Photolysis of gramicidin A channels in lipid bilayers

We have tested the hypothesis that peptide tryptophan groups can control the ionic conductance of transmembrane channels. We report here that single gramicidin A channels change conductance state when the peptide tryptophans are flash photolyzed with ultraviolet light. The current flow through planar lipid bilayers containing multiple gramicidin A channels decreases irreversibly when exposed to ultraviolet light. The current-loss action spectrum peaks sharply at the 280 nm absorption maximum of the gramicidin A tryptophans. Gramicidin channel sensitivity to ultraviolet light is found to be about 20-fold higher than that of frog node sodium channels which is even more than expected based on the high tryptophan content of gramicidin. Channels which survive an ultraviolet light exposure exist in a wide variety of different low-conductance forms. The broad distribution of the single channel conductance of these partially photolyzed channels is attributable to the loss of different combinations of the dimer's normal complement of eight tryptophans per channel. Flash photolysis of single channels results in discrete conductance state changes. Partially photolyzed single channels manifest a further conductance cascade when exposed to a second flash of ultraviolet light. Analysis of the photolysis conductance turn-off process indicates that gramicidin A is a multistate electrochemical unit where the peptide tryptophan groups can modulate the flow of ions through the transmembrane channel.     

Small iminium ions block gramicidin channels in lipid bilayers.

Guanidinium and acetamidinium, when added to the bathing solution in concentrations of approximately 0.1M, cause brief blocks in the single channel potassium currents from channels formed in planar lipid bilayers by gramicidin A. Single channel lifetimes are not affected indicating that the channel structure is not modified by the blockers. Guanidinium block durations and interblock times are approximately exponential in distribution. Block frequencies increase with guanidinium concentration whereas block durations are unaffected. Increases in membrane potential cause an increase in block frequency as expected for a positively charged blocker but a decrease in block duration suggesting that the block is relieved when the blocker passes through the channel. At low pH, urea, formamide, and acetamide cause similar blocks suggesting that the protonated species of these molecules also block. Arginine and several amines do not block. This indicates that only iminium ions which are small enough to enter the channel can cause blocks in gramicidin channels.     

Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.     

Voltage-dependent block by saxitoxin of sodium channels incorporated into planar lipid bilayers.

We have previously studied single, voltage-dependent, saxitoxin-(STX) blockable sodium channels from rat brain in planar lipid bilayers, and found that channel block by STX was voltage-dependent. Here we describe the effect of voltage on the degree of block and on the kinetics of the blocking reaction. From their voltage dependence and kinetics, it was possible to distinguish single-channel current fluctuations due to blocking and unblocking of the channels by STX from those caused by intrinsic channel gating. The use of batrachotoxin (BTX) to inhibit sodium-channel inactivation allowed recordings of stationary fluctuations over extended periods of time. In a range of membrane potentials where the channels were open greater than 98% of the time, STX block was voltage-dependent, provided sufficient time was allowed to reach a steady state. Hyperpolarizing potentials favored block. Both association (blocking) and dissociation (unblocking) rate constants were voltage-dependent. The equilibrium dissociation constants computed from the association and dissociation rate constants for STX block were about the same as those determined from the steady-state fractional reduction in current. The steepness of the voltage dependence was consistent with the divalent toxin sensing 30-40% of the transmembrane potential.     

Voltage-dependent capacitance in lipid bilayers made from monolayers.

Electrocompression has been measured in lipid bilayers made by apposition of two monolayers. The capacitance C(V), as a function of membrane potential, V, was found to be well described by C(V) = C(O) [1 + alpha(V + delta psi)2] where C(O) is the capacitance at V = O, alpha is the fractional increase in capacitance per square volt, and delta psi is the surface potential difference. In lipid bilayers made from monolayers alpha has a value of 0.02 V-2, which is ca. 500-fold smaller than the value found in solvent containing membranes. In asymmetric bilayers made of one neutral and one negatively charged monolayer, delta psi values were found to be those expected from independent measurements of surface charge density. If the fractional increase in capacitance found here is a good approximation to that of biological membranes, nonlinear capacitative charge displacement derived from electrostriction is expected to be less than 1% of the total gating charge displacement found in squid axons.     

