Temperature-dependent properties of gramicidin A channels

doi:10.1016/0005-2736(74)90037-6

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 367, Issue 2, 29 October 1974, Pages 127-133

https://doi.org/10.1016/0005-2736(74)90037-6 Get rights and content

Abstract

The temperature dependence of the mean life-time and of the conductance Λ of single ion channels induced by gramicidin A in lipid bilayer membranes has been measured. In a second series of experiments, the rate constants of formation (k_R) and dissociation (k_D) of the conducting channel have been determined at different temperatures from electrical relaxation experiments. It has been found that the channel formation proceeds according to a second-order reaction in the whole temperature range (10–40 °C). Furthermore, the mean life-time of the channel approximately agreed with the reciprocal of the dissociation rate constant. Both findings are consistent with the view that the channel consists of a dimer of gramicidin A. From the temperature dependence of Λ, k_R, and k_D the activation energies of ion migration through the channel (E_Λ) as well as the activation energy of formation (E_R) and dissociation (E_D) of the dimer may be calculated. The magnitude of E_D (17 kcal/mole) is consistent with the assumption that dissociation involves the breakage of several hydrogen bonds. The value of E_R (20 kcal/mole) is tentatively explained by the energy required for the structural rearrangement of the lipid matrix in the vicinity of the channel.

Gramicidin A, a linear, hydrophobic pentadecapeptide isolated from Bacillus brevis, creates cation-selective channels in lipid bilayer membranes [1–12]. The structure of the channel, as proposed by Urry [13–15], consists of a helix which is formed by head-to-head association of two gramicidin A monomers in the membrane. The β⁶_3,3-helix has in its center a 4 · 10⁻² μm wide tunnel which is lined with the oxygen atoms of the amide carbonyls. The dimerisation hypothesis is supported by the finding that the covalent dimer which is formed by chemical linkage of two monomers at the formyl ends is even more effective in inducing ion permeability than normal gramicidin A [7, 11]. Further evidence for the dimer mechanism was obtained recently by studying the kinetics of channel formation by electrical relaxation experiments [12]. The results of the relaxation measurements indicate that the channel formation is a second-order reaction; furthermore, they allow a numerical evaluation of the rate constants of formation (k_R) and dissociation (k_D) of the dimer. The aim of the present study was to obtain some insight into the thermodynamics of channel formation. For this purpose relaxation experiments as well as single-channel conductance measurements were performed at different temperatures. From the experimental results the activation energies for the formation and dissociation of the dimer, the activation energy for the migration of the ion through the channel, and the enthalpy of dimerisation may be obtained.

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Cited by (111)

