobic fluorescent dyes (Rhodamine 6G, Safranine O, Pyronine B) is much less potentiated by digitonin-lysis of the outer membrane than that by polyamines or adriamycin. This situation may be explained by impermeability of the anion-selective channels in the outer mitochondrial membrane to large cations and by the ability of hydrophobic (but not polar or amphipathic) ions to directly permeate lipid bilayers.
Animals↗
Fusion of the mitochondrial outer membrane: use in forming large, two-dimensional crystals of the voltage-dependent, anion-selective channel protein.
Phospholipase A2 induces crystallization of the channel protein, VDAC (also called mitochondrial porin), in the outer membrane of Neurospora crassa mitochondria. The channel crystals formed in native membranes typically contain a few hundred unit cells. To increase the size of these membrane crystals for low-contrast electron microscopic imaging and diffraction studies, fusion of the isolated mitochondrial outer membranes was attempted before and after phospholipase treatment. Successful fusion of the untreated membranes was achieved by a procedure involving slow dehydration at acid pH. Single crystals of channels obtained by subsequent action of soluble phospholipase A2 on fused mitochondrial outer membranes may contain several thousand unit cells.
Crystallization↗
Structure of the mitochondrial outer membrane channel derived from electron microscopy of 2D crystals.
A structural model for the channel in the mitochondrial outer membrane is presented, derived from electron microscopic studies of two-dimensional crystals and inferences from the primary structure of the 30-kDa polypeptide which forms the channel. The channel is represented as a cylindrical beta-barrel, with a carbon backbone diameter of 3.8 nm. The axial projection of the cylinder is divided radially into four sectors by four interchannel contact points. These sectors are characterized in terms of their interactions with lipid and macromolecular ligands, and in terms of the presence or absence of exposed basic amino acids.
Crystallization↗
Properties of channels in the mitochondrial outer membrane.
Patch-clamping studies with native outer mitochondrial membranes show a complex behavior. In the range of potentials in which the polarity of the pipette is positive, the behavior resembles that of VDAC incorporated into bilayers. Accordingly, there is a decrease in conductance with voltage. An involvement of VDAC is also supported by responses of the patches to the presence of polyanion or treatment with succinic anhydride, both of which affect VDAC. In contrast, in the negative range of potential, the conductance of the patches generally increases with the magnitude of the voltage. The increase in conductance shows a biphasic time course which is consistent with a model in which channels are first activated (first phase) and then assembled into larger high-conductance channels (second phase). A variety of experiments support the notion that an assembly takes place. The time course of the conductance increase is consistent with formation of the second-phase channels from 6 +/- 1 subunits.
Animals↗
Kinetics of voltage-induced conductance increases in the outer mitochondrial membrane.
The kinetics of the increase in conductance in the outer mitochondrial membrane induced by patch-clamping at various negative potentials (pipette inside negative) are reported. The changes are biphasic, a rapid increase is followed by a slowly developing larger change. The results can be predicted by a model in which an initial activation of channels is followed by their assembly into highly conducting channels. The model suggests that five to seven activated subunits form each high-conductance channel.
Animals↗
Enhanced uptake of spermidine and methylglyoxal-bis(guanylhydrazone) by rat liver mitochondria following outer membrane lysis.
Isolated rat liver mitochondria rapidly bound the 14C-labeled organic cations spermidine, a physiologically important polyamine, and methylglyoxal-bis(guanylhydrazone) (MGBG), an anticancer drug. This rapid, Mg2+-sensitive, respiration-independent binding is assumed to involve adsorption to anionic surface groups. A slower progressive uptake of the organic cations exhibited respiration dependence, indicating that it involves transport across the inner mitochondrial membrane into the matrix compartment. Addition of digitonin, to lyse the outer mitochondrial membrane, caused an increase in the mitochondrial content of the organic cations and enhanced the rate of progressive, respiration-dependent cation uptake. The data are consistent with the interpretation that the outer mitochondrial membrane limits access of the organic cations, spermidine and MGBG, to the inner mitochondrial membrane. This conclusion is supported also by published data indicating that outer membrane lysis enhances inhibitory effects of the organic cations on mitochondrial respiration. The uptake of spermidine by mitochondria was inhibited by MGBG.
Animals↗
Lateral segregation of sterol and channel proteins in the mitochondrial outer membrane induced by phospholipase A2: evidence from negative-stain electron microscopy using filipin.
The channel protein in the mitochondrial outer membrane of Neurospora crassa aggregates laterally into crystalline arrays by the action of phospholipase A2. When mitochondrial outer membranes are reacted with filipin and examined by negative-stain electron microscopy, filipin-sterol complexes are found everywhere on the membranes except on the crystalline channel arrays. This suggests that the channel-rich membrane domains may have a relatively low content of accessible sterol. It is proposed that in vitro segregation of protein and lipid membrane components by phospholipase A2 may reflect a mechanism by which the endogenous enzyme organizes the native mitochondrial membrane into functional domains.
Filipin↗
Evidence for a novel voltage-activated channel in the outer mitochondrial membrane.
Patch-clamp studies of the outer mitochondrial membrane indicate a voltage-dependent increase in conductance for potentials positive relative to the exterior of the mitochondrion. The time course of the conductance changes is consistent with an activation of channels. Voltage pulse experiments suggest that the activation phenomenon corresponds to assembly of the channels from subunits with disassembly occurring after recovery of the original conductance. Effects of temperature and concanavalin A on the voltage-induced conductances are also consistent with a channel assembly model.
Animals↗
Importance of the mitochondrial outer membrane channel as a model biological channel.
The channels of the mitochondrial outer membrane represent a useful model for studies into the mechanisms underlying phenomena of voltage-dependent gating and ion selectivity.
Intracellular Membranes↗
Electron microscopy and image analysis of the mitochondrial outer membrane channel, VDAC.
The channel protein in the outer membrane of Neurospora 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.
Intracellular Membranes↗
Molecular genetics of the VDAC ion channel: structural model and sequence analysis.
The voltage-dependent anion-selective channel of the outer mitochondrial membrane provides a unique system in which to study the molecular basis of voltage gating of ion flow. We have cloned and sequenced a cDNA coding for this protein in yeast. From the derived amino acid sequence, we have generated a preliminary model for the secondary structure of the protein which suggests that the protein forms a "beta-barrel" type structure. Comparison of the VDAC amino acid sequence with that of the bacterial porins has indicated that the two classes of molecules appear to be unrelated.
Amino Acid Sequence↗