Ion channels formed by amphipathic helical peptides

Channel forming peptides (CFPs) are amphipathic peptides, of length ca. 20 residues, which adopt an -helical conformation in the presence of lipid bilayers and form ion channels with electrophysiological properties comparable to those of ion channel proteins. We have modelled CFP channels as bundles of parallel trans-bilayer helices surrounding a central ion-permeable pore. Ion-channel interactions have been explored via accessible surface area calculations, and via evaluation of changes in van der Waals and electrostatic energies as a K⁺ ion is translated along the length of the pore. Two CFPs have been modelled: (a) zervamicin-A1-16, a synthetic apolar peptaibol related to alamethicin, and (b) -toxin from Staphylococcus aureus. Both of these CFPs have previously been shown to form ion channels in planar lipid bilayers, and have been shown to have predominantly helical conformations. Zervamicin-A1-16 channels were modelled as bundles of 4 to 8 parallel helices. Two related helix bundle geometries were explored. K⁺channel interactions have been shown to involve exposed backbone carbonyl oxygen atoms. -Toxin channels were modelled as bundles of 6 parallel helices. Residues Q3, D11 and D18 generate favourable K⁺-channel interactions. Rotation of W15 about its C-C bond has been shown to be capable of occluding the central pore, and is discussed as a possible model for sidechain conformational changes in relation to ion channel gating.     

The properties of ion channels formed by zervamicins

The zervamicins (Zrv) are a family of 16 residue peptaibol channel formers, related to the 20 residue peptaibol alamethicin (Alm), but containing a higher proportion of polar sidechains. Zrv-1113 forms multi-level channels in planar lipid (diphytanoyl phosphatidylcholine) bilayers in response to cis positive voltages. Analysis of the voltage and concentration dependence of macroscopic conductances induced by Zrv-IIB suggests that, on average, channels contain ca. 13 peptide monomers. Analysis of single channel conductance levels suggests a similar value. The pattern of successive conductance levels is consistent with a modified helix bundle model in which the higher order bundle are distorted within the plane of the bilayer towards a torpedo shaped cross-section. The kinetics of intro-burst switching between adjacent conductance levels are shown to be approximately an order of magnitude faster for Zrv-IIB than for Alm. The channel forming properties of the related naturally occurring peptaibols, Zrv-Leu and Zrv-IC, have also been demonstrated, as have those of the synthetic apolar analogue Zrv-Al-16. The experimental studies on channel formation are combined with the known crystallographic structures of Zrv-Al-16 and Zrv-Leu to develop a molecular model of Zrv-II3 channels.Abbreviations Alm Alamethicin - Zrv Zervamicin - CFP Channel forming peptide - Aib -aminoisobutyric acid Correspondence to: M. S. P. Sansom     

The roles of serine and threonine sidechains in ion channels: a modelling study

The ion channel of the nicotinic acetylcholine receptor (nAChR) is believed to be lined by transmembrane M2 helices. A 4-8-12 sequence motif, comprising serine (S) or threonine (T) residues at positions 4, 8 and 12 of M2, is conserved between different members, anion and cation selective, of the nAChR superfamily. Parallel bundles of 4-8-12 motif-containing helices are considered as simplified models of ion channels. The relationship between S and T sidechain conformations and channelion interactions is explored via evaluation of interaction energies of K⁺ and of Cl^– ions with channel models. Energy calculations are used to determine optimal ₂ (C-C\-O-H) values in the presence of K⁺ or Cl^– ions. 4-8-12 motif-containing bundles may form favourable interactions with either cations or anions, dependent upon the ₂ values adopted. Parallel-helix and tilted-helix bundles are considered, as are heteromeric models designed to mimic the Torpedo nAChR. The main conclusion of the study is that conformational flexibility at ₂ enables both S and T residues to form favourable interactions with anions or cations. Consequently, there is apparently no difference between S and T residues in their interactions with permeant ions, which suggests that the presence of T vs. S residues within the 4-8-12 motif is not a major mechanism whereby anion/cation selectivity may be generated. The implications of these studies with respect to more elaborate models of nAChR and related receptors are considered.Abbreviations nAChR, GluR, NMDA-R, 5HT₃-R, GABA_AR, GlyR nicotinic acetylcholine, glutamate, NMDA, 5HT₃, GABA_A and glycine receptors, respectively - PhTx philanthotoxin - M2 second membrane-spanning helix of receptor-channel subunits     

