Structure of the membrane channel porin from Rhodopseudomonas blastica at 2.0 A resolution.

The crystal structure of a membrane channel, homotrimeric porin from Rhodopseudomonas blastica has been determined at 2.0 A resolution by multiple isomorphous replacement and structural refinement. The current model has an R-factor of 16.5% and consists of 289 amino acids, 238 water molecules, and 3 detergent molecules per subunit. The partial protein sequence and subsequently the complete DNA sequence were determined. The general architecture is similar to those of the structurally known porins. As a particular feature there are 3 adjacent binding sites for n-alkyl chains at the molecular 3-fold axis. The side chain arrangement in the channel indicates a transverse electric field across each of the 3 pore eyelets, which may explain the discrimination against nonpolar solutes. Moreover, there are 2 significantly ordered girdles of aromatic residues at the nonpolar/polar borderlines of the interface between protein and membrane. Possibly, these residues shield the polypeptide conformation against adverse membrane fluctuations.     

Voltage-dependent gating properties of the channel formed byE. coli hemolysin in planar lipid membranes

Escherichia coli hemolysin forms cation selective, ion-permeable channels of large conductance in planar phospholipid bilayer membranes. The pore formation mechanism is voltage dependent resembling that of some colicins and of diphtheria toxin: pores open when negative voltages are applied and close with positive potentials. The pH dependence of this gating process suggests that it is mediated by a negative fixed charge present in the lumen of the pore. A simple physical model of how the channel opens and closes in response to the applied voltage is given.     

Probing the volume changes during voltage gating of Porin 31BM channel with nonelectrolyte polymers

To probe the volume changes of the voltage-dependent anion-selective channel (VDAC), the nonelectrolyte exclusion technique was taken because it is one of the few existing methods that may define quite accurately the rough geometry of lumen of ion channels (in membranes) for which there is no structural data.Here, we corroborate the data from our previous study [FEBS Lett. 416 (1997) 187] that the gross structural features of VDAC in its highest conductance state are asymmetric with respect to the plane of the membrane, and state that this asymmetry is not dependent on sign of voltage applied. Hence, the plasticity of VDAC does not play a role in the determination of lumen geometry at this state and the asymmetry is an internal property of the channel.We also show that the apparent diameter of the cis segment of the pore decreases slightly from 2 to 1.8 nm when the channel's conductance decreases from its high to low state. However, the trans funnel segment undergoes a more marked change in polymer accessible volume. Specifically, its larger diameter decreases from ∼4 to 2.4 nm. Supposing the channel's total length is 4.6 nm, the apparent change in channel volume during this transition is estimated to be about 10 nm³, i.e. about 40% of the channel's volume in the high conductance state.     

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

Summary 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.     

Conduction and blocking properties of a predominantly anion-selective channel from human platelet surface membrane reconstituted into planar phospholipid bilayers

We have investigated the basic properties of a predominantly anion-selective channel derived from highly purified human platelet surface membrane. Single channels have been reconstituted into planar phospholipid bilayers by fusion of membrane vesicles and recorded under voltage-clamp conditions. The channel is found to have the following properties: (i) Channel activity occurs in bursts of openings separated by long closed periods. (ii) The current-voltage relationship is nonlinear. Channel current is seen to rectify, with less current flowing at positive than at negative voltages. Rectification may be due to asymmetric block by HEPES/Tris buffers. In 450 mM KCl, 5 mM HEPES/Tris, pH 7.2, the single channel conductance at -40 mV is approximately 160 pS and at +40 mV is approximately 90 pS. (iii) The conductance-concentration relationship follows a simple saturation curve. Half maximal conductance is achieved at a concentration of approximately 1000 mM KCl, and the curve saturates at a conductance of approximately 500 pS. (iv) Reversal potentials interpreted in terms of the Goldman-Hodgkin-Katz equation indicate a Cl: K permeability ratio of 4:1. (v) The channel accepts all of the halides as well as a number of other anions. The following sequence of relative anion permeabilities (in the presence of K+) is obtained: F- less than acetate- less than gluconate- less than Cl- less than Br- less than I- less than NO3- less tha SCN-.(vi) Cations as large as TEA+ are permeant. (vii) Current through the channel is blocked in the presence of DIDS, SITS and ATP, but not by Zn2+.     

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

Summary 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.     

