Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH

Ion-conducting channels formed in lipid bilayers by diphtheria toxin are highly pH dependent. Among other properties, the channel's single channel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH-dependent single channel properties as that of the intact toxin. This region corresponds to an a-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two -helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysine has no effect on the DT channel's single channel conductance or selectivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3 cis/7.2 trans (in 1 m KCl), consistent with this group interacting electrostatically with ions in the channel. The cation selectivity of these mutant channels is also reduced from that of wild-type channels, a direction consistent with residue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 (in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0 cis/7.2 trans (with a high concentration of permeant buffer in the cis compartment) imply that residue 352 is on or near the trans side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. The trans location of position 352 severely constrains the number of possible membrane topologies for this region.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).     

Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.

The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.     

Electrical properties and molecular architecture of the channel formed by Escherichia coli hemolysin in planar lipid membranes

A 107 kDa hemolysin from Escherichia coli is able to open pores in lipid membranes. By studying its interaction with planar phospholipid bilayers we have derived some structural information on the organization of the pore. We measured the current-voltage characteristic and the ion selectivity of the channel both in neutral membranes, made of egg phosphatidylcholine (PC) and in negatively charged membranes, made of a 1:1 mixture of PC with phosphatidylserine (PS). Experiments were performed varying both the pH and the salt concentration of the bathing KCl solution. In neutral membranes the pore is ohmic and its conductance increases almost linearly with the salt concentration. The channel is cation-selective at high pH but nearly unselective at low pH. We interpret these results in terms of a minimal model based on classical electro-diffusional theories assuming that the pore is wide and bears a negative charge at its entrances. In membranes containing the acidic lipid the current-voltage curve is non-linear in such a way to suggest that the trans (but not the cis) entrance of the pore is affected by the surface potential of the membrane. Applying our model we find that the trans and cis entrances are located, respectively, about 0.5 nm and more than 5 nm apart from the plane of the membrane. We confirmed the asymmetric disposition of the channel by enzymatic digestion of preformed pores. This was effective only when the enzyme was applied on the cis side.     

Alteration of the pH-dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis.

Colicin E1 is a soluble, bacteriocidal protein that forms voltage-gated channels in planar lipid bilayers. The channel-forming region of the 522-amino acid protein is near the COOH terminus, and contains a 35-amino acid hydrophobic segment which is presumed to be important in interacting with the membrane. We have used site-directed mutagenesis in the region immediately upstream from the hydrophobic segment to construct several functional colicin mutants in which a wild-type residue was replaced with a cysteine. We also replaced the only naturally occurring cysteine in the molecule, Cys-505, with alanine, so that synthetically introduced cysteines could unambiguously serve as targets for chemical modification. All of the replacements reported here (at positions 449, 459, 473, 505, and some combinations) resulted in a channel that had an ion selectivity (K+ versus Cl-) identical to wild type at low pH. At higher pH, however, one of these mutations, which replaced the negatively charged aspartate at position 473 (the upstream boundary of the hydrophobic segment), resulted in a channel that was less cation-selective than was wild type. When the introduced Cys-473 was reacted with iodoacetic acid, which inserted a COOH group close to the position of the missing aspartate COOH, wild-type ion selectivity was restored, suggesting that the greater cation selectivity of the wild-type channel was directly produced by the negative charge at Asp-473. By comparing the ion selectivity of the Cys-473 mutant channel to that of the wild type as a function of the pH on the cis and trans sides of the membrane, it was possible to locate residue 473 close to the cis side. Locating in this manner the positions in the channel of particular residues places important constraints on channel model building.     

Relationship of membrane sidedness to the effects of the lipophosphoglycan of Leishmania donovani on the fusion of influenza virus.

Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N-terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20-amino-acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane-inserted peptide from alpha-helix to beta-structure. The beta-structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA-expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature-promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.     

Alphavirus 6K proteins form ion channels

Ross River virus and Barmah Forest virus are Australian arboviruses of the Alphavirus genus. Features of alphavirus infection include an increased permeability of cells to monovalent cations followed by virion budding. Virally encoded ion channels are thought to have a role in these processes. In this paper, the 6K proteins of Ross River virus and Barmah Forest virus are shown to form cation-selective ion channels in planar lipid bilayers. Using a novel purification method, bacterially expressed 6K proteins were inserted into bilayers with a defined orientation (i.e. N-terminal cis, C-terminal trans). Channel activity was reversibly inhibited by antibodies to the N and C termini of 6K protein added to the cis and trans baths, respectively. Channel conductances varied from 40-800 picosiemens, suggesting that the protein is able to form channels with a range of possible oligomerization states.     

