We have discussed in some detail a variety of experimental studies which were designed to elucidate the conformational and dynamic properties of gramicidin and alamethicin. Although the behavior of these peptides is by no means fully characterized, these studies have already permitted aspects of ion channel activity to be understood in molecular terms. Studies with gramicidin in a variety of organic solutions have revealed conformational heterogeneity of this peptide; at least five major isomers exist, several of which have been characterized in detail using NMR spectroscopy and X-ray crystallography. When added to lipid membranes gramicidin undergoes a further conformational conversion. Although the conformation of gramicidin in membranes is not as well characterized as the solution conformation(s) and an X-ray structure is not yet available, detailed data, particularly from solid-state NMR studies, continue to become available and a right-handed beta 6.3 helical conformation of the peptide backbone is now generally accepted. Two of these beta 6.3 helices joined at their N-termini are believed to form the conducting channel. The conformational behavior of the side-chains of gramicidin in the membrane-bound form is not well established and several NMR, CD, fluorescence and theoretical studies are now focussed on this. Although the side-chains do not directly contact the permeating ions, they can have distinct effects on conductance and selectivity by altering the electrostatic environment sensed by the ion. The dynamics of both side-chain and backbone conformations of gramicidin appear critical to a detailed understanding of the ion transport process in this channel. As the description of the membrane-bound conformation of gramicidin becomes more detailed, simulations of ion transport using computational methods are likely to improve and will further our understanding of the processes of ion transport. As well as internal motion of the backbone and side-chains, gramicidin undergoes rotational and translational motion in the plane of the membrane. These motions do not appear to be essential for the process of ion transport but can affect channel lifetime since lifetime is determined by the rate of association and dissociation of gramicidin monomers. Gramicidin-membrane interactions are also likely to be involved in the frequency of occurrence of channel subconductance states, the frequency of channel flickering and fundamentally in the stability of the membrane-bound gramicidin conformation. Alamethicin forms channels in membranes which are strongly voltage-dependent. The molecular origin of voltage-dependent conductances has been a fundamental problem in biophysics for many years.(ABSTRACT TRUNCATED AT 400 WORDS)

Model ion channels: gramicidin and alamethicin.

Thermodynamics of Heat Activation of Single Capsaicin Ion Channels VR1

The various details provided by physical methods have been integrated into a coherent picture of the cation transport process. The free energy profile describes simultaneously the thermodynamic and kinetic factors that govern conductance. Localization of ion binding sites near each end of the channel by NMR and X-ray crystallographic techniques demonstrates that there is an energy barrier separating the two ends. There is no a priori reason to suspect any barrier to ion entry into the channel, and recent molecular modeling computations confirm this intuition. Therefore, the channel is best described as a two-site, one-barrier channel. However, the movement of ions across the central barrier over a distance of 1.9 nm is really a multistep process best described as Brownian motion (even the short steps from one pair of carbonyls to the next is more diffusive than activated for ions as large as Na+), the entry step is probably best considered as a compound step requiring correction for interfacial polarization and diffusion limitation, the binding sites in the channel ought not be considered to be in equilibrium with the bath, and quasi-knock off behavior (binding of a second ion at the entry facilitates release of a first ion from the exit) is probably the rule at physiological permeant ion concentrations and higher. Progress will probably focus on channel kinetics. The channel backbone probably undergoes conformational changes as the ions pass which, according to solid state NMR results, may last long enough to provide the channel with a sort of memory and which may give rise to excess single channel noise. These conformational changes are further reflected in shifts in side chain positions which, in turn, may be partly responsible for changes in the single-channel lifetime, which appears to be exquisitely sensitive to side chain-lipid interactions. Reasonable explanations for the impermeance of divalent cations, anions, and medium-sized organic cations such as guanidinium have proven elusive at first, but it now appears that divalent cations bind water too tightly, anions bind water in an orientation that produces unfavorable contacts between water oxygens and peptide carbonyl oxygens at the channel entry, and iminium ions bind strongly to the flexible peptide carbonyls at the channel entry.

The use of physical methods in determining gramicidin channel structure and function.

Solvent history dependence of gramicidin A conformations in hydrated lipid bilayers.

In previous work from our laboratory, 5-10% of the channels formed by [Val1]gramicidin A have conductances that fall outside the narrow range that conventionally has defined the standard gramicidin channel [e.g., see Russell et al. (1986) Biophys. J. 49, 673]. Reports from other laboratories, however, show that up to 50% of [Val1]gramicidin channels have conductances that fall outside the range for standard channels [e.g., see Prasad et al. (1986) Biochemistry 25, 456]. This laboratory-to-laboratory variation in the distribution of gramicidin single-channel conductances suggests that the conductance variants are induced by some environmental factor(s) [Busath et al. (1987) Biophys. J. 51, 79]. In order to test whether extrinsic agents can induce such conductance heterogeneity, we examined the effects of nonionic or zwitterionic detergents upon gramicidin channel behavior. In phospholipid bilayers, detergent addition induces many changes in gramicidin channel behavior: all detergents tested increase the channel appearance rate and average duration; most detergents decrease the conductance of the standard channel; and all but one of the detergents increase the conductance heterogeneity. These results show that the conductance heterogeneity can result from environmental perturbations, thus providing a possible explanation for the laboratory-to-laboratory variation in the heterogeneity of gramicidin channels. In addition, the differential detergent effects suggest possible mechanisms by which detergents can induce the conformational perturbations that result in gramicidin single-channel conductance variations.

Induction of conductance heterogeneity in gramicidin channels.

A study of the temperature dependence of gramicidin A conductance of K+ in diphytanoyllecithin/n-decane membranes shows the plot of In (single channel conductance) as a function of reciprocal temperature to be nonlinear for the most probable set of conductance, states. These results are considered in terms of a series of barriers, of the dynamics of channel conformation,vis-a-vis the peptide libration mechanism, and of the effect of lipid viscosity on side chain motions again as affecting the energetics of peptide libration.

Temperature dependence of single channel currents and the peptide libration mechanism for ion transport through the gramicidin A transmembrane channel.

R. D. Harris

Papers

Temperature dependence of single channel currents and the peptide libration mechanism for ion transport through the gramicidin A transmembrane channel.