Alamethicin and related peptaibols — model ion channels

Peptaibols are considered as models of those ion channels which consist of a bundle of transbilayer helices surrounding a central pore. X-Ray diffraction and NMR studies have yielded high resolution structures for several peptaibols. In conjunction with other spectroscopic investigations and molecular dynamics simulations, these studies suggest that peptaibols form proline-kinked -helices, and that there may be hinge-bending movement of the helix in the region of the central proline residue. The amphipathicity of peptaibol helices is analyzed in relation to their channel-forming properties. Studies of the interactions of peptaibols with lipid bilayers suggest that they are helical when in a membrane-like environment, and that the helix orientation relative to the bilayer is sensitive to the peptaibol: lipid ratio, and to the degree of hydration of the bilayer. Electrical studies reveal that many peptaibols form multiple-conductance level channels in a voltage-dependent fashion. Analysis of conductance levels provides support for the barrel stave model of channel formation, whereby different conductance levels correspond to different numbers of monomers in a helix bundle. Alternative models for voltage-activation are discussed, and the roles of molecular dipoles and of hinge-bending in this process are considered. Two molecular models for an N = 6 bundle of alamethicin helices are presented and their electrostatic properties analyzed. The relevance of studies of peptaibols to channel and transport proteins in general is considered.Abbreviations Aib -amino-isobutyric acid - Alm alamethicin - ATR-FTIR attenuated total reflection Fourier transform infrared - CD circular dichroism - CFP channel-forming peptide - Chol cholesterol - diPhyPC diphytanoyl phosphatidylcholine - DMPC dimyristoyl phosphatidylcholine - DOPC dioleoyl phosphatidylcholine - DOPE dioleoyl phosphatidylethanolamine - DPPS dipahnitoyl phosphatidylserine - DTPC ditetradecyl phosphatidylcholine - HSM hydrophilic surface map - I-V current-voltage - MLV multilamellar vesicle - nAChR nicotinic acetylcholine receptor - P:L protein-to-lipid ratio - POPC palmitoyloleoyl phosphatidylcholine - SUV small unilamellar vesicle - Zrv zervamicin     

Assembly of microfibrils in vivo and in vitro from (1»3)-β-d-glucan synthesized by protoplasts of Saccharomyces cerevisiae

Polymer chains of (13)--d-glucan were dissolved with 1 M NaOH at 4° C from native microfibrillar protoplast nets. The chains associated into microfibrils during NaOH neutralization or dialysis. In contrast to the native microfibrils which are of uniform width individually (10 to 20 nm) and arranged in flat bundles, the microfibrils formed in vitro showed no band formation and consisted of fibrous spindle-shaped subunits of variable width or loose elementary fibrils about 1.7 nm wide. X-ray diagrams of native nets indicated a fairly high crystallinity and were different for wet and dry specimens. They corresponded to those of paramylon. Precipitated glucans produced diagrams different from the former and revealing a lower crystallinity especially with the dry samples.The X-ray pattern, combined with other data, allowed the precipitated microfibrils to be identified as aggregates of molecular strands composed each of three intertwined helical glucan chains. Since these triple helical chains are about 1.7 nm wide the elementary fibrils of this width can represent only single triple-helical strands. These helices have 7 glucose residues per turn and therefore a low symmetry which explains the poor crystallizing properties. The 7 membered helix represents a basic difference with the well crystallized native glucan which is built of highly symmetrical triple helices with 6 glucose residues per turn. Since 6₁ helical conformation is not formed in vitro at normal temperatures its generation in vivo must be due to the action of synthesizing enzymes at the protoplast membrane. The intertwining of these helices and crystallization of the strands are determined by their symmetry and physical properties of the chains. This characterizes the native microfibrils as products of self-assembly of enzymegenerated 6₁ helices.     

Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers

Polystyrene culture dishes and polystyrene microcarriers were coated with Pronectin-F and poly-l-lysine (polylysine), either alone or in combination. Pronectin-F is a recombinant peptide containing repeats of the RGD cell-attachment sequence from fibronectin. Polylysine is a polymer ofl-lysine. Pronectin-F supported attachment of Madin-Darby Canine Kidney (MDCK) cells at concentrations as low as 0.025 g/cm² of surface area. The cells rapidly spread after attachment. Polylysine at concentrations of 0.05–0.5 g/cm² also supported cell attachment but cells did not rapidly spread after attachment to this substrate. Higher concentrations of polylysine could not be used because of toxicity. When the two peptides were used in conjunction, MDCK cells attached and spread at lower peptide concentrations than they did when either substrate was used alone. These findings suggest that recombinant Pronectin-F alone or in conjunction with a cationic polymer could be a useful replacement for materials such as gelatin or collagen which are currently used as microcarrier surfaces.     

An investigation into the N- and C-capping effects of glycine in cavitand-based four-helix bundle proteins

The capping efficiency of glycine on cavitand-based synthetic four-helix bundles was investigated. Glycine, a common C-capping amino acid, has always been included as a C-terminal residue in our de novo peptides, although the exact contribution of the glyince cap to the overall stability and structure of the caviteins had not previously been examined. The uncapped proteins were found to be less helical according to their CD spectra. In addition, the H/D exchange experiments suggested that the uncapped caviteins were more conformationally flexible. Capped and uncapped caviteins exhibited similar values of unfolding. Overall, it can be concluded that glycine caps are useful, as they reduce helical unravelling and enhance helicity, and thus, glycine will be included as a C-terminal residue in future de novo peptide sequences.     

Helical kink and channel behaviour: a comparative study with the peptaibols alamethicin,trichotoxin and antiamoebin

Kinks or bends introduced in peptides and proteins by helical distorter residues such as proline, other imino acids and glycine, especially when these are in close proximity in the sequence, are increasingly recognized as playing an essential role in the gating of channel-forming peptides as well as of physiological ion channels. Peptaibols are useful simple models for the much more complex biological ion channels, especially voltage-gated ones. In this short review, we compare the monomeric structures of three selected peptaibols (alamethicin, trichotoxin and antiamoebin) that widely differ with regards their near-central kink angles and dipolar moment orientations. These structural features are then shown to be correlated to the different patterns of channel activity, both at the macroscopic and single-channel levels of investigation.Presented at the British Biophysical Society–Société Française de Biophysique joint meeting Ion channels: from Biophysics to disorders, held in May 2003, Rennes, France     

Zum Nachweis tritiierter Assimilate in den Siebröhren von Cucurbita

Summary After application of D-glucose-6-T to small areas of leaf surfaces of Cucurbita, radioactivity — mainly sucrose-T — could exclusively be localized by histoautoradiography (dry-technique) within single sieve tubes of exporting bundles. The centre of activity corresponded to the slime-plugs (in crosssection-autoradiograms) of the sieve tubes and to the sieve plates (in longitudinal sections). It is concluded that only the sieve tubes are the conducting channels for the long-distance transport of those substances which are labeled with tritium in these experiments.     