Voltage-dependent conductance induced by alamethicin-phospholipid conjugates in lipid bilayers.

Alamethicin, a linear 20-amino acid antibiotic, forms voltage-dependent channels in lipid bilayer membranes. We show here that alamethicin-phospholipid conjugates can be prepared by photolysis of unilamellar vesicles containing alamethicin and a phosphatidylcholine analogue with a carbene precursor at the end of the C-2 fatty acyl chain. This result indicates that at least a portion of the alamethicin molecule is in contact with the hydrocarbon moiety of the membrane in the absence of an applied voltage. Furthermore, the alamethicin-phospholipid photoproduct is able to induce a voltage-gated conductance similar to that of natural alamethicin. The importance of these results in terms of mechanisms for channel gating is discussed.     

The effects of gramicidin on electroporation of lipid bilayers.

The effects of the channel-forming peptide gramicidin D (gD) on the conductance and electroporation thresholds of planar bilayer lipid membranes, made of the synthetic lipid 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 200-900 mV) rectangular voltage pulses of 15 ms duration were used to perturb the bilayers and monitor the transmembrane conductance. Electroporation voltage thresholds were found, and conductance was recorded before and after electroporation. Gramicidin was added to the system in peptide/lipid ratios of 1:10, 000, 1:500, and 1:15. The addition of gD in a ratio of 1:10,000 had no effect on electroporation, but ratios of 1:500 and 1:15 significantly increased the thresholds by 16% (p < 0.0001) and 40% (p < 0.0001), respectively. Membrane conductance before electroporation was measurable only after the addition of gD and increased monotonically as the peptide/lipid ratio increased. The effect of gD on the membrane area expansivity modulus (K) was tested using giant unilamellar vesicles (GUVs). When gD was incorporated into the vesicles in a 1:15 ratio, K increased by 110%, consistent with the increase in thresholds predicted by an electromechanical model. These findings suggest that the presence of membrane proteins may affect the electroporation of lipid bilayers by changing their mechanical properties.     

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

Reconstituted lipid bilayers of dimyristoylphosphatidylcholine (DMPC) and gramicidin A' have been prepared by cosolubilizing gramicidin and DMPC in one of three organic solvent systems followed by vacuum drying and hydration. The conformational state of gramicidin as characterized by 23Na NMR, circular dichroism, and solid state 15N NMR is dependent upon the cosolubilizing solvent system. In particular, two conformational states are described; a state in which Na+ has minimal interactions with the polypeptide, referred to as a nonchannel state, and a state in which Na+ interacts very strongly with the polypeptide, referred to as the channel state. Both of these conformations are intimately associated with the hydrophobic core of the lipid bilayer. Furthermore, both of these states are stable in the bilayer at neutral pH and at a temperature above the bilayer phase transition temperature. These results with gramicidin suggest that the conformation of membrane proteins may be dictated by the conformation before membrane insertion and may be dependent upon the mechanism by which the insertion is accomplished.     

Conductance channels in neutral lipid bilayers

Summary A model channel for the conduction of ions (positive or negative but not both) through a lipid bilayer is presented. The transition-state theory is used to relate the current with voltage and ionic concentrations. Sites within the channel are considered to act cooperatively so that the ion is subjected to a ligand field in which it has complete freedom along the channel axis. The ions in the channel are treated as an ionic gas. Effects due to space-charges within the channel arising from the conducting ions are considered whereas surface-charge effects are neglected.The ionic specificity of the channel is indicated and the theory compared to that in which equilibrium free energy changes are the dominant influence.     

