Alamethicin

In a further example of pore formation in artificial bilayers, the properties of alamethicin appear to be rather more complex than those for gramicidin and polyenes. 

The IfV curves for lipid bilayers containing alamethicin are linear over only a small potential range [28], but then conductance increases in proportion to the 6th power of the potential. Apparently, conductance also increases with the 6th power of the salt concentration and the 6th power of the alamethicin concentration. 

When alamethicin is examined by the techniques used for observing single channels, complex step-like current fluctuations are seen in which not all the current levels are equally probable and not all the current steps are of equal size [29]. Hie molecular basis of the events which underlie this behaviour is not fully resolved. The results have been interpreted in ways which suggest that interaction occurs between the individual channels, perhaps by some form of clustering. Presumably then clusters of one size are more probable than others, and also the formation of clusters may alter the conductance of individual channels. Both of these properties would produce the sort of behaviour seen with alamethicin channels. 

EM (excitability-inducing material) and BWSV (black widow spider venom) 

Thus far, pore formers with known chemical constitution have been discussed, and clearly these are of greater importance for understanding the microarchitecture of the conducting sites. There are a number of other substances whose structure is unknown, but since they are of biological origin it is sometimes argued that similar substances may be present in animal cell membranes. 

Remarkably, nearly half of the amino acid components are L-a-amino-isobutyric acid residues (Aib), an amino acid not present in ribosome-manufactured proteins (p. 8). Peptides containing Aib-residues have been recognized to form a-helices particularly readily, an explanation of the pore forming properties of alamethicins and of similar natural and artificial polypeptides. In the natural analogs alanine or valine is found to be replaced by Aib closely related natural compounds are among others, suzukacillin, trichotoxin (mould products), and less similar, a component of bee venom, the 25-peptide mellitin. In the synthesis of peptides containing a-amino-isobutyric acid certain difficulties are encountered due to the poor steric accessibility of the amino—as well as of the carboxyl group. 

Note that the ferrocenoyl group is bound to the C terminus via ester formation. 

In the reduced (neutral) form, both ferrocenoyl alamethicin derivatives are shown to form voltage-dependent channels in planar lipid bilayers at positive potentials, with conductance properties similar to unmodified alamethicin. When they were incorporated in the lipid bilayer, oxidation of the ferrocenoyl peptide ALM-CO-CpFe(Cp-COOH) results in a shorter lived channel whereas the oxidation of ALM-CO-Fc causes a time-dependent elimination of channel-openings, which can be restored by increasing the trans-bilayer potential. Pretreatment of the ferrocenoyl peptides with oxidizing agents alters their single-chaimel properties in a qualitatively similar manner. 

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Bechinger, B. Structure and functions of channel-forming peptides Magainins, cecropins, melittin, and alamethicin. /. Memhr. Biol. 1997, 756, 197-211. 

A similar effect has been observed for alamethicin I and II, hemocyanin, antiamoebin I and other substances. Great interest in the behaviour of these substances was aroused by the fact that they represent simple models for ion channels in nerve cells. 

Many of these materials form voltage-gated transmembrane ion channels, i.e. they exhibit non-linear current voltage curves, unlike the polyene antibiotics and gramicidin A which have linear current voltage curves. Of the peptaibol antibiotics the most thoroughly investigated substance to date is alamethicin 201 203>. 

Fig. 22. The Fox and Richards model of the alamethicin transmembrane channel shown diagrammatically. Interruption of the oc-helical hydrogen bonding by the Pro-14 residue is signified by representation of each monomer as two cylindrical sections. The stippled spheres at the mouth of the channel represent the Glu-18, the spheres at the centre the Gln-7, and the spheres at the top the Gln-19 residues. Fox, R. O., Richards, F. M. Reprinted by permission from Nature 300, 325 (1982). Copyright Macmillan Journals Limited...

A variety of hypothetical models have been proposed for the alamethicin pores. These generally invoke the bent helix monomer conformation, hydrogen bonding between adjacent helical monomers, and structural features compatible with the voltage dependence of the channels 201-204). Unfortunately experimental evidence is insufficient to distinguish between these models at present. 

Fisher, M.B., Campanale, K., Ackermann, B.L. et al. (2000) In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin. Drug Metabolism and Disposition The Biological Fate of Chemicals, 28, 560-566. 

The amphipathic a-helical class of host defense peptides is the most abundant and most well-characterized class. Upon interaction with the hydrophobic membrane environment, the largely unstructured peptide adopts an amphipathic ct-helical conformation with one helical face containing the majority of the hydrophobic residues, the opposite containing a large proportion of the polar residues. These peptides are often short (<40 amino acids), devoid of cysteine residues, and found to be unstructured or linear in nonhydrophobic environments. Peptides found within this class include the antimicrobial peptide alamethicin, bee venom melittin, the magainins, and the human cathelicidin LL-37. ... 

When alamethicin is added to a ternary vesicle system comprising PDA, phospholipid, and lipopolysaccharide (LPS), the addition of polymyxin, an LPS-binding antibiotic, sensitizes the vesicles to alamethicin (Katz et al. 2003). Cholesterol-containing PDA liposomes have been used to colorimetrically detect streptolysin O, a cholesterol-dependent pore-forming toxin (Ma and Cheng 2005). 

Ion channel-forming alamethicin is a potent elicitor of volatile biosynthesis and tendril coiling. Cross talk between jasmonate and salicylate signaling in lima bean. Plant Physiology 125 369-377. 

Alamethicin (from Tricoderma viridae). Recrystd from MeOH. [Panday et al. JACS 99 8469 7977]. 

CNMR investigations of several cyclic and acyclic peptide antibiotics and toxins have been reported in the literature, e.g., gramicidin S-A (cyclo(Phe-Pro-Val-Orn-Leu)2) [176, 801, 872 875], viomycin (cyclopeptide constituted of L-serine, at, I-L-di-aminopropionic acid, /J-L-lysine and viomycidine) [876], bacitracin A (Ile-Cys-Leu-D-Glu-Ile-Lys-n-Orn-Ile) [815], alamethicin (Ac-Aib-Pro-Val-Aib-Val-Aib-Aib-Ala-Aib-... 

Transmembrane channels represent a special type of multi-unit effector allowing the passage of ions or molecules through membranes by a flow or site-to-site hopping mechanism. They are the main effectors of biological ion transport. Natural and synthetic peptide channels (gramicidin A, alamethicin) allowing the transfer of cations have been studied [6.66-6.68]. 

Phospholipid-bilayer membranes with valinomycin, gramicidin D, alamethicin Membrane conductance [29]... 

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