The nature of the voltage-dependent conductance induced by alamethicin in black lipid membranes

This The peptide also affects the electrical properties of planar artificial lipid membranes in a unique and specific way: minute concentrations of ALA induce voltage-dependent conductances that mimic the action potential phenomena observed in nerve membranes. These effects were observed by Mueller and Rudin (6) and have been studied also by other groups (7-9). It is now generally believed that several ALA molecules aggregate in the membrane to form ion-conducting channels (8,(10)(11)(12).Despite a number of physicochemical (13, 14), conformational (15-18), and chemical (17-22) studies, the primary structure of ALA remains unknown. Currently, there are two proposed sequences: a cyclic (ALA-c) and an open chain (ALA-o) peptide structure (Fig. 1). The former, proposed by Payne et al. (19), has been confirmed by Ovchinnikov et al. (20), while the latter, proposed by Martin and Williams (21,22), found the support of the Jung group (17). The two proposals agree on a sequence of 15 consecutive residues (3-17 according to ALA-o numbering). In ALA-c the y-carboxyl group of Glu'8 is in amide linkage with Pro2 to form a cyclic structure. In

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confidence: 58%

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confidence: 99%

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confidence: 99%

See 1 more Smart Citation

Synthesis of a 19-residue peptide with alamethicin-like activity.

Gisin¹,

Kobayashi²,

Hall³

1977

“…2 B and C. Rather, an increase in the pore formation rate seems to occur, contrary to the situation with 1,2-DOPC/decane membranes (15). Because an increase in the pore formation rate with raised ALS0 concentration has been reported (15), we think that the structural modifications associated with the lipid phase transition lead to domains that are more concentrated in AL30 than the homogeneous lipid membrane above t. AL30-induced conductance depends on the 9th to 10th power of ALS0 concentration (15,19,20), which would easily explain the effect described above.…”

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confidence: 63%

Lipid phase transition in planar bilayer membrane and its effect on carrier- and pore-mediated ion transport.

Boheim¹,

Hanke²,

Eibl³

1980

127

Using mixed-chain lipids, we have recorded cooling and heating curves of planar bilayer membranes while they passed the lipid phase transition range. With unmodified planar bilayers, spontaneous current fluctuations are observed near the lipid phase transition temperature (tc t 290C). This effect coincides with the expected and measured decrease in membrane capacitance. Carrier (valinomycin)-modified planar bilayers exhibit near tc an abrupt change from a high-conducting state above tc to the state ofbare membrane conductance below tc. In contrast to this behavior, planar bilayers modified by pore-forming antibiotics (gramicidin A, alamethicin) do not show any peculiar effect at tc. However, at 22-230C a pronounced maximum in pore-induced conductance is seen. Whereas the gramicidin A pore abruptly stops stepwise fluctuations below 16'C, with alamethicin a few long-lasting pore and pore state fluctuations persist down to 10'C. It is suggested that the carrier may freeze out into the membrane/water interface. The effects observed with pore-forming substances, on the other hand, are interpreted in terms of lateral phase separation into pure lipid and lipid/antibiotic domains. Phase changes are well-known phenomena in artificial lipid/ water systems (1) and biological systems (2). These phase transitions, which may play a role in triggering biological processes (3), can be induced by temperature changes or by interaction of ions with charged membrane lipids (4, 5). A great number of publications report on phase transition phenomena in pure and protein-loaded vesicles and liposomes. Due to the instability of planar bilayer membranes in the solid state, there are so far only two reports on electrochemical measurements in the freezing and melting range of planar lipid bilayer membranes. Experiments carried out on membranes from a 1:1 (wt/wt) mixture of dipalmitoylglycerol and distearoylglycerol in n-decane led to the interpretation that ion carriers became frozen and thus immobile within the membrane phase (6). On the other hand, the ionic conductance induced by the pore-former gramicidin was found to remain unchanged at the transition temperature tc of 41'C. Recently, ion-conducting channels were reported to appear in unmodified planar bilayer membranes at the phase tr of 59°C (7). Membranes were formed from a 1,2-distearoyl-glycero-3-phosphocholine/decane solution. There is also a paper, based on optical reflectivity measurements on membranes from monostearoylglycerol in n-hexadecane, which demonstrated an ;70% increase in membrane thickness when the system was cooled below the tc of 55°C (8).Using saturated mixed-chain lipids with a tc of ;290C, we succeeded in forming virtually solvent-free planar bilayer membranes below and above tc. In this paper we report our investigations on pure and ionophore-modified planar bilayers of this type in the 10-40°C temperature range.

“…A large liposome just after formation ofa seal is shown in Fig. 2 We have tested this possibility by studying the behavior of these putative isolated bilayer patches in the presence of the channel-forming antibiotics alamethicin and gramicidin, both of which require a lipid bilayer for activity (22,23 Tor-pedomembrane extracts and excess lipids, current records from isolated bilayer patches were quite noisy, with a variety of unidentified channel-like phenomena present. In an attempt to obtain quieter patches, we purified the AcChoR from these extracts by using AcChoR affinity chromatography (20).…”

Section: Resultsmentioning

confidence: 99%

Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.

Tank¹,

Miller²,

Webb³

1982

Small unilamellar vesicles formed from purified phospholids by detergent/dialysis methods may be enlarged to 30-,um diameter by freezing and thawing. Very-high-resistance seals were formed by applying a glass micropipette to the surface of these large liposomes, and single bilayer "patches" of membrane were isolated from the liposome surface while remaining sealed to the micropipette. The exogenous channel-forming peptides gramicidin and alamethicin induced characteristic single-channel fluctuation behavior in these excised patches held under voltageclamp conditions. Large liposomes were formed from the small unilamellar vesicles made from cholate extracts of Torpedo electroplax plasma membrane vesicles. Isolated patches formed from these reconstituted membranes displayed current fluctuations due to single voltage-gated Cl channels from non-innervated-face membranes; the properties of these Cl1 channels are identical to those observed in planar bilayer membranes after direct insertion from native membranes. This liposome-patch method combines the advantages ofmembrane protein incorporation into liposomes with high-resolution electrical recording methods and may provide a generally applicable approach to the study ofintegral membrane channel proteins after solubilization and reconstitution.In any complete biochemical study of a membrane transport system, it is necessary to solubilize the protein of interest, manipulate it in the solubilized state, and then reinsert it into a "reconstituted" membrane in which its solute-transport activity can be assayed. This "solubilization/reconstitution" approach has been applied to many classes of membrane transport systems (1, 2), with the single exception ofthe ion channel proteins of higher organisms. Only one ion channel protein, the nicotinic acetylcholine receptor (AcChoR), has been reconstituted (3-5). Ion channels have been resistant to classical reconstitution work because the small unilamellar vesicles (SUVs) used in such work present great difficulties in the measurement of channel-mediated ion fluxes; a "typical" SUV containing a channel of50-pS conductance would completely equilibrate its ion content in several milliseconds, far faster than the time resolution of the conventional vesicle flux assay (1, 6). A further problem is that many ion channels are controlled by membrane voltage, which cannot be reliably maintained constant during such assays. Methods do exist for inserting ion channels into planar bilayer membranes, which may be voltage clamped and subjected to electrical current assays (7-10), but these methods, unlike vesicle reconstitution methods, are not yet generally applicable; they work well for some channels but not for others (6).In this paper, we report that the solubilization/reconstitution method for inserting membrane proteins into SUVs can be combined with a high-resolution electrical assay technique to study a reconstituted integral membrane ion channel-the voltage-gated Cl-channel of Torpedo electroplax. The method is shown schemati...