The structure of the gramicidin A transmembrane channel

Abstract

Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across lipid bilayer membranes by forming channels. It has been proposed that the conducting channel is a dimer which is in equilibrium with nonconducting monomers in the membrane. To directly test this model in several independent ways, we have prepared and purified a series of gramicidin C derivatives. All of these derivatives are fully active analogs of gramicidin A, and each derivative has a useful chromophore esterified to the phenolic hydroxyl of tyrosine #11.Simultaneous conductance and fluorescence measurements on planar lipid bilayer membranes containing dansyl gramicidin C yielded four conclusions: 1) A plot of the logarithm of the membrane conductance versus the logarithm of the membrane fluorescence had a slope of 2.0 * 0.3, over a concentration range for which nearly all the gramicidin was monomeric. Hence, the active channel is a dimer of the nonconducting species. 2) In a membrane in which nearly all of the gramicidin was dimeric, the number of channels was approximately equal to the number of dimers. Thus, most dimers are active channels and so it should be feasible to carry out spectroscopic studies of the conformation of the transmembrane channel.3) The association constant for dimerization is more than 1,000-fold larger in a glycerolester membrane with 26 &hydrocarbon thickness than in a 47 &glycerolester membrane. The dimerization constant in a 48 a-phosphatidyl choline membrane was 200 times larger than in a 47 &glycerolester membrane, showing that it depends on the type of lipid as well as on the thickness of the hydrocarbon core. 4) We were readily able to detect 1 O-I4 mole cmP2 of dansyl gramicidin C in a bilayer membrane, which corresponds to 60 fluorescent molecules per square pm. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates and receptors in planar bilayer membranes.An alternative method of determining the number of molecules of gramicidin in the channel is to measure the fraction of hybrid channels present in a mixture of 2 chemically different gramicidins. The single-channel conductance of p-phenylazobenzene-sulfonyl ester gramicidin C (PABS gramicidin C) was found to be 0.68 that of gramicidin A. In membranes containing a mixture of these 2 gramicidins, a hybrid channel was evident in addition to 2 pure channels. The hybrid channel conductance was 0.82 that of gramicidin A. Fluorescence energy transfer from dansyl gramicidin C to diethylamino-phenylazobenzene-sulfonyl ester gramicidin C (DPBS gramicidin C), provided an independent way to measure the fraction of hybrid channels on liposomes. For both techniques the fraction of hybrid channels was found to be 2ad where a2 and d2 were the fractions of the 2 kinds of pure channels. This result strongly supports a dimer channel and the hybrid data excludes the possibility of a William Veatch is now at the Department of Pharmacology, Harvard Medical School, Boston...

Formation of ion channels by a negatively charged analog of gramicidin a

“…A likely explanation of this intermediate peak is that it results from hybrid pores which are formed by association of a gramicidin monomer with a O-pyromellitylgramicidin monomer. Such hybrid pores have also been observed by W. Veatch (1976) and with a chemically modified gramicidin C. The finding that a mixture of two different gramicidin species yields, in addition to channels corresponding to the pure compounds, a single probability peak at intermediate conductances further supports the dimer model of the gramicidin channel.…”

Pressure effects on alamethicin conductance in bilayer membranes

“…It is well established that the conducting unit in alamethicin-doped membranes is an oligomer containing on the order of 10 monomeric units. This clearly represents a more complex situation than, for example, that of gramicidin (Veatch, 1976), where the conducting unit is known to be a dimer. In choosing alamethicin for this initial study we have reasoned that, precisely because of this greater complexity, larger activation and/or reaction volumes would be likely, leading to more readily resolved pressure effects.…”