Summary. Pore formation of alamethicin has been studied by the analysis of steadystate fluctuations of single-pore conductances. An aggregation model is proposed where transitions to the next higher or lower pore state occur by uptake or release of one monomer. It is assumed that alamethicin forms an elongated loop in the bilayer. The main voltage-dependent step is the insertion of this monomer into the membrane after complexation with a cation. This mechanism is equivalent to dipole orientation in an electric field. Pore formation is restricted by the energy required to enlarge the channel in the membrane.Since Mueller and Rudin (1968) reported on action potentials and oscillations induced by alamethicin in black lipid membranes, considerable progress has been made in examining the properties of this macrocyclic polypeptide antibiotic. The observation of discrete conductance steps has given evidence that certain substances are able to create ion-permeable pores in lipid bilayers. These pores open and close in a statistically random manner. Bean, Shepherd, Chan and Eichner (1969) and Ehrenstein, Lecar and Nossal (1970) described the pore formation properties of excitability inducing material (EIM). Hladky and Haydon (1970) demonstrated conductance fluctuations induced by gramicidin A in black lipid films. Alamethicin has been shown by Gordon and Haydon (1972) and in an extensive report by Eisenberg, Hall and Mead (1973) to be a pore former, too. In contrast to EIM and gramicidin A, which mainly show fluctuations between two conductance states, the alamethicin pore exhibits up to five conductance levels in bacterial phosphatidylethanolamine-n-decane membranes (Eisenberg et al., 1973) and up to seven levels in glyceryl monooleaten-decane membranes (Gordon & Haydon, 1972).While the primary structure of EIM is not yet known, a series of ~LD-helices for gramicidin A dimer pores has been proposed by Urry (1971) and 18.

Single calcium dependent potassium channels from cultured rat myoballs have been studied with the patch clamp technique, and current records subjected to statistical analysis. From the dependence of the mean open state probability on the internal calcium concentration, two calcium ions are required to open the channel. The open state and closed state lifetime distributions reveal that the usual activation model is not applicable to these channels. They are consistent with a two step gating mechanism that involves both activation by calcium and blockade by a calcium-sensitive gate.

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The electrical properties of an alamethicin multi-pore system have been studied by voltage-jump current-relaxation experiments (this paper) and by autocorrelation and spectral analysis (following paper). With these methods a slow time constant and a fast time constant were observed which differ by about one to three orders of magnitude depending on the experimental conditions. Steady-state current and time constants were analyzed as functions of voltage, alamethicin concentration and temperature. Within experimental error the data obtained with these different methods are in good agreement. The experimentally measured relation between the voltage and alamethicin concentration dependence of the slow relaxation time fits into a model of an alamethicin pore which adopts consecutive pore states and which decays only from the lowest state. It indicates that the uptake of one alamethicin molecule by the existing pore and, in formal equivalence, the transfer of about one positive elementary charge across the membrane are associated with the transition from a given pore conductance state to the next higher state. From the voltage and alamethicin concentration dependence of the pore formation rate evidence shows that a hexameric preaggregate exists at the membrane interface out of which two to three molecules are simultaneously inserted into the membrane to form the pore nucleus. The effects of different voltage pretreatment on the experimentally determined parameters have been investigated and are discussed in detail.Alamethicin has been shown by Mueller and Rudin (1968), Baumann and Mueller (1974) and Mueller (1976a, b) to be a model substance which induces excitability phenomena in artificial lipid membranes. A way to elucidate the underlying molecular mechanism of alamethicin action has been opened through single channel investigations by Gordon and Haydon (1972)

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.