Alan Finkelstein

Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria

SummaryWe have incorporated into planar lipid bilayer membranes a voltage-dependent, anion-selective channel (VDAC) obtained fromParamecium aurelia. VDAC-containing membranes have the following properties: (1) The steady-state conductance of a many-channel membrane is maximal when the transmembrane potential is zero and decreases as a steep function of both positive and negative voltage. (2) The fraction of time that an individual channel stays open is strongly voltage dependent in a manner that parallels the voltage dependence of a many-channel membrane. (3) The conductance of the open channel is about 500 pmho in 0.1 to 1.0m salt solutions and is ohmic. (4) The channel is about 7 times more permeable to Cl− than to K+ and is impermeable to Ca++. The procedure for obtaining VDAC and the properties of the channel are highly reproducible.VDAC activity was found, upon fractionation of the paramecium membranes, to come from the mitochondria. We note that the published data on mitochondrial Cl− permeability suggest that there may indeed be a voltage-dependent Cl− permeability in mitochondria.The method of incorporating VDAC into planar lipid bilayers may be generally useful for reconstituting biological transport systems in these membranes.

The gramicidin a channel: A review of its permeability characteristics with special reference to the single-file aspect of transport

SummaryGramicidin A forms univalent cation-selective channels of ≈4 Å diameter in phospholipid bilayer membranes. The transport of ions and water throughout most of the channel length is by a singlefile process; that is, cations and water molecules cannot pass each other within the channel. The implications of this single-file mode of transport for ion movement are considered. In particular, we show that there is no significant electrostatic barrier to ion movement between the energy wells at the two ends of the channel. The rate of ion translocation (e.g., Na+ or Cs+) through the channel between these wells is limited by the necessity for an ion to move six water molecules in single file along with it; this also limits the maximum possible value for channel conductance. At all attainable concentrations of NaCl, the gramicidin A channel never contains more than one sodium ion, whereas even at 0.1M CsCl, some channels contain two cesium ions. There is no necessity to postulate more than two ion-binding sites in the channel or occupancy of the channel by more than two ions at any time.

Single-length and double-length channels formed by nystatin in lipid bilayer membranes

SummaryNystatin forms two types of channels in sterol-containing planar bilayer membranes. One type is formed when it is added to onlyone side of the membrane: the other is formed when it is added toboth sides of the membrane. The relative permeability of these channels to nonelectrolytes (urea and glycerol) is identical. The sensitivity of membranes to the one-sided action of nystatin is critically dependent on their thickness; in particular, membranes made from monoglycerides with more than 18 carbon atoms in their acyl chain are insensitive to nystatins one-sided action. These data are consistent with a model in which the two types of channels formed by nystatin have essentially identical structures, except that the channel formed by its two-sided action is twice the length of that formed by its one-sided action, because it is a tail-to-tail dimer of the latter.

The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers

The heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayer membranes. Channel activity is confined to the N‐terminal half of this chain; the C‐terminal half is inactive. Channel activity is stimulated by low pH (4.5–5.5) on the cis side (the side to which protein is added), neutral pH on the opposite (trans) side, and cis positive voltages. These findings are strikingly similar to those previously reported for analogous fragments of diphtheria and tetanus toxins.

Weak-acid uncouplers of oxidative phosphorylation. Mechanism of action on thin lipid membranes

Abstract Weak-acid uncouplers of oxidative phosphorylation such as p -trifluoromethoxycarbonylcyanidephenylhydrazone, tetrachloro-2-trifluoromethylbenzimidazole, m -chlorocarbonylcyanidephenylhydrazone, and 2,4-dinitrophenol can increase the conductance of thin lipid membranes by several orders of magnitude. In this high conductance state these membranes appear to be ideally selectively permeable to H + or OH − . We suggest, however, that the primary charge carrier in the membrane is neither H + or OH − , but rather a dimer formed between the undissociated and dissociated form of the weak acid, and we show that all of the data on the action of these weak acids on thin lipid membranes are consistent with this picture.