Molecular Mechanism for Gramicidin Dimerization and Dissociation in Bilayers of Different Thickness
2019, Biophysical Journal
Citation Excerpt :
The bilayer-spanning gramicidin channel forms by transmembrane dimerization of two nonconducting monomers (1,2), and the antiparallel dimer structure is stabilized by six hydrogen bonds at the formyl-N-termini (3–5). Each hydrogen bond contributes ∼4 kBT to the channel stability (6–9), indicating that gramicidin channel dissociation occurs on a timescale far beyond microseconds. The measured gramicidin channel lifetimes are sensitive to the lipid bilayer thickness: dependent on the lipid composition, the channel lifetimes may vary by an order of magnitude in response to subnanometer changes of lipid bilayer thickness (10,11).
Membrane protein functions can be altered by subtle changes in the host lipid bilayer physical properties. Gramicidin channels have emerged as a powerful system for elucidating the underlying mechanisms of membrane protein function regulation through changes in bilayer properties, which are reflected in the thermodynamic equilibrium distribution between nonconducting gramicidin monomers and conducting bilayer-spanning dimers. To improve our understanding of how subtle changes in bilayer thickness alter the gramicidin monomer and dimer distributions, we performed extensive atomistic molecular dynamics simulations and fluorescence-quenching experiments on gramicidin A (gA). The free-energy calculations predicted a nonlinear coupling between the bilayer thickness and channel formation. The energetic barrier inhibiting gA channel formation was sharply increased in the thickest bilayer (1,2-dierucoyl-sn-glycero-3-phosphocholine). This prediction was corroborated by experimental results on gramicidin channel activity in bilayers of different thickness. To further explore the mechanism of channel formation, we performed extensive unbiased molecular dynamics simulations, which allowed us to observe spontaneous gA dimer formation in lipid bilayers. The simulations revealed structural rearrangements in the gA subunits and changes in lipid packing, as well as water reorganization, that occur during the dimerization process. Together, the simulations and experiments provide new, to our knowledge, insights into the process and mechanism of gramicidin channel formation, as a prototypical example of the bilayer regulation of membrane protein function.
pH-Dependent properties of ion channels formed by N-terminally glutamate substituted gramicidin A in planar lipid bilayers
2017, Biochimica et Biophysica Acta - Biomembranes
The N-terminally glutamate substituted analogue of the pentadecapeptide gramicidin A [Glu1]gA has been previously described as a low-toxic uncoupler of mitochondrial oxidative phosphorylation and neuroprotector. Here, we studied ion channel-forming activity of this peptide in planar bilayer lipid membranes (BLMs). [Glu1]gA exhibited an ability to induce both macroscopic current and single channels in a broad pH range, albeit with a lower potency than the parent gramicidin A (gA). Single-channel recordings in 1 M KCl at pH about 4 showed channel openings of one type with the conductance (about 26 pS), similar to that of gA, and the lifetime (40 ms), much shorter than that of gA. By contrast, two populations of channels were found at pH 9, one of which had much longer duration (several seconds) and lower conductance (3.5–10 pS). Autocorrelation function of the current noise of [Glu1]gA revealed a marked shift towards longer correlation times upon alkalinization. The sensitized photoinactivation technique also revealed substantial differences in [Glu1]gA conducting properties at alkaline and acidic pH, in particular deceleration of the photoinactivation kinetics and a sharp decrease in its amplitude upon alkalinization. A double-logarithmic plot of the concentration dependence of [Glu1]gA-induced BLM conductance had the slope of about 3, which pointed to peptide aggregation in the membrane. The data were discussed in relation to pH-dependent aggregation of [Glu1]gA, resulting from deprotonation of the glutamate side chain at alkaline pH.
Lipid bilayer technologies in ion channel recordings and their potential in drug screening assay
2013, Sensors and Actuators, B: Chemical
Citation Excerpt :
Most of the LOC devices developed for lipid bilayer formation use the pore forming antibiotic gramicidin or bacterial toxin α-haemolysin to demonstrate proof-of-concept [46,51,56,57,60,66,75,77–80]. Gramicidin and α-haemolysin are usually preferred because of their well characterized ion channel functions [86–89]. In addition, insertion of these proteins into lipid bilayer membranes is relatively easier as compared to eukaryotic ion channels.