The sting. Melittin forms channels in lipid bilayers

Melittin, a toxin of bee venom, is a cationic polypeptide composed of 26 amino acids. The six residues of the C-terminal end are polar and 19 of the 20 residues of the N-terminal end are hydrophobic. Exposure of the lecithin bilayer to melittin results in the formation of channels that are more permeable to anions that to cations. Unilateral addition of melittin produces a voltage-dependent increase in membrane conductance when the side where the polypeptide is present in made positive but not when it is made negative. At a fixed voltage, the conductance increases with the fourth power of the melittin concentration in the aqueous phase. At a fixed peptide concentration, the conductance increases approximately e-fold per 6-mV increase in the electrical potential difference across the membrane. These results suggest that four melittin monomers are needed to form a channel and, furthermore, that a minimum of four equivalent electronic charges need to be displaced by the electrical field to explain the voltage dependence of the conductance.     

Delta-endotoxins form cation-selective channels in planar lipid bilayers.

Delta-endotoxins CryIA(c) and CryIIIA, two members of a large family of toxic proteins from Bacillus thuringiensis, were each allowed to interact with planar lipid bilayers and were analyzed for their ability to form ion-conducting channels. Both of these toxins made clearly resolved channels in the membranes and exhibited several conductance states, which ranged from 200 pS to about 4000 pS (in 300 mM KCl). The channels formed by both toxins were highly cation-selective, but not ideally so. The permeability ratio of K+ to Cl- was about 25 for both channels. The ability of these proteins to form such channels may account for their toxic action on sensitive cells, and suggests that this family of toxins may act by a common mechanism.     

Optimizing and characterizing alignment of oriented lipid bilayers containing gramicidin D.

31P NMR spectroscopy and optical microscopy have been used to characterize samples of gramicidin D in oriented lipid bilayers. Correlations have been made between the defect structures observed under crossed polarizers by optical microscopy and characteristic features of 31P NMR spectra. The sample preparation protocol has been improved using these techniques to achieve minimal dispersion of the bilayer normal and minimal amounts of unoriented sample. The molar ratio of gramicidin to dimyristoyl-phosphatidylcholine, the extent of hydration, and the cosolubilizing solvent system were used as the protocol variables. While hydration level and solvent system had profound effects on the sample orientation the molar ratio did not. However, the 31P chemical shift anisotropy is very sensitive to the molar ratio and can be used as an in situ method for determining the molar ratio.     

Theoretical analysis of hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

We present a quantitative analysis of the effects of hydrophobic matching and membrane-mediated protein-protein interactions exhibited by gramicidin embedded in dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) bilayers (Harroun et al., 1999. Biophys. J. 76:937-945). Incorporating gramicidin, at 1:10 peptide/lipid molar ratio, decreases the phosphate-to-phosphate (PtP) peak separation in the DMPC bilayer from 35.3 A without gramicidin to 32.7 A. In contrast, the same molar ratio of gramicidin in DLPC increases the PtP from 30.8 A to 32.1 A. Concurrently, x-ray in-plane scattering showed that the most probable nearest-neighbor separation between gramicidin channels was 26.8 A in DLPC, but reduced to 23.3 A in DMPC. In this paper we review the idea of hydrophobic matching in which the lipid bilayer deforms to match the hydrophobic surface of the embedded proteins. We use a simple elasticity theory, including thickness compression, tension, and splay terms to describe the membrane deformation. The energy of membrane deformation is compared with the energy cost of hydrophobic mismatch. We discuss the boundary conditions between a gramicidin channel and the lipid bilayer. We used a numerical method to solve the problem of membrane deformation profile in the presence of a high density of gramicidin channels and ran computer simulations of 81 gramicidin channels to find the equilibrium distributions of the channels in the plane of the bilayer. The simulations contain four parameters: bilayer thickness compressibility 1/B, bilayer bending rigidity Kc, the channel-bilayer mismatch Do, and the slope of the interface at the lipid-protein boundary s. B, Kc, and Do were experimentally measured; the only free parameter is s. The value of s is determined by the requirement that the theory produces the experimental values of bilayer thinning by gramicidin and the shift in the peak position of the in-plane scattering due to membrane-mediated channel-channel interactions. We show that both hydrophobic matching and membrane-mediated interactions can be understood by the simple elasticity theory.     