Ion and nonelectrolyte permeability properties of channels formed in planar lipid bilayer membranes by the cytolytic toxin from the sea anemone,Stoichactis helianthus

SummaryWhen present at nanomolar concentrations on one side of a lipid bilayer membrane,helianthus toxin (a protein of mol wt≈16,000) increases enormously membrane permeability to ions and nonelectrolytes by forming channels in the membrane. Membranes containing sphingomyelin are especially sensitive to toxin, but sphingomyelin isnot required for toxin action. Conductance is proportional to about the 4th power of toxin concentration. Single channel conductances are approximately 2×10−10 mho in 0.1m KCl. Toxin-treated membranes are more permeable to K+ and Na+ than to Cl− and SO4=, but the degree of selectivity is pH dependent. Above pH 7 membranes are almost ideally selective for K+ with respect to SO4=, whereas below pH 4 they are poorly selective. The channels show classical molecular sieving for urea, glycerol, glucose, and sucrose — implying a channel radius >5 Å. In symmetrical salt solutions above pH 7, theI–V characteristic of the channel shows significant rectification: below pH 5 there is very little rectfication. Because of the effects of pH on ion selectivity and channel conductance, and also because of the rectification in symmetrical salt solutions and the effect of pH on this, we conclude that there are titratable negative charge groups in the channel modulating ion permeability and selectivity. Since pH changes on the side containing the toxin are effective whereas pH changes on the opposite side are not, we place these negative charges near the mouth of the channel facing the solution to which toxin was added.

Transmembrane insertion of the colicin Ia hydrophobic hairpin.

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wildtype colicin Ia.

Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent ion selectivity

SummaryThe E1 subgroup (E1, A, Ib, etc.) of antibacterial toxins called colicins are known to form voltage-dependent channels in planar lipid bilayers. The genes for colicins E1, A and Ib have been cloned and sequenced, making these channels interesting models for the widespread phenomenon of voltage dependence in cellular channels. In this paper we investigate ion selectivity and channel size—properties relevant to model building. Our major finding is that the colicin E1 channel is large, having a diameter ofat least 8 Å at its narrowest point. We established this from measurements of reversal potentials for gradients formed by salts of large cations or large anions. In so doing, we exploited the fact that the colicin channel is permeable to both cations and anions, and its relative selectivity to them is a functions and anions, and its relative selectivity to them is a function of pH. The channel is anion selective (Cl− over K+) in neutral membranes, and the degree of selectivity is highly dependent on pH. In negatively charged membranes, it becomes cation selective at pHs higher than about 5. Experiments with pH gradients cross the membrane suggest that titratable groups both within the channel lumen and near the channel ends affect the selectivity. Individual E1 channels have more than one open conductance state, all displaying comparable ion selectivity. Colicins A and Ib also exhibit pH-dependent ion selectivity, and appear to have even larger lumens than E1.

Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DTs N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191–386) and its C-terminal R domain (amino acids 387–535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (Il–2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes α-helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.

The channel formed in planar lipid bilayers by the protective antigen component of anthrax toxin

Anthrax toxin consists of three proteins: edema factor (EF, 89 kDa), lethal factor (LF, 90 kDa), and protective antigen (PA, 83 kDa). The former two gain access to the cytosol, where they exert their respective toxic effects on a cell, only in binary combination with PA. The proposed pathways of EF and LF transport consists of (i) PA attaching to a membrane receptor; (ii) its proteolytic cleavage into two fragments, of which the larger, 63 kDa piece (PA63) remains attached to the receptor; (iii) either EF or LF binding to PA63; (iv) the complex undergoing endocytosis, and EF or LF being translocated into the cytosol from an acidic vesicle compartment. In planar phospholipid bilayers, PA63 (but not whole PA) forms cation-selection channels; the channel-forming activity of PA63 dramatically increases when the pH of the solution to which it was added is lowered. Tetraalkylammonium ions block the PA63 channel by binding to a site within the channel lumen. Analysis of this blocking phenomenon reveals that these ions can pass through the channel from one side of the membrane to the other and that the diameter of the channel is about 12 A. The N-terminal 30 kDa end of EF, which contains the region of EF that binds to PA63, interacts with the PA63 channel in a voltage-dependent manner. The nature of the voltage-gating suggests that this binding fragment of EF can enter and block the channel and even pass through it, but further evidence will be required to establish this.