Ion channels are expressed in every tissue and are important for many physiological processes. Malfunction of ion channels can lead to diseases in many tissues. All these make ion channels ideal targets for drug development. Many techniques are employed to study ion channel functions including the lipid bilayer technique. Lipid bilayer membranes are being synthesized in vitro to mimic natural cell membranes. Ion channels are then incorporated into the bilayer membrane for ion channels activity recordings. The use of Lab-on-a-Chip (LOC) technologies to form lipid bilayer has made lipid bilayer technique efficient for ion channel recordings and has brought great potential for this technique to be scaled up to a high throughput platform for ion channel drug screening. In this review we discuss the use of lipid bilayer for ion channel studies, conventional methods and current LOC technologies on lipid bilayer formations, comparison of lipid bilayer high throughput platform to automated patch clamp system, and finally the potential of lipid bilayer platforms in drug screening assay.
The Gramicidin Dimer Shows Both EX1 and EX2 Mechanisms of H/D Exchange
2009, Journal of the American Society for Mass Spectrometry
We describe the use of H/D amide exchange and electrospray ionization mass spectrometry to study, in organic solvents, the pentadecapeptide gramicidin as a model for protein self association. In methanol-OD, all active H's in the peptide exchange for D within 5 min, indicating a monomer/dimer equilibrium that is shifted towards the fast-exchanging monomer. H/D exchange in n-propanol-OD, however, showed a partially protected gramicidin that slowly converts to a second species that exchanges nearly all the active hydrogens, indicating EX1 kinetics for the H/D exchange. We propose that this behavior is the result of the slower rate of unfolding in n-propanol compared with that in methanol. The rate constant for the unfolding of the dimer is the rate of disappearance of the partially protected species, and it agrees within a factor of two with a value reported in literature. The rate constant of dimer refolding can be determined from the ratio of the rate constant for unfolding and the affinity constant for the dimer, which we determined in an earlier study. The unfolding activation energy is 20 kcal mol⁻¹, determined by performing the exchange experiments as a function of temperature. To study gramicidin in an even more hydrophobic medium than n-propanol, we measured its H/D exchange kinetics in a phospholipids vesicle and found a different H/D amide exchange behavior. Gramicidin is an unusual peptide dimer that can exhibit both EX1 and EX2 mechanisms for its H/D exchange, depending on the solvent.
Fluorometric immunoassay based on pH-sensitive dye-encapsulating liposomes and gramicidin channels
2007, Analytical Biochemistry
Citation Excerpt :
Consequently, the small amount of gramicidin in an open state and/or the use of multilamellar liposomes are responsible for the slow attainment of the steady fluorescence intensity. The gramicidin channel is permeable to monovalent cations, such as H+ and Na+, but is not permeable to divalent cations [38,42]. The effect of monovalent and divalent cations on fluorescence of BCECF (1.0 mM) entrapped in biotin–liposomes at pH 5.5 was investigated.
This article describes a new method for direct fluorometric immunoassay with a liposome array using pH-sensitive dye (BCECF [2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein])-encapsulating liposomes immobilized on an avidin slip and gramicidin channels. The liposomes were composed of phosphatidylcholine (PC), cholesterol (Chol), biotinylated phosphatidylethanolamine (B-cap-PE), and recognition sites (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(2,4-dinitrophenyl) [DNP-PE], Fab′ fragment of anti-substance P, and Fab′ of anti-neurokinin A). The addition of gramicidin induced release of H⁺ ions from the inner solution (pH 5.5) to the outer one (pH 7.8), enhancing fluorescence of BCECF (1.0 mM) encapsulated in liposome. The binding of an analyte (anti-dinitrophenyl [anti-DNP], avidin, substance P, or neurokinin A) to the membrane-bound recognition sites caused further enhancement of fluorescence of BCECF due to a local distortion of the bilayer structure that affects the channel kinetics of gramicidin. The intensity of fluorescence from the immobilized liposomes 60 min after the addition of gramicidin (10 ng/ml) increased with an increase in the concentration of anti-DNP ranging from 1.2 × 10⁻⁸ to 1.2 × 10⁻⁶ g/ml, avidin ranging from 1.0 × 10⁻⁸ to 1.0 × 10⁻⁶ g/ml, substance P ranging from 1.0 × 10⁻⁸ to 1.0 × 10⁻⁶ g/ml, and neurokinin A ranging from 1.0 × 10⁻⁸ to 1.0 × 10⁻⁶ g/ml. The direct fluorometric immunoassay with a liposome array is simple and easy to carry out. The intensity of fluorescence emitted from the immobilized liposomes is directly measured after incubation with a sample solution and a gramicidin solution in sequence without washing steps. The assay allows simultaneous quantification of multiple components without labeling of antibody or antigen with a fluorescent tag. The liposome-based assay is discussed in terms of principle, sensitivity, and selectivity.
Sensitized photoinactivation of minigramicidin channels in bilayer lipid membranes
2007, Biochimica et Biophysica Acta - Biomembranes
The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dimer of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double β^5.7-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded β^6.3-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer.

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Temperature-dependent properties of gramicidin A channels

Abstract

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

Biochim. Biophys. Acta