Intermolecular functional coupling between phosphoinositides and the potassium channel KcsA

Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total membrane lipids but modulate the functions of various membrane proteins, including ion channels, which play important roles in cell signaling. KcsA, a prototypical K⁺ channel that is small, simple, and easy to handle, has been broadly examined regarding its crystallography, in silico molecular analysis, and electrophysiology. It has been reported that KcsA activity is regulated by membrane phospholipids, such as phosphatidylglycerol. However, there has been no quantitative analysis of the correlation between direct lipid binding and the functional modification of KcsA, and it is unknown whether PIPns modulate KcsA function. Here, using contact bubble bilayer recording, we observed that the open probability of KcsA increased significantly (from about 10% to 90%) when the membrane inner leaflet contained only a small percentage of PIPns. In addition, we found an increase in the electrophysiological activity of KcsA correlated with a larger number of negative charges on PIPns. We further analyzed the affinity of the direct interaction between PIPns and KcsA using microscale thermophoresis and observed a strong correlation between direct lipid binding and the functional modification of KcsA. In conclusion, our approach was able to reconstruct the direct modification of KcsA by PIPns, and we propose that it can also be applied to elucidate the mechanism of modification of other ion channels by PIPns.     

An anion channel of sarcoplasmic reticulum incorporated into planar lipid bilayers: Single-channel behavior and conductance properties

Summary An anion channel of sarcoplasmic reticulum vesicle has been incorporated into planar lipid bilayers by means of a fusion method and its basic properties were investigated. Analysis of fusion processes suggested that one SR vesicle contained approximately one anion channel. The conductance of this channel has several substates and shows a flickering behavior. The occupation probability of each substate was voltage dependent, which induced an inward rectification of macroscopic currents. Further, the anion channel was found to have the following properties. (1) The single-channel conductance is about 200 pS at 100mm Cl^–. (2) The channel does not select among monovalent anions but SO ₄ ^2– hardly permeates through the channel. (3) SO ₄ ^2– added to thecis side (the side to which SR vesicles were added) inhibits Cl^– current competitively in a voltage-dependent manner. (4) An analysis of this voltage dependence suggests that the binding site of SO ₄ ^2– is located at about 36% of the way across the channel from thecis entrance.     

Electrochemical study of ion channel behavior in incorporated poly L-glutamate bilayer lipid membranes

The lipid layer membranes were fabricated on the glassy carbon electrode (GC) and demonstrated to be bilayer lipid membranes by impedance spectroscopy. The formation of incorporated poly L-glutamate bilayer lipid membrane was achieved. The ion channel behavior of the incorporated poly L-glutamate membrane was determined. When the stimulus calcium cations were added into the electrolyte, the ion channel was opened immediately and exhibited distinct channel current. Otherwise, the ion channel was closed. The cyclic voltammogram at the GC electrode coated with incorporated poly L-glutamate DMPC film response to calcium ion is very fast compared with that at the GC electrode coated only with DMPC film. Ion channel current is not dependent on the time but on the concentration of calcium. The mechanism of the ion channel formation was investigated.     

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 -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.     

Mechanism of sugar transport through the sugar-specific LamB channel ofEscherichia coli outer membrane

Summary Lipid bilayer experiments were performed with the sugar-specific LamB (maltoporin) channel ofEscherichia coli outer membrane. Single-channel analysis of the conductance steps caused by LamB showed that there was a linear relationship between the salt concentration in the aqueous phase and the channel conductance, indicating only small or no binding between the ions and the channel interior. The total or the partial blockage of the ion movement through the LamB channel was not dependent on the ion concentration in the aqueous phase. Both results allowed the investigation of the sugar binding in more detail, and the stability constants of the binding of a large variety of sugars to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the sugars. The channel was highly cation selective, both in the presence and absence of sugars, which may be explained by the existence of carbonyl groups inside the channel. These carbonyl groups may also be involved in the sugar binding via hydrogen bonds. The kinetics of the sugar transport through the LamB channel were estimated relative to maltose by assuming a simple one-site, two-barrier model from the relative rates of permeation taken from M. Luckey and H. Nikaido (Proc. Natl. Acad. Sci. USA 77:165–171 (1980a)) and the stability constants for the sugar binding given in this study.     

Pore formation by complement in the outer membrane of Gram-negative bacteria studied with asymmetric planar lipopolysaccharide/phospholipid bilayers

Summary The interaction of complement with an asymmetric planar lipopolysaccharide/phospholipid bilayer system as a model for the lipid matrix of the outer membrane of Gram-negative bacteria has been studied. The addition of whole human serum to the aqueous solution at the lipopolysaccharide side of the asymmetric membrane resulted in a rapid increase of the bilayer conductance in discrete steps, indicating the formation of transmembrane pores, which were not observed in the case of pure phospholipid membranes. The amplitudes of the discrete conductance steps varied over a range of more than one order of magnitude. The mean single step conductance was (0.39±0.24) nS for a subphase containing (inmm): 100 KCl, 5 MgCl₂ and 5 HEPES buffer. The steps were grouped into bursts of typically 9±3 events per burst and the conductance change within one burst was (8.25±4.00) nS.The pore-forming activity of serum at the asymmetric membrane system was independent of the presence of specific antibodies against the lipopolysaccharide but was dependent on calcium ions. Furthermore, the pore-forming activity required complement component C9.A model for the mode of pore formation by complement is proposed: The complement pore is generated in discrete steps by insertion of C9 monomers into the membrane and their irreversible aggregation to water-filled channels with a diameter of approximately 7 nm assuming a circular geometry.     