Circular dichroism analysis of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin

Analysis of the far-ultraviolet circular dichroism spectrum of bovine blood coagulation factor IX reveals the presence of approximately 14% helical structures 26% -sheets, 20% -turns, and 40% coils. These values are essentially the same for the activation products of this zymogen, factor IXa and factor IXa. Similar analysis for bovine factor X permits calculation of these secondary structural as approximately 11% helices, 31% -structures, 22% -turns, and 36% random structures. Bovine prothrombin contains approximately 12% helical structures, 35% -structures, 24% -turns, and 29% coils. None of these values is substantially altered as a result of increase of thepH from 7.4 to 10.5, or upon addition of Ca²⁺ to a concentration of at least 20 mM. Analysis of the near-ultraviolet spectra of factor IX and prothrombin suggests that several aromatic amino acid residues and the disulfide bond present in their -carboxyglutamic acid-containing regions are exposed to solvent and are perturbed by the abovepH adjustment and Ca²⁺ addition. Similar effects are observed in the case of factor X; in addition, the Trp residue at the amino terminus of the heavy chain appears to be influenced by the abovepH alteration. The results reported in this paper show that these vitamin K-dependent blood coagulation proteins are similar in their ordered secondary structures, which are dominated by -sheets and -turns. Their overall secondary structures are not influenced by Ca²⁺ binding and are stable to alkalinepH changes. However, these same environmental alterations appear to be effective probes of aromatic residues in the -carboxyglutamic acid regions.     

Folding versatility of the C-terminal tetrapeptide amide sequence of the lipopeptaibol antibiotics trichodecenin and trichogin

Summary An X-ray diffraction analysis ofZ-l-Leu-Aib-Gly-l-Ile-l-Leu-OMe, containing the N-acylated tetrapeptide amide sequence-l-Leu-Aib-Gly-l-Ile-, showed that in the crystal state the carbonyl group preceding thel-Leu¹ residue acts as the acceptor of two C=OH–N intramolecular H-bonds, which give rise to an-l-Leu¹-Aib²-type-III' -turn and an-l-Leu¹-Aib²-Gly³-l-Ile⁴--turn, respectively. A second (type-I') -turn encompasses the-Aib²-Gly³-sequence. This is the third type of folding motif known for that tetrapeptide sequence, considering also those already published for the C-terminal segment of the lipopeptaibol antibiotics trichodecenin I and trichogin A IV.     

Studies on the putative role ofγ-glutamyl transpeptidase in intestinal transport of amino acids in Atlantic salmon

Summary The -glutamyl cycle is considered to function in the membrane transport of amino acids, particularly glutamine and cysteine. When groups of Atlantic salmon were fed either a control diet containing 45% crude protein or an amino acid diet (of similar overall amino acid composition but containing elevated levels of glutamine and cysteine) for 16 weeks, weight gains were significantly greater in the former group than in those given the amino acid diet. There were no significant differences between treatments in -glutamyl transpeptidase (GT) activity in the proximal intestine; in distal intestine there was significantly more activity in control fish. Mean levels of GSH were higher in tissues (pyloric caeca, distal intestine and kidney) of amino acid diet fish than in those of control fish. Glutamine was less effective as a -glutamyl acceptor than several other amino acids when tested with salmon caecal GT. There were no morphological adaptations to the two feeds. Nutrient uptake studies showed an increased uptake of glutamine, but decreased uptakes of proline and methionine in proximal intestine of salmon fed amino acid diet. Much the greater part of the glutamine uptake, even at high concentrations was shown to be by Na⁺ dependent processes. There is no evidence that GT itself is Na⁺ dependent. The results do not support the view that the -glutamyl cycle and GT in particular are involved in the transport of amino acids in the intestine and are discussed in this context.Abbreviations GT -glutamayl transpeptidase - GSH reduced glutathione     

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

Summary All the backbone ¹H and ¹⁵N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by ¹⁵N-edited two-dimensional NMR experiments on selectively ¹⁵N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly ¹⁵N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the -sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.     

