Conformation of sequential polypeptide poly(Leu-Leu-D-Phe-Pro) and formation of ion channel across bilayer lipid membrane.

Sequential polypeptide, poly(Leu-Leu-D-Phe-Pro), containing a part of beta-turn sequence in gramicidin S, was synthesized and investigated as a model for ion channels. Sequential peptides, Boc-(Leu-Leu-D-Phe-Pro)n-OBzl1 (n = 1-4), were also synthesized to acquire conformational information about this polypeptide. From the analyses by NMR, CD, and IR measurements, intramolecular hydrogen bonds were found in the sequential peptides with n larger than two and Boc-(Leu-Leu-D-Phe-Pro)3-OBzl was deduced to adopt a 3(10)-helical conformation. Poly(Leu-Leu-D-Phe-Pro) was also suggested to have this conformation. With the addition of this polymer to oxidized cholesterol membrane, current-voltage response across the membrane was observed. Stepwise fluctuation of current was recorded under a positive electric field to support the channel formation. This polymer might form bundles of 3(10)-helices across the bilayer lipid membrane to pass through the ion.     

Kinetics of macrotetrolide-induced ion transport across lipid bilayer membranes

Ion transport across lipid bilayer membranes in the presence of macrotetrolide antibiotics has been studied by stationary conductance and nonstationary relaxation methods. The results are discussed on the basis of a carrier model which has already been successfully applied to valinomycin induced ion transport. Again a kinetic analysis has been performed from which the single rate constants of the carrier model could be derived. In addition the equilibrium constant of complex formation in the aqueous phase could be determined. Measurements have been made for 4 macrotetrolides, for several ions and for various chain lengths of the lipids molecules composing the membrane.     

Phloretin-induced changes in ion transport across lipid bilayer membranes

Phloretin, the aglucone derivative of phlorizin, increases cation conductance and decreases anion conductance in lipid bilayer membranes. In this paper we present evidence that phloretin acts almost exclusively by altering the permeability of the membrane interior and not by modifying the partition of the permanent species between the membrane and the bulk aqueous phases. We base our conclusion on an analysis of the current responses to a senylborate, and the cation complex, peptide PV-K+. These results are consistent with the hypothesis that phloretin decreases the intrinsic positive internal membrane potential but does not modify to a great extent the potential energy minima at the membrane interfaces. Phloretin increases the conductance for the nonactin-K+ complex, but above 10(-5) M the steady- state nonactin-K+ voltage-current curve changes from superlinear to sublinear. These results imply that, above 10(-5) M phloretin, the nonactin-5+ transport across the membrane becomes interfacially limited.     

The movement of ion pairs across bilayer lipid membranes.

If a polyhalide concentration gradient exists across a bilayer lipid membrane (BLM), ion pair movement occurs. The term ion pair indicates a lipid soluble complex of cation and anion with stoichiometry dictated by the respective charges. In a mixture of metal halide (MX_n, X = I, Cl, Br) and iodine, the ion pair is of the form M(I₂X)_n. The flux of ion pairs was monitored by measuring the flow of metal ions or polyhalide ions across the BLM. The flux of ion pairs across the BLM depended on cation crystal radius, fluidity of the membrane, strength of the ion pair complex and on the osmotic gradient (i.e., there exists a coupling between water and ion pair fluxes). The relationship between ion pairing and the electrical conductivity of BLM is briefly discussed.     

