Freezing and Melting of Lipid Bilayers and the Mode of Action of Nonactin, Valinomycin, and Gramicidin

Krasne, Sally; Eisenman, George; Szabó, Gábor

doi:10.1126/science.174.4007.412

Cited by 310 publications

(115 citation statements)

References 27 publications

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“…5, the effect of nigericin also decreased as the incubation temperature was lowered. This was consistent with previous findings in this and other experimental systems (Krasne et al, 1971 ;Krulwich et al, 1978;Racker & Hinkle, 1974).…”

Section: Eflect Of External P H On Nigericin-induced Deathsupporting

confidence: 83%

Nigericin-induced Death of an Acidophilic Bacterium

Guffanti

Davidson

Mann

et al. 1979

Journal of General Microbiology

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201At an external pH of 3.5, nigericin (which catalyses an electroneutral H+/K+ exchange) abolished the transmembrane proton gradient (ApH) of Bacillus acidocaldarius, causing a rapid acidification of the cytoplasm from approximately pH 6.0 to pH 3.5. A pronounced loss of viability and fine-structural changes rapidly followed treatment with nigericin. A marked decline in respiration and an even more rapid decrease in cytoplasmic ATP were observed. Activity of at least one cytoplasmic enzyme decreased more slowly. There was no generalized loss in the integrity of the cytoplasmic membrane, as assayed by permeability to inulin or Na+ or by release of ultraviolet light-absorbing compounds. The loss of viability upon treatment with carbonyl cyanide m-chlorophenylhydrazone was similar to that observed with nigericin, so proton influx alone, rather than together with K+ efflux, was probably involved in the death of the organism. Moreover, acidification of the cytoplasm rather than abolition of the ApH was the lethal event, since no loss of viability was observed when the ApH was abolished by elevation of the external pH. I N T R O D U C T I O NIn accord with Mitchell's chemiosmotic hypothesis (196 1, 1963), bacteria generate, via respiration and/or ATP hydrolysis, a protonmotive force generally consisting of a transmembrane pH gradient (ApH, exterior acid) and a transmembrane electrical potential (A@, exterior positive). The protonmotive force, in turn, energizes a variety of energyrequiring processes, as reviewed by Harold (1977). When we became interested in the protonmotive force in acidophilic bacteria, it was with the expectation that such organisms would maintain extremely large pH gradients across the membrane. Thus, bacteria which could grow at external pH values of 2 to 3 would presumably require cytoplasmic pH values much closer to neutrality in order to maintain viability. The consistently large ApH resulting from this requirement would, moreover, probably preclude the existence of any appreciable A~, exterior positive. In fact, the occurrence of a 'reversed' A+, i.e. interior positive, seemed a likely adaptation in such organisms.Our own studies with Bacillus acidocaldarius (Krulwich et al., 1978) and those of others with different acidophiles (Hsung & Haug, 1975, 1977Searcy, 1976) have indeed shown that such organisms maintain a cytoplasmic pH of greater than 5, thus generating large ApH values, and concomitantly exhibiting reversed A+ values. The assumption that organisms would need to maintain cytoplasmic pH values much closer to neutrality than a highly acidic milieu appeared validated. Thus, if the large ApH in an acidophile such as B. acidocaldarius were abolished so that rapid acidification of the cytoplasm occurred, viability of the organism would be threatened. In the study presented here, nigericin was used to abolish the ApH in B. acidocazdarius and the ensuing death of the organism was monitored and characterized. The results indicated that the constraints imposed by a lower cytoplasmic pH limit...

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Section: Eflect Of External P H On Nigericin-induced Deathsupporting

confidence: 83%

Nigericin-induced Death of an Acidophilic Bacterium

Guffanti

Davidson

Mann

et al. 1979

Journal of General Microbiology

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“…In going beyond the temperature range accessible experimentally to Krasne et al (6), we also observed freezing effects with the gramicidininduced membrane conductance (Fig. 2A).…”

mentioning

confidence: 56%

“…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.…”

mentioning

confidence: 92%

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

Boheim¹,

Hanke²,

Eibl³

1980

Proc. Natl. Acad. Sci. U.S.A.

127

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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.

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“…That biologically relevant transport processes can occur in rigid systems has recently been shown by Krasne et al [71], where it was demonstrated that the ion translocating antibiotic, gramicidin, was able to mediate potassium ion transport in both "solid" as well as "liquid" black lipid membranes.…”

Section: X-raymentioning

confidence: 89%