Blocking of the beta-latrotoxin channels in bimolecular lipid membranes by antibodies

Asymmetric ion channels are formed in a bimolecular lipid membrane by beta-latrotoxin (LT) introduced to one (cis) side of the membrane. LT-specific antibodies added to the opposite (trans) side of the membrane block the current through the LT channels when a negative potential is applied to the cis side, no blockade is observed at positive potentials. LT-specific antibodies do not block the channel current when added to the cis compartment after removal of LT. LT-unspecific immunoglobulins have no influence on LT channel conductance.     

Sidedness of reconstituted calcium channels from muscle transverse tubules as determined by D600 and D890 blockade.

The verapamil-type calcium antagonist, D600, and its charged quaternary derivative, D890, were used to assess the sidedness of blockade in single calcium channels reconstituted from purified transverse tubules of skeletal muscle. Spontaneous single channel openings were induced with the agonist Bay-K8644 and recordings were made in a two-chamber planar bilayer setup so that drugs could be delivered to either side of the channel. Micromolar drug addition resulted in a greater than 10-fold decrease in probability of open channel events (po) without a significant change in single channel currents. Changes in po occurred in parallel with changes in mean open time and both parameters could be titrated with a similar IC50. At pH 7.2, cis or trans D600 blocked with an IC50 of 5 microM but for D890 the IC50 was cis 3 microM and trans greater than 75 microM (cis is the intracellular-equivalent side as defined by the voltage-dependent activation). The asymmetry of D890 blockade indicates that the drug can readily gain access to the blocking site from the aqueous phase adjacent to the inner but not extracellular end of the channel.     

Histidine ligand protonation and redox potential in the rieske dioxygenases: role of a conserved aspartate in anthranilate 1,2-dioxygenase

The Rieske dioxygenase, anthranilate 1,2-dioxygenase, catalyzes the 1,2-dihydroxylation of anthranilate (2-aminobenzoate). As in all characterized Rieske dioxygenases, the catalytic conversion to the diol occurs within the dioxygenase component, AntAB, at a mononuclear iron site which accepts electrons from a proximal Rieske [2Fe-2S] center. In the related naphthalene dioxygenase (NDO), a conserved aspartate residue lies between the mononuclear and Rieske iron centers, and is hydrogen-bonded to a histidine ligand of the Rieske center. Engineered substitutions of this aspartate residue led to complete inactivation, which was proposed to arise from elimination of a productive intersite electron transfer pathway [Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837]. Substitutions of the corresponding aspartate, D218, in AntAB with alanine, asparagine, or glutamate also resulted in enzymes that were completely inactive over a wide pH range despite retention of the hexameric quaternary structure and iron center occupancy. The Rieske center reduction potential of this variant was measured to be approximately 100 mV more negative than that for the wild-type enzyme at neutral pH. The wild-type AntAB became completely inactive at pH 9 and exhibited an altered Rieske center absorption spectrum which resembled that of the D218 variants at neutral pH. These results support a role for this aspartate in maintaining the protonated state and reduction potential of the Rieske center. Both the wild-type and D218A variant AntABs exhibited substrate-dependent rapid phases of Rieske center oxidations in stopped-flow time courses. This observation does not support a role for this aspartate in a facile intersite electron transfer pathway or in productive substrate gating of the Rieske center reduction potential. However, since the single turnovers resulted in anthranilate dihydroxylation by the wild-type enzyme but not by the D218A variant, this aspartate must also play a crucial role in substrate dihydroxylation at or near the mononuclear iron site.     

Ion selectivity of colicin E1: Modulation by pH and membrane composition

Colicin E1 is a plasmid-encoded bacteriocidal protein which, though water soluble when secreted by its host bacterium, spontaneously interacts with planar lipid bilayers to form voltage-gated ion channels. In asolectin bilayers, the preference for anions over cations exhibited by these channels at low pH can be reversed by raising the pH on either side of the membrane. When incorporated into membranes composed of either of the two zwitterionic lipids, bacterial phosphatidylethanolamine and diphytanoyl phosphatidylcholine, colicin E1 channels were nearly ideally anion selective in the limit of low pH and moderately cation selective at the high pH limit. In phosphatidylcholine membranes, however, the response of these channels to changes in pH exhibited a pattern of behavior peculiar to this lipid. If the side of the membrane on which the protein had been introduced (the cis side) was exposed to pH 4.0, all the channels in the bilayer, whether opened or closed, became refractory to further changes in pH. This irreversibility has been interpreted as evidence that the selectivity of colicin E1 is under the control of a pH-sensitive conformational change. Protonation of groups on the cis side of the membrane appear to be essential to the conversion to the anion-selective state. These groups are rendered kinetically inaccessible to the aqueous phase when the transition takes place in phosphatidylcholine membranes.     

Mutations at Arginine 352 Alter the Pore Architecture of CFTR

Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA⁺, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.     

