Alamethicin pore

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. 

It is known that the helical conformation of alamethicin would provide a channel too small to allow passage of ions, since it has been calculated that the interior of an a-helix of poly-L-alanine has an energy barrier of approximately 840 kJ mol for passage of protons The alamethicin pores arise from circular a regates of jKirallel... 

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. 

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). 

Alamethicin exerts bacteriostatic, fungicidal, cytostatic, and hemolytic effects. The most important property of the alamethicins is the formation of potential-dependent ion-conducting pores in lipid membranes as a model for the conduction of nerve impulses 227 b). 

In order to cover glucuronidation reactions in incubations in microsomal fractions several modifications have been applied in order to optimize conditions. These comprise longer incubation times than necessary for oxidative reactions by cytochrome P450s, and use of modifiers, both to overcome the latency in activity due to the diffusional barriers of the endoplasmatic reticulum (Coughtrie and Fisher 2003 Csala et al. 2004). Modifiers used are detergents or the pore-forming peptide alamethicin (Fisher 2000). Also disruption of cells by sonication is applied (Ethell 1998). 

Although several studies on linear amphipathic a-helical AMPs showed the formation of transmembrane pores probably through the barrel-stave mechanism, only a few of them could be confirmed. Among them are pardaxin, alamethicin, and the helix a5 of the 5-endotoxin [87]. 

The use of microsomes along with UDPGA as a cofactor assay to measure UGT enzyme activity has been hampered historically by the fact that this enzymatic activity in microsomes is often in a latent form and requires activation by physical or detergent-induced disruption of the membrane matrices. Recently, a generic method involving the addition of the pore-forming peptide alamethicin to overcome the latency exhibited by this enzyme system has been described.99 The inclusion of alamethicin seems to provide a more consistent method of assessing UGT enzyme activity. 

The peptaibols, a class of small, naturally occurring peptides, have been used as models of ion channels which consist of a bundle of transmembrane helices surrounding a central pore. Perhaps the most thoroughly studied member of this class of peptides is alamethicin. Alamethicin is a 20-residue linear peptide from the fungus Trichoderma viride. The sequence of this hydrophobic peptide is shown below ... 

Since the early 1970s, most investigators agree that alamethicin monomers form a water-filled conducting pore like the staves of a barrel, and this assumption is consistent with most ion conduction data (23-25). However, conduction experiments provided no clues for the nonconduction state, either... 

Figure 4. Multistate conductance shown by alamethicin on a planar bilayer membrane with applied potential of 210 mV (top) and on frog sarcolemmal membrane with — 110-mV resting potential (bottom). Current bursts begin at A and continue until B. The different levels observed are not integral multiples of unit current conductance, which implies different states of the pore. (Upper figure reproduced with permission from reference 41. Copyright 1972 Elsevier. Lower figure reproduced with permission from reference 302. Copyright 1979 Macmillan Magazines.)...

In the case of human microsome samples, pretreatment of the protein with alamethicin, a pore-forming peptide, is known to increase enzyme activity (II). This reagent is excluded from the SN-38 glucuronidation assay presented here because alamethicin has almost no effect on the glucuronidation activity of the COS-1 membrane fractions. 

Fig. 1. Comparison of the current noise due to a carrier with that due to a pore. (A) Current fluctuations in the presence of valinomycin with lithium (which is poorly transported) and the rubidium (which is well transported) as the cation. Noise increases at high frequencies because the empty carrier must return after carrying one ion across the membrane. (From [8].) (B) Current fluctuations in the presence of alamethicin. Noise decreases at high frequencies because the channels open and close at a limited rate. (From [7].)...

Of greater interest, because it may indicate how potassium pores are formed and controlled in mammalian physiology, is alamethicin, a linear peptide from the fungus Trichoderma viride. It has 20 peptide-linked components which, except for the terminal L-phenylalaninol, are all amino acids eight of them are a-aminoisobutyric acid residues, and the others are normal protein constituents. X-ray crystal structure analysis shows that the residues facing in one direction are hydrophilic and those of opposite situation are lipophilic. 

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. 

G. Boheim, H.A. Kolb, Analysis of the multi-pore system of alamethicin in a lipid membrane. I. Voltage-jump current-relaxation measurements, J. Membrane Biol. 38 99-150 (1978)... 

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