Gliding motility of Peloploca spp., and their distribution in the sediment and water column of a eutrophic lake

Observations indicating gliding motility in the gas-vacuolate, filamentous organism Peloploca were made using microcapillary tubes. Tubes containing semi-solid agar, incubated in sediment cores gave good enrichments of Peloploca spp. The organisms, which had the form of helical bundles of filaments, were seen to corkscrew through the agar at up to 2–3 m s^-1.The vertical distribution of Peloploca spp. in the sediment and water column of a eutrophic lake was examined periodically during summer stratification. The organisms were confined to anoxic conditions in the sediment prior to stratification. With increasing anoxia in the hypolimnion, the population shifted upwards in the sediment, and towards the end of stratification, in the most reducing conditions, appeared in the lower hypolimnion. Anaerobically incubated sediment cores also showed the movement of the Peloploca population from sediment into the overlying water.It is suggested that the gliding motility and helical morphology of Peloploca bundles enable them to migrate through sediment in response to oxygen and Eh gradients, in addition to their use of gas vacuoles to adjust their position in the water column. The taxonomic implications of gliding motility in Peloploca spp. are discussed.     

Seepage patterns, pore water, and aquatic plants: hydrological and biogeochemical relationships in lakes

This study documented linkages between lakeshore seepage fluxes, pore water chemistry, and aquatic plants in several lakes of the Adirondack Mountains region of New York, USA. Three replicate stations were set up along each of four different lake shorelines. From June through September 1998 and from snowmelt in April through August 1999, seepage flux was measured with seepage meters. Throughout this time period, lake surface water and pore water chemistry were monitored weekly to biweekly. At each station, leaf tissue chemistry of the water lily Nuphar variegatum was measured once in each year. Sediment chemistry and plant abundance were also measured once in 1998. We found that pore water concentrations of base cations, iron, and zinc were related to the direction, magnitude, and variability of seepage fluxes. Concentrations of base cations, iron, and zinc were both highest and most variable where seepage was low (0 to 50mLm^–2h^–1) in contrast to being more stable where seepage was highest and variable (–608 to 612mLm^–2h^–1). Leaf tissue chemistry and plant abundance were also related to seepage patterns. N. variegatum leaves had elevated zinc content at stations with low average discharge. Knowledge of seepage patterns helped to explain spatial patterns of elevated trace metal content in both pore water and plant tissues. Our work suggests that the hydrological process of lakeshore seepage exerts important controls on lakeshore biogeochemistry.     

Modelling of transmembrane α-helix bundles

A strategy for modelling transmembrane -helix bundles has been investigated. Results concerning the rotational orientations of the helices are described and perspectives for extensions of the method are discussed.     

Gating processes of channels induced by colicin A,its C-terminal fragment and colicin E1 in planar lipid bilayers

The dependence on pH and membrane potential of the pore formed by colicin A and its C-terminal 20 kDa fragment has been measured using planar lipid bilayers. The single channel conductance of the pore formed by both colicin A and the fragment increases with pH with an apparent pK of 6.0. At pH 5.0 the gating by membrane potential of the channels formed by either colicin A or its fragment is identical. At the same pH, quite similar pore properties were found when using the related bacteriocin, colicin E₁. In agreement with previous studies, these data indicate that the protein structure containing the lumen of the pore resides in the 20 kDa C-terminal part of the colicin A and favours the recently proposed model, based on protein sequence analysis, which proposes that colicin A, E₁ and I_B C-terminal domains are folded in the same three-dimensional structure. However, it is also shown that colicin A and not its C-terminal fragment undergoes a pH dependent transition between an acidic and a basic form of the pore with an apparent pK of 5.3. The two forms of the pore differ by their gating charge but not by the channel size. These results suggest that there is a pH dependent association between the C-terminal domain carrying the lumen of the pore and another domain of the molecule which affect the pore sensitivity to membrane potential.     