Redox regulation of CLIC1 by cysteine residues associated with the putative channel pore

Chloride intracellular channels (CLICs) are putative pore-forming glutathione-S-transferase homologs that are thought to insert into cell membranes directly from the cytosol. We incorporated soluble, recombinant human CLIC1 into planar lipid bilayers to investigate the associated ion channels, and noted that channel assembly (unlike membrane insertion) required a specific lipid mixture. The channels formed by reduced CLIC1 were similar to those previously recorded from cells and "tip-dip" bilayers, and specific anti-CLIC1 antibodies inhibited them. However, the amplitudes of the filtered single-channel currents were strictly regulated by the redox potential on the "extracellular" (or "luminal") side of the membrane, with minimal currents under strongly oxidizing conditions. We carried out covalent functional modification and site-directed mutagenesis of this controversial ion channel to test the idea that cysteine 24 is a critical redox-sensitive residue located on the extracellular (or luminal) side of membrane CLIC1 subunits, in a cysteine-proline motif close to the putative channel pore. Our findings support a simple structural hypothesis to explain how CLIC1 oligomers form pores in membranes, and suggest that native channels may be regulated by a novel mechanism involving the formation and reduction of intersubunit disulphide bonds.     

pH alterations "reset" Ca2+ sensitivity of brain Na+ channel 2, a degenerin/epithelial Na+ ion channel, in planar lipid bilayers

Members of the degenerin/epithelial Na(+) channel superfamily of ion channels subserve many functions, ranging from whole body sodium handling to mechanoelectrical transduction. We studied brain Na(+) channel 2 (BNaC-2) in planar lipid bilayers to examine its single channel properties and regulation by Ca(2+). Upon incorporation of vesicles made from membranes of oocytes expressing either wild-type (WT) BNaC-2 or BNaC-2 with a gain-of-function (GF) point mutation (G433F), functional channels with different properties were obtained. WT BNaC-2 resided in a closed state with short openings, whereas GF BNaC-2 was constitutively activated; a decrease in the pH in the trans compartment of the bilayer activated WT BNaC-2 and decreased its permeability for Na(+) over K(+). Moreover, these maneuvers made the WT channel more resistant to amiloride. In contrast, GF BNaC-2 did not respond to a decrease in pH, and its amiloride sensitivity and selectivity for Na(+) over K(+) were unaffected by this pH change. Buffering the bathing solutions with EGTA to reduce the free [Ca(2+)] to <10 nm increased WT single channel open probability 10-fold, but not that of GF BNaC-2. Ca(2+) blocked both WT and GF BNaC-2 in a dose- and voltage-dependent fashion; single channel conductances were unchanged. A drop in pH reduced the ability of Ca(2+) to inhibit these channels. These results show that BNaC-2 is an amiloride-sensitive sodium channel and suggest that pH activation of these channels could be, in part, a consequence of H(+) "interference" with channel regulation by Ca(2+).     

Heparin influence on alpha-staphylotoxin formed channel

The effects of heparin on ion channels formed by Staphylococcus aureus alpha-toxin (ST channel) in lipid bilayers were studied under voltage clamp conditions. Heparin concentrations as small as 100 pM induced a sharp dose-dependent increase in channel voltage sensitivity. This was only observed when heparin was added to the negative-potential side of lipid bilayers in the presence of divalent cations. Divalent cations differ in their efficiency: Zn2+>Ca2+>Mg2+. The apparent positive gating charge increased 2-3-fold with heparin addition as well as with acidification of the bathing solution. 'Free' carboxyl groups and carboxyl groups in ion pairs of the protein moiety are hypothesized to interact with sulfated groups of heparin through divalent cation bridges. The cis mouth of the channel (that protrudes beyond the membrane plane on the side of ST addition and to which voltage was applied) is less sensitive to heparin than the trans-mouth. It is suggested that charged residues which interact with heparin at the cis mouth of ST channels and which contribute to the effective gating charge at negative voltage may be physically different from those at the trans mouth and at positive voltage.     

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.     

Probing the mechanism of fusion in a two-dimensional computer simulation

A two-dimensional (2D) model of lipid bilayers was developed and used to investigate a possible role of membrane lateral tension in membrane fusion. We found that an increase of lateral tension in contacting monolayers of 2D analogs of liposomes and planar membranes could cause not only hemifusion, but also complete fusion when internal pressure is introduced in the model. With a certain set of model parameters it was possible to induce hemifusion-like structural changes by a tension increase in only one of the two contacting bilayers. The effect of lysolipids was modeled as an insertion of a small number of extra molecules into the cis or trans side of the interacting bilayers at different stages of simulation. It was found that cis insertion arrests fusion and trans insertion has no inhibitory effect on fusion. The possibility of protein participation in tension-driven fusion was tested in simulation, with one of two model liposomes containing a number of structures capable of reducing the area occupied by them in the outer monolayer. It was found that condensation of these structures was sufficient to produce membrane reorganization similar to that observed in simulations with "protein-free" bilayers. These data support the hypothesis that changes in membrane lateral tension may be responsible for fusion in both model phospholipid membranes and in biological protein-mediated fusion.     