Millimeter microwave effect on ion transport across lipid bilayer membranes

The effects of millimeter microwaves in the frequency range of 54–76 GHz on capacitance and conductance of lipid bilayer membranes (BLM) were studied. Some of the membranes were modified by gramicidin A and amphotericin B or by tetraphenylboron anions (TPhB_?). The millimeter microwaves were pulse-modulated (PW) at repetition rates ranging from 1 to 100 pps, PW at 1000 pps, or unmodulated continuous waves (CW). The maximum output power at the waveguide outlet was 20 mW. It was found that CW irradiation decreased the unmodified BLM capacitance by 1.2% ± 0.5%. At the same time, membrane current induced by TPhB transport increased by 5% ± 1%. The changes in conductance of ionic channels formed by gramicidin A and amphotericin B were small (0.6% ± 0.4%). No “resonance-like” effects of mm-wave irradiation on membrane capacitance, ionic channel currents, or TPhB transport were detected. All changes in membrane capacitance and currents were independent of the modulation employed and were equivalent to heating by approximately 1.1 °C. © 1995 Wiley-Liss, Inc.     

Modulation of a gated ion channel admittance in lipid bilayer membranes.

A future class of amperometric biosensors may utilize gated ion channels such as acetylcholine and glutamate receptors as chemical detection components. In this study, bilayer lipid membranes containing voltage-dependent anion channels (VDAC) were used to model an ion-channel-based biosensor which could continuously monitor AC amperometric changes resulting from induced changes in channel conductance. The in-phase and quadrature components of the induced alternating membrane current were monitored as a function of the applied DC offset voltage which was superimposed on the sinusoidal test voltage. The accuracy and sensitivity of the AC-measured VDAC response was dependent on the magnitude of the AC test voltage relative to the DC offset necessary for channel closure. The VDAC channel appears to be a suitable model protein for AC impedance-based biosensor fabrication.     

Structural investigation of poly-L,D-proline, a synthetic ion channel across bilayer membranes: crystal structure and molecular conformation of two tetra-D,L-proline derivatives

The crystal structures and molecular conformations of two tetraproline derivatives with alternating configurations Boc(D -Pro,L -Pro)₂OH and Boc(D -Pro,L -Pro)₂OCH₃ are investigated in connection with the ability of the homologous polymer to selectively increase (as an ion channel) the ion permeability across bilayer membranes. Both tetramers are characterized by the cis-trans alternating conformation of the peptide bonds, which formally transforms in a turn of the poly-D ,L -proline channel after a cis-trans change of the central peptide residue.     

The passage of an ion through a synthetic ion channel

The simulated system consisted of a fatty acid bilayer membrane dividing two electrolyte layers each containing ions, and a channel composed of linked 15-crown-5 ether rings. The Na⁺ and F^?  ions in the aqueous electrolyte layers were too large to enter the channel, but the Li⁺ ions entered and were transported. Conditions that optimised the passive, electric-field-induced transport of Li⁺ ions through the channel were investigated. It was calculated and rationalised that the higher the numerical value of the electrostatic charge on the oxygen atoms of the crown ether rings, the more easily does the channel convey the Li⁺ ions.     

A synthetic peptide forms voltage-gated porin-like ion channels in lipid bilayer membranes

Design of simple protein structures represents the essential first step toward novel macromolecules and understanding the basic principles of protein folding. Our work focuses on the ion channel formation and structure of peptides having a repeated pattern of glycine residues. Investigation of the ion channel properties of a glycine repeat peptide, VSLGLSIGFSVGVSIGWSFGRSRG revealed the formation of porin-like high conductance, multimeric, non-selective voltage-gated channels in phospholipid bilayer membranes. ATR-IR and CD spectroscopic studies showed an anti-parallel beta sheet structure in membranes. The formation of porin-like ion channels by a beta sheet peptide suggests spontaneous assembly into a beta barrel structure through oligomerization as in pore forming bacterial toxins. The present work is the first example of a short synthetic peptide mimicking the pore characteristics of a complex beta barrel protein and demonstrates that smaller peptides are capable of mimicking the complex functional properties of natural ion channels. This will have implications in understanding the folding of beta sheet proteins in membranes, the mechanism of two state voltage gating, and the role of glycine residues in beta barrel proteins.     

Effect of 3-phenylindole on lipophilic ion and carrier-mediated ion transport across bilayer lipid membranes

The physical effects of 3-phenylindole, an antimicrobial compound which interacts