Oligo-riboprobes

Summary In situ hybridisation detection of mRNAs using riboprobes has become a widely used technique. However, the identification of cells producing closely-related yet distinct mRNAs is difficult with the usual size probes. More-over, it is not always easy to obtain the required cDNA essential for cRNA probe synthesis. To avoid these problems, we have used synthetic oligodeoxynucleotides to generate short, single stranded RNA probes (oligo-riboprobes). These probes can be labelled to very high (10⁹ cpm/g) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from preprotachykinin cDNA.     

Mechanisms of amyloid beta protein-induced modification in ion transport systems: implications for neurodegenerative diseases

1. Alzheimer's disease (AD) is a neurodegenerative disorder that affects the cognitive function of the brain. Pathological changes in AD are characterized by the formation of amyloid plaques and neurofibrillary tangles as well as extensive neuronal loss. Abnormal proteolytic processing of amyloid precursor protein (APP) is the central step that leads to formation of amyloid plaque, neurofibrillary tangles, and neuronal loss.2. The plaques, which accumulate extracellularly in the brain, are composed of aggregates and cause direct neurotoxic effects and/or increase neuronal vulnerability to excitotoxic insults. The aggregates consist of soluble pathologic amyloid beta peptides AP[1–42] and AP[1–43] and soluble nonpathologic AP[1–40]. Both APP and AP interact with ion transport systems. AP induces a wide range of effects as the result of activating a cascade of mechanisms.3. The major mechanisms proposed for AP-induced cytotoxicity involve the loss of Ca²⁺ homeostasis and the generation of reactive oxygen species (ROS). The changes in Ca²⁺ homeostasis could be the result of (1) changes in endogenous ion transport systems, e.g. Ca²⁺ and K⁺ channels and Na⁺/K⁺-ATPase, and membrane receptor proteins, such as ligand-driven ion channels and G-protein-driven releases of second messengers, and (2) formation of heterogeneous ion channels.4. The consequences of changes in Ca²⁺-homeostasis-induced generation of ROS are (a) direct modification of intrinsic ion transport systems and their regulatory mechanisms, and (b) indirect effects on ion transport systems via peroxidation of phospholipids in the membrane, inhibition of phosphorylation, and reduction of ATP levels and cytoplasmic pH.5. We propose that in AD, AP with its different conformations alters cell regulation by modifying several ion transport systems and also by forming heterogeneous ion channels. The changes in membrane transport systems are proposed as early steps in impairing neuronal function preceding plaque formation. We conclude that these changes damage the membrane by compromising its integrity and increasing its ion permeability. This mechanism of membrane damage is not only central for AD but also may explain other malfunctioned protein-processing–related pathologies.     

Modeling the secondary structures of the peptaibols antiamoebin I and zervamicin II modified with<Emphasis Type="SmallCaps"> D</Emphasis>-amino acids and proline analogues

Antiamoebin I (AAM-I) and zervamicin II (Zrv-IIB) are peptaibols that exert antibiotic activity through the insertion/disruption of cell membranes. In this study, we investigated how the folding of these peptaibols are affected when some of their native residues are replaced with proline analogues and asymmetrical D-α,α-dialkyl glycines (two classes of noncanonical amino acids). Systematic substitutions of native Aib, Pro, Hyp, and Iva residues were performed to elucidate the folding properties of the modified peptaibols incorporating noncanonical residues. The secondary structure of a peptaibol influences its ability to incorporate into membranes and therefore its function. Our findings reveal that native Zrv-IIB unfolds considerably in water. The presence of Iva and the noncanonical proline analogue cis-3-amino-L-proline (ALP) in both peptaibols induces helical structures. Inserting asymmetric glycines such as α-methyl-D-leucine (MDL) and α-methyl-D-phenylalanine (MDP) into the peptaibols induces folding. This preorganization in water may help to overcome the energy barrier required for peptaibol insertion into the membrane, as well as to facilitate the formation of transmembrane channels.

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Graphical abstract AAM-I and Zrv-IIB peptidomimetics carrying MDL and ALP noncanonical amino acids, exhibiting improved helical secondary structure in water