The dependence of the conductance and lifetime of gramicidin channels on the thickness and tension of lipid bilayers

The lifetimes of channels formed by natural gramicidin and its dimeric analog in monoglyceride lipid bilayers of various compositions were investigated. The bilayer surface tension was altered by changing the length of the monoglycerides' fatty acid chain or the chain length of hydrocarbon solvent by isomerization or saturation of the lipid, by varying the amount of solvent in the bilayer, and by changing the salt composition of the aqueous solutions. The logarithms of mean channel lifetimes were found to be proportional to the surface tension of the membrane irrespective of how the surface tension was changed. In contrast, no simple relationship between channel conductance and surface tension or bilayer thickness was found.     

Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers.

Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two populations of conductive events, one in the 0.1-0.5 pA range, and one in the 1.0-5.0 pA range, whereas nearly all events caused by poly-L-alanine were in the 0.1-0.5 pA range at an applied voltage of +60 mV. The channel-like activity appeared to switch between conductive and nonconductive states, with most open-times in the range of 50-200 ms. We conclude that hydrophobic polyamino acids produce proton-conducting defects in lipid bilayers that may be used to model functional proton channels in biological membranes.     

Experimental evidence for hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

Hydrophobic matching, in which transmembrane proteins cause the surrounding lipid bilayer to adjust its hydrocarbon thickness to match the length of the hydrophobic surface of the protein, is a commonly accepted idea in membrane biophysics. To test this idea, gramicidin (gD) was embedded in 1, 2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1, 2-myristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at the peptide/lipid molar ratio of 1:10. Circular dichroism (CD) was measured to ensure that the gramicidin was in the beta6.3 helix form. The bilayer thickness (the phosphate-to-phosphate distance, or PtP) was measured by x-ray lamellar diffraction. In the Lalpha phase near full hydration, PtP is 30.8 A for pure DLPC, 32.1 A for the DLPC/gD mixture, 35.3 A for pure DMPC, and 32.7 A for the DMPC/gD mixture. Gramicidin apparently stretches DLPC and thins DMPC toward a common thickness as expected by hydrophobic matching. Concurrently, gramicidin-gramicidin correlations were measured by x-ray in-plane scattering. In the fluid phase, the gramicidin-gramicidin nearest-neighbor separation is 26.8 A in DLPC, but shortens to 23.3 A in DMPC. These experiments confirm the conjecture that when proteins are embedded in a membrane, hydrophobic matching creates a strain field in the lipid bilayer that in turn gives rise to a membrane-mediated attractive potential between proteins.     

Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers.

Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.     

Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers.

Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA dimers. g(max) and K(D), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its dimer. In GMO bilayers, however, proton affinities of gA and the dioxolane-dimer were significantly lower (K(D) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and 2150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers.     

Reconstitution of vacuolar ion channels into planar lipid bilayers.

Vacuolar ion channels were characterized after reconstitution into planar lipid bilayers. (1) Channel activity was observed after incorporation of tonoplast-enriched microsomal membranes, purified tonoplast membranes or of solubilized tonoplast proteins. (2) Channels of varying single-channel conductances were detected after reconstitution. In symmetrical 100 mmol l-1 KCl, conductances between 1 and 110 pS were frequently measured; the largest number of independent reconstitution events was seen for single-channel conductances of 16-25 pS (28 experiments), 30-42 pS (26), 49-56 pS (15) and 64-81 pS (15). Channel current usually increased linearly with voltage. (3) In asymmetrical solutions, cation-, non-selective and, for the first time for the tonoplast, anion-selective channels were detected. Ca(2+)-dependent regulation of channel opening was not observed in our reconstitution system. (4) Permeability was also observed for Cl-, NO3-, SO4(2-) and phosphate. (5) After fractionation of tonoplast proteins by size exclusion chromatography, ion channel activity was recovered in specific fractions. (6) Some of these fractions catalyzed sulfate transport after reconstitution into liposomes. The results suggest that different channels are active at the tonoplast membrane at a larger number than has been concluded from previous work.