Gating processes of channels induced by colicin A,its C-terminal fragment and colicin E1 in planar lipid bilayers

The dependence on pH and membrane potential of the pore formed by colicin A and its C-terminal 20 kDa fragment has been measured using planar lipid bilayers. The single channel conductance of the pore formed by both colicin A and the fragment increases with pH with an apparent pK of 6.0. At pH 5.0 the gating by membrane potential of the channels formed by either colicin A or its fragment is identical. At the same pH, quite similar pore properties were found when using the related bacteriocin, colicin E₁. In agreement with previous studies, these data indicate that the protein structure containing the lumen of the pore resides in the 20 kDa C-terminal part of the colicin A and favours the recently proposed model, based on protein sequence analysis, which proposes that colicin A, E₁ and I_B C-terminal domains are folded in the same three-dimensional structure. However, it is also shown that colicin A and not its C-terminal fragment undergoes a pH dependent transition between an acidic and a basic form of the pore with an apparent pK of 5.3. The two forms of the pore differ by their gating charge but not by the channel size. These results suggest that there is a pH dependent association between the C-terminal domain carrying the lumen of the pore and another domain of the molecule which affect the pore sensitivity to membrane potential.     

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A.

Synthetic peptides patterned after the predicted transmembrane sequence of botulinum toxin A were used as tools to identify an ion channel-forming motif. A peptide denoted BoTxATM, with the sequence GAVILLEFIPEIAI PVLGTFALV, forms cation-selective channels when reconstituted in planar lipid bilayers. As predicted, the self-assembled conductive oligomers express heterogeneous single-channel conductances. The most frequent openings exhibit single-channel conductance of 12 and 7 pS in 0.5 M NaCl, and 29 and 9 pS in 0.5 M KCl. In contrast, ion channels are not formed by a peptide of the same amino acid composition as BoTxATM with a scrambled sequence. Conformational energy calculations show that a bundle of four amphipathic alpha-helices is a plausible structural motif underlying the measured pore properties. These studies suggest that the identified module may play a functional role in the ion channel-forming activity of intact botulinum toxin A.     

Toad bladder amiloride-sensitive channels reconstituted into planar lipid bilayers

In the present study we used established methods to obtain apical membrane vesicles from the toad urinary bladder and incorporated these membrane fragments to solvent-free planar lipid bilayer membranes. This resulted in the appearance of a macroscopic conductance highly sensitive to the diuretic amiloride added to the cis side. The blockage is voltage dependent and well described by a model which assumes that the drug binds to sites in the channel lumen. This binding site is localized at about 15% of the electric field across the membrane. The apparent inhibition constant (K(0)) is equal to 0.98 microM. Ca2+, in the micromolar range on the cis side, is a potent blocker of this conductance. The effect of the divalent has a complex voltage dependence and is modulated by pH. At the unitary level we have found two distinct amiloride-blockable channels with conductances of 160 pS (more frequent) and 120 pS. In the absence of the drug the mean open time is around 0.5 sec for both channels and is not dependent on voltage. The channels are cation selective (PNa/PCl = 15) and poorly discriminate between Na+ and K+ (PNa/PK = 2). Amiloride decreases the lifetime in the open state of both channels and also the conductance of the 160-pS channel.     

Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

Plasma membrane was purified from roots of rye (Secale cereale L. cv. Rheidol) by aqueous-polymer two-phase partitioning and incorporated into planar bilayers of 1-palmitoyl-2-oleoyl phosphatidylethanolamine by stirring with an osmotic gradient. Since plasmamembrane vesicles were predominantly oriented with their cytoplasmic face internal, when fused to the bilayer the cytoplasmic side of channels faced the trans chamber. In asymmetrical (cis:trans) 280100 mM KCl, five distinct K⁺-selective channels were detected with mean chord-conductances (between +30 and -30 mV; volyages cis with respect to trans) of 500 pS, 194 pS, 49 pS, 21 pS and 10 pS. The frequencies of incorporation of these K⁺ channels into the bilayer were 48, 21, 50, 10 and 9%, in the order given (data from 159 bilayers). Only the 49 pS channel was characterized further in this paper, but the remarkable diversity of K⁺ channels found in this preparation is noteworthy and is the subject of further study. In symmetrical KCl solutions, the 49 pS channel exhibited non-ohmic unitary-current/voltage relationships. The chord-conductance (between +30 and-30 mV) of the channel in symmetrical 100 mM KCl was 39 pS. The unitary current was greater at positive voltages than at corresponding negative voltages and showed considerable rectification with increasing positive and negative voltages. This would represent inward rectification in vivo. Gating of the channel was not voltage-dependent and the channel was open for approx. 80% of the time. Presumably this is not the case in vivo, but we are at present uncertain of the in vivo controls of channel gating. The distribution of channel-open times could be approximated by the sum of two negative exponential functions, yielding two open-state time constants (_o, the apparent mean lifetime of the channel-open state) of 1.0 ms and 5.7 s. The distribution of channel-closed times was best approximated by the sum of three negative exponential functions, yielding time constants (_c, the apparent mean lifetime of the channel-closed state) of 1.1 ms, 51 ms and 11 s. This indicates at least a five-state kinetic model for the activity of the channel. The selectivity of the 49 pS channel, determined from both reversal potentials under biionic conditions (100 mM KCl100 mM cation chloride) and from conductance measurements in symmetrical 100 mM cation chloride, was Rb⁺ K⁺ > Cs⁺ > Na⁺ > Li⁺ > tetraethylammonium (TEA⁺). The 49 pS channel was reversibly inhibited by quinine (1 mM) but TEA⁺ (10 mM), Ba²⁺ (3 mM), Ca²⁺ (1 mM), 4-aminopyridine (1 mM) and charybdotoxin (3 M) were without effect when applied to the extracellular (cis) surface.Abbreviations and Symbols GHK Goldman-Hodgkin-Katz - I/V current/voltage - PEG polyethyleneglycol - P_o probability o f the channel being open - TEA⁺ tetraethylammonium - _c apparent mean lifetime of the channel-closed state - _o apparent mean lifetime of the channel-open state P.J.W. was supported by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Professor E.A.C. MacRobbie and M.T. by the Glaxo Junior Research Fellowship at Churchill College, Cambridge. We thank Dr. D.T. Cooke (AFRC, Long Ashton Research Station, University of Bristol, UK) and Ms. J. Marshall (University of York, UK) for their advice and assistance with the aqueous-polymer two-phase partitioning of plasma membrane from rye roots, Mr. J. Banfield and Miss P. Parmar (University of Cambridge, UK) for technical assistance and Professor E.A.C. MacRobbie, Dr. G. Thiel (University of Cambridge, UK), Dr. M.R. Blatt (Wye College, University of London, UK), Dr. D. Sanders and Dr. E. Johannes (University of York, UK) for helpful discussions.     

Properties of single- and double-barreled Cl channels of shark rectal gland in planar bilayers

Chloride channels from the apical plasma membrane fraction of rectal gland of Squalus acanthias were characterized by incorporation into planar bilayers in the presence of cAMP-PK/ATP. In a total of 80 bilayer preparations, 21 Cl-selective channels were observed as single channels and 13 as pairs. This was a significantly greater number of double Cl channels than expected from a binomial distribution. The double Cl channels were divided into two groups based on kinetic and voltage-dependent behavior. One group had properties identical to the single channels (gb1) while the other was consistent with a double-barreled channel (gb2) with coordinated activity between proto-channels. The single-channel slope conductances of gb1 and gb2 from -60 to +20 mV with a 250/70 mM KCl gradient were 41 and 75 pS, respectively. With symmetrical 250 mM KCl, the I-V relation of gb1 showed outward rectification with 47.8 +/- 6.6 pS at cis negative potentials and 68.9 +/- 6.1 pS at cis positive potentials. gb1 was open from 70 to 95% at all electrochemical potentials from -80 to +40 mV. gb2 was steeply voltage dependent between -80 and -20 mV. Both gb1 and gb2 were insensitive to Ca (from 100 nm to 1 microM), blocked by 0.1 mM DIDS and highly selective for chloride. These data suggest that double-barreled Cl channels are related to the family of small, outwardly rectifying Cl channels of epithelial membranes.     