Ion selectivity of colicin E1: II. Permeability to organic cations

Channels formed by colicin E1 in planar lipid bilayers have large diameters and conduct both cations and anions. The rates at which ions are transported, however, are relatively slow, and the relative anion-to-cation selectivity is modulated over a wide range by the pH of the bathing solutions. We have examined the permeability of these channels to cationic probes having a variety of sizes, shapes, and charge distributions. All of the monovalent probes were found to be permeant, establishing a minimum diameter at the narrowest part of the pore of approximately 9 A. In contrast to this behavior, all of the polyvalent organic cations were shown to be impermeant. This simple exclusionary rule is interpreted as evidence that, when steric restrictions require partial dehydration of an ion, the structure of the channel is able to provide a substitute electrostatic environment for only one charged group at time.     

Structural and functional properties of colicin B

Colicin B was isolated in pure form from cells of Escherichia coli that contained the colicin activity and immunity genes cloned on a multi-copy plasmid. Active colicin B consisted of a single polypeptide with Mr of about 60,000. The sequence of 44 amino acids from the amino-terminal portion is presented. The isoelectric point of the protein was at 4.5. Colicin B inhibited the membrane potential-dependent transport of proline and enhanced the uptake of alpha-methylglucoside via the phosphoenolpyruvate-dependent phosphotransferase system. Colicin B formed small, ion permeable channels with an average single-channel conductance of 13.7 pS (1 pS = 10(-12) siemens) in 1 M KCl. Channel formation was voltage-dependent in the pH range between 4.5 and 6. At pH 7 the channels were voltage independent. Voltage-dependent channels were only formed when the trans compartment (the protein was added to the cis compartment) was negative by at least 70 mV. Evidence for an asymmetric single channel conductance was obtained. With KCl a hyperbolic conductance-concentration relationship was observed. The conductance for monovalent cations was minimal for Li+ and was maximal for NH+4. The single channel conductance of colicin B was larger than that of colicin A as judged from lipid bilayer experiments under otherwise identical conditions.     

Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules

Few methods exist for obtaining the internal dimensions of transmembrane pores for which 3-D structures are lacking or for showing that structures determined by crystallography reflect the internal dimensions of pores in lipid bilayers. Several approaches, involving polymer penetration and transport, have revealed limiting diameters for various pores. But, in general, these approaches do not indicate the locations of constrictions in the channel lumen. Here, we combine cysteine mutagenesis and chemical modification with sulfhydryl-reactive polymers to locate the constriction in the lumen of the staphylococcal alpha-hemolysin pore, a model protein of known structure. The rates of reaction of each of four polymeric reagents (MePEG-OPSS) of different masses towards individual single cysteine mutants, comprising a set with cysteines distributed over the length of the lumen of the pore, were determined by macroscopic current recording. The rates for the three larger polymers (1.8, 2.5, and 5.0 kD) were normalized with respect to the rates of reaction with a 1.0-kD polymer for each of the seven positions in the lumen. The rate of reaction of the 5.0-kD polymer dropped dramatically at the centrally located Cys-111 residue and positions distal to Cys-111, whether the reagent was applied from the trans or the cis side of the bilayer. This semi-quantitative analysis sufficed to demonstrate that a constriction is located at the midpoint of the pore lumen, as predicted by the crystal structure, and although the constriction allows a 2.5-kD polymer to pass, transport of a 5.0-kD molecule is greatly restricted. In addition, PEG chains gave greater reductions in pore conductance when covalently attached to the narrower regions of the lumen, permitting further definition of the interior of the pore. The procedures described here should be applicable to other pores and to related structures such as the vestibules of ion channels.     

Location of the permeation pathway in the recombinant type 1 inositol 1,4,5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) forms ligand-regulated intracellular Ca(2+) release channels in the endoplasmic reticulum of all mammalian cells. The InsP(3)R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP(3)R pore. Mutant InsP(3)Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; P(Ca)/P(Cs) = 6.3). These mutant channels bound InsP(3), but ligand occupancy did not regulate the constitutively open pore (P(o) > 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398-2589) near the COOH terminus of the protein forms the InsP(3)R pore. Further, we have produced a constitutively open InsP(3)R pore mutant that is ideal for future site-directed mutagenesis studies of the structure-function relationships that define Ca(2+) permeation through the InsP(3)R channel.