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 channelin vitro raises the possibility that VDAC may perform a regulatory rolein 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.     

An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial␣membrane

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high‐resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure‐guided mutations demonstrate the importance of both types of protein–lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.     

Using mutagenesis to study potassium channel mechanisms

The voltage-activated K⁺ channels are members of an ion channel family that includes the voltage-activated Na⁺ and Ca²⁺ channels. These ion channels mediate the transmembrane ionic currents that are responsible for the electrical signals produced by cells. The recent cloning of numerous voltage-activated K⁺ channels has made it possible to combine molecular-genetic and biophysical methods to study K⁺ channel mechanisms. These mutagenesis-function studies are beginning to provide new information about the architecture of K⁺ channel proteins and how they form a voltage-gated, K⁺-selective pore.     

Statistical properties of ion channel records. Part II: estimation from the macroscopic current

Macroscopic ion channel current is the summation of the stochastic records of individual channel currents and therefore relates to their statistical properties. As a consequence of this relationship, it may be possible to derive certain statistical properties of single channel records or even generate some estimates of the records themselves from the macroscopic current when the direct measurement of single channel currents is not applicable. We present a procedure for generating the single channel records of an ion channel from its macroscopic current when the stochastic process of channel gating has the following two properties: (I) the open duration is independent of the time of opening event and has a single exponential probability density function (pdf), (II) all the channels have the same probability to open at time t. The application of this procedure is considered for cases where direct measurement of single channel records is difficult or impossible. First, the probability density function (pdf) of opening events, a statistical property of single channel records, is derived from the normalized macroscopic current and mean channel open duration. Second, it is shown that under the conditions (I) and (II), a non-stationary Markov model can represent the stochastic process of channel gating. Third, the non-stationary Markov model is calibrated using the results of the first step. The non-stationary formulation increases the model ability to generate a variety of different single channel records compared to common stationary Markov models. The model is then used to generate single channel records and to obtain other statistical properties of the records. Experimental single channel records of inactivating BK potassium channels are used to evaluate how accurately this procedure reconstructs measured single channel sweeps.     

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 ( 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 genein 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.     

The number of subunits comprising the channel formed by the T domain of diphtheria toxin.

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as glucosamine(+) and NAD(-), it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an NH(2)-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric.     

Regulation of K+ flow by a ring of negative charges in the outer pore of BKCa channels. Part II: Neutralization of aspartate 292 reduces long channel openings and gating current slow component

Neutralization of the aspartate near the selectivity filter in the GYGD pore sequence (D292N) of the voltage- and Ca(2+)-activated K+ channel (MaxiK, BKCa) does not prevent conduction like the corresponding mutation in Shaker channel, but profoundly affects major biophysical properties of the channel (Haug, T., D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese. 2004. J. Gen. Physiol. 124:173-184). Upon depolarizations, the D292N mutant elicited mostly gating current, followed by small or no ionic current, at voltages where the wild-type hSlo channel displayed robust ionic current. In fact, while the voltage dependence of the gating current was not significantly affected by the mutation, the overall activation curve was shifted by approximately 20 mV toward more depolarized potentials. Several lines of evidence suggest that the mutation prevents population of certain open states that in the wild type lead to high open probability. The activation curves of WT and D292N can both be fitted to the sum of two Boltzmann distributions with identical slope factors and half activation potentials, just by changing their relative amplitudes. The steeper and more negative component of the activation curve was drastically reduced by the D292N mutation (from 0.65 to 0.30), suggesting that the population of open states that occurs early in the activation pathway is reduced. Furthermore, the slow component of the gating current, which has been suggested to reflect transitions from closed to open states, was greatly reduced in D292N channels. The D292N mutation also affected the limiting open probability: at 0 mV, the limiting open probability dropped from approximately 0.5 for the wild-type channel to 0.06 in D292N (in 1 mM [Ca2+]i). In addition to these effects on gating charge and open probability, as already described in Part I, the D292N mutation introduces a approximately 40% reduction of outward single channel conductance, as well as a strong outward rectification.