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: the role of protein translocation

Summary The voltage-dependent channel formed in planar lipid bilayers by colicin E1, or its channel-forming C-terminal fragments, is susceptible to destruction by the nonspecific protease pepsin under well-defined conditions. In particular, pepsin acts only from thecis side (the side to which colicin has been added) and only upon channels in the closed state. Channels in the open state are refractory to destruction bycis pepsin, and neither open nor closed channels are destroyed bytrans pepsin. Colicin E1 channels are normally turned on bycis positive voltages and turned off bycis negative voltages. For large (>80 mV) positive voltages, however, channels inactivate subsequent to opening. Associated with the inactivated state, some channels become capable of being turned on bycis negative voltages and turned off bycis positive voltages, as if the channel-forming region of the molecule has been translocated across the membrane. Consistent with this interpretation is the ability now oftrans pepsin to destroy these reversed channels when they are closed, but not when they are open, whereascis pepsin has no effect on them in either the open or closed state. Our results indicate that voltage gating of the E1 channel involves translocation of parts of the protein across the membrane, exposing different domains to thecis andtrans solutions in the different channel states.     

Water state and diffusion through lipid bilayers: Effect of hydration degree

The dependences of adsorbed water state (obtained from the variations in ¹H NMR spectra with the angle between the bilayer normal and magnetic field direction) and water diffusion along the bilayer normal (measured using pulsed field gradient ¹H NMR) on hydration degree have been studied in macroscopically oriented bilayers of dioleoylphosphatidylcholine. The angle dependences of the shape of NMR spectrum are qualitatively different only for water concentrations higher and lower than that achieved by hydration from saturated vapors (χ_eq, about 23%). At concentrations lower than χ_eq, all water in the sample either makes the hydration shells of the lipid polar heads or is in fast exchange with the shell water, so the spin-echo signal from water is detected only within a narrow range of angles close to the magic angle, 54.7°. At concentration exceeding χ_eq, the spin-echo signal from water is retained at all orientations, suggesting that a portion of water between bilayers (quasi-free water) slowly exchanges with water bound to the polar heads. There is an inverse dependence of the coefficient of water self-diffusion through the bilayer system on the hydration degree, which is described in the Tanner model with account of water self-diffusion in the hydrophobic part of the bilayer. Bilayer permeability, distribution coefficient of molecules between aqueous and lipid phases, and water self-diffusion coefficient in the hydrophobic region of the bilayer are estimated.     

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: translocation of regions outside the channel-forming domain

Summary C-terminal fragments of colicin E1, ranging in mol wt from 14.5 to 20kD, form channels with voltage dependence and ion selectivity qualitatively similar to those of whole E1, placing an upper limit on the channel-forming domain. Under certain conditions, however, the gating kinetics and ion selectivity of channels formed by these different E1 peptides can be distinguished. The differences in channel behavior appear to be correlated with peptide length. Enzymatic digestion with trypsin of membrane-bound E1 peptides converts channel behavior of longer peptides to that characteristic of channels formed by shorter fragments. Apparently trypsin removes segments of protein N-terminal to the channel-forming region, since gating behavior of the shortest fragment is little affected by the enzyme. The success of this conversion depends on the side of the membrane to which trypsin is added and on the state, open or closed, of the channel. Trypsin modifies only closed channels from thecis side (the side to which protein has been added) and only open channels from thetrans side. These results suggest that regions outside the channel-forming domain affect ion selectivity and gating, and they also provide evidence that large protein segments outside the channel-forming domain are translocated across the membrane with channel gating.     

Gating properties of channels formed by colicin Ia in planar lipid bilayer membranes

Summary Colicin Ia forms voltage-dependent channels when incorporated into planar lipid bilayers. A membrane containing many Colicin Ia channels shows a conductance which is turned on when high positive voltages (>+10 mV) are applied to thecis side (side to which the protein is added). The ionic current flowing through the membrane in response to a voltage step shows at first an exponential and then a linear rise with time. The relationship between the steady-state conductance, achieved immediately after the exponential portion, and voltage is S-shaped and is adequately fit by a Boltzmann distribution. The time constant () of the exponential is also dependent on voltage, and the relation between these two parameters is asymmetric aroundV _o (voltage at which half of the channels are open). In both cases the steepness of the voltage dependence, a consequence of the number of effective gating particles (n) present in the channel, is greatly influenced by the pH of the bathing solutions. Thus, increasing the pH leads to a reduction inn, while acidic pH's have the opposite effects. This result is obtained either by changing the pH on both sides of the membrane or on only one side, be itcis orrans. On the other hand, changing pH on only one side by addition of an impermeant buffer fails to induce any change inn. At the single-channel level, pH had an effect both on the unitary conductance, doubling it in going from pH 4.5 to 8.2, as well as on the fraction of time the channels stay open,F _(v). For a given voltage,F _(v) is clearly diminished by increasing the pH. This titration of the voltage sensitivity leads to the conclusion that gating in the Colicin Ia molecule is accomplished by charged amino-acid residues present in the protein molecule. Our results also support the notion that these charged groups are inside the aqueous portion of the channel.     

Direct AFM observation of saposin C-induced membrane domains in lipid bilayers: from simple to complex lipid mixtures

Saposin C (Sap C) is a small glycoprotein required by glucosylceramidase (GCase) for hydrolysis of glucosylceramide to ceramide and glucose in lysosomes. The molecular mechanism underlying Sap C stimulation of the enzyme activation is not fully understood. Here, atomic force microscopy (AFM) has been used to study Sap C-membrane interactions under physiological conditions. First, to establish how Sap C-membrane interactions affect membrane structure, lipid bilayers containing zwitterionic and anionic phospholipids were used. It was observed that Sap C induced two types of membrane restructuring effects, i.e., the formation of patch-like domains and membrane destabilization. Bilayers underwent extensive structural reorganization. To validate the biological importance of the membrane restructuring effects, interaction of Sap C with lipid bilayers composed of cholesterol, sphingomyelin, and zwitterionic and anionic phospholipids were studied. Although similar membrane restructuring effects were observed, Sap C-membrane interactions, in this case, were remarkably modulated and their effects were restricted to a limited area. As a result, nanometer-sized domains were formed. The establishment of a model membrane system will allow us to further study the dynamics, structure and mechanism of the Sap C-associated membrane domains and to examine the important role that these domains may play in enzyme activation.     

Theory of passive proton conductance in lipid bilayers

The large permeability of lipid bilayers to protons compared to other small ions calls for a special proton transport mechanism. At the present time, only mechanisms involving transient hydrogen-bonded chains of water can account for the experimental result that the conductance is nearly independent of pH. Three models involving transient hydrogen-bonded chains are discussed, including an outline of the kinetic calculations that lead to predictions of current versus voltage drop and current versus pH differences. These calculations can be compared to experiment to determine which, if any, of these models pertains to lipid bilayers.     

Phloretin and phloretin analogs: Mode of action in planar lipid bilayers and monolayers

Summary Phloretin and other neutral phloretin-like molecules are able to decrease the electrostatic potential within neutral lipid bilayers and monolayers. The relationship between the change in the dipole potential and the aqueous concentration of the molecule is well described by a Langmuir isotherm. From the Langmuir isotherm, the apparent dissociation constants (K _D ^A ) and the maximum dipole potential change ( _max) are obtained for the different phloretin-like molecules tested. Considering the phloretin analogs as derivatives of acetophenone containing two kinds of substituents, one on the benzene ring and another on the carbon chain, it is found that (a)K _D ^A is related to the hydrophobicity of the compound and is also a function of the position of the hydroxyl substituent in the ring; (b) from the dependence ofK _D ^A on the length of the acyl chain, it is estimated that the free-energy change is 650 cal/mole CH₂; (c) _max is not a simple function of the dipole moment of the molecule but depends on the substituent on the carbon chain and on the position and number of hydroxyl groups on the benzene ring; (d) phloretin adsorption parameters are a function of membrane lipid composition. The results are discussed in terms of the effect of these compounds on chloride transport in red blood cells.     

Anion channels in the sea urchin sperm plasma membrane

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO₃^? > CNS^? > Br^? > CI^?. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4′-diisothiocyano-2,2′ -stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS. © 1993 Wiley-Liss, Inc.     

Contribution of aqueous interphases to the permeability barrier of lipid bilayers for non-electrolytes

Equilibrium dialysis experiments are used to measure excluded volumes for the non-electrolyte permeant [U-¹⁴C] erythritol in lipid bilayer systems. The data indicate amounts of water associated with the lipid membranes which correspond with amounts calculated from calorimetric measurements.The membrane systems can be described as composite elements consisting of the lipid bilayers and adjacent water layers on both sides. The finding that the permeant is excluded indicates that the water layers contribute to the permeability barrier.The mean thickness of the water layers is about 6 Å for planar bilayers in multilayered liposomes and 10 Å for curved bilayers in sonicated vesicles. Next to the difference in thickness of the water layers differences in interfacial adsorption between the two systems are apparent.     

Investigation of the mechanism of action of novel amphipathic peptides: Insights from solid-state NMR studies of oriented lipid bilayers

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by ³¹P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

A carboxy-terminal fragment of colicin Ia forms ion channels

A carboxy-terminal, 18 kD fragment of colicin Ia, a bacterial toxin, forms ion channels in artificial phospholipid bilayers. This fragment, which comprises a quarter of the intact 70 kD molecule, is resistant to extensive protease digestion and probably constitutes a structural domain of the protein. The ion channels formed by the 18 kD fragment are functionally heterogeneous, having conductances that range from 15 to 30 pS at positive voltages and from 70 to 250 pS at negative voltages, and open lifetimes that range from at least 25 msec to 5 sec. In contrast, ion channels formed by whole colicin Ia open only at negative voltages, at which their conductances range from 6 to 30 pS, and their open lifetimes range from 1 sec to 3 min. Additionally, the open state of the 18 kD fragment channel is characterized by noisy fluctuations in current, while the open state of the whole molecule ion channel is often marked by numerous, stable subconductance states. Since the properties of the fragment channel differ substantially from those of the whole molecule channel, we suggest that portions of the molecule outside of the 18 kD fragment are involved in forming the whole molecule ion channel.     

Nanoscale analysis of supported lipid bilayers using atomic force microscopy

During the past 15 years, atomic force microscopy (AFM) has opened new opportunities for imaging supported lipid bilayers (SLBs) on the nanoscale. AFM offers a means to visualize the nanoscale structure of SLBs in physiological conditions. A unique feature of AFM is its ability to monitor dynamic events, like bilayer alteration, remodelling or digestion, upon incubation with various external agents such as drugs, detergents, proteins, peptides, nanoparticles, and solvents. Here, we survey recent progress made in the area.     

Conformational order of the hydrocarbon chains in lipid bilayers. A raman spectroscopic study

Raman spectra of dihexadecylphosphatidic acid (DHPA) and of dimyristoylphosphatidylcholine (DMPC) and its longer chain homologues have been obtained as a function of temperature in order to study the conformational order of the hydrocarbon chains in lipid bilayers. The frequency of the longitudinal acoustical (LA) vibration band is evaluated in terms of the length of all-trans chain segments. In the ordered phase, the chains are found to be overwhelmingly in the all-trans conformation. In the fluid phase, definite all-trans segments occur predominantly, the length of which coincides with the extension of the order parameter plateau known from deuterium magnetic resonance (DMR). The frequency of the skeletal optical (SO) trans vibration band leads to the same result, if evaluated under the assumption of vibrational decoupling by gauche bands in the fluid phase, thus lending support to this assumption. The intensity of this band determined from the band area increases linearly with chain length in the ordered phase and is independent of chain length in the fluid phase. Evaluating the intensity for the length of all-trans segments, the same result for the chain conformation is obtained as derived from the frequencies, with the additional information that the length of the all-trans segments in the fluid phase does not vary with chain length.     

Permeability of bilayers composed of mixtures of saturated phospholipids

Liposomes composed of an equimolar binary mixture of phospholipids were formed from a series of saturated phosphatidylcholines (PC) and phosphatidylethanolamines (PE). Mixtures were chosen such that the two phospholipids differed either in terms of head group alone, chain length alone, or both head group and chain length. Cation effluxes, both with and without ionophores (nigericin and valinomycin) were measured over a range of temperatures that encompassed the regions of phase separation for these different lipid mixtures. There was a good correlation between the temperatures at which permeability maxima and phase separation occur. For phospholipid mixtures with the same acyl chain but different head groups (PC vs. PE), the PC component ‘controls’ permeability. For mixtures of PCs differing in chain length, the short chain lipid dominates the permeability pattern particularly if the chain lengths are sufficiently different. Lipids differing in both head group and chain length give rise to more complex permeability patterns. The results of the present study are interpreted in terms of a model in which one of the lipid components of the mixture may specifically congregate at defects between co-existing phases and thus ‘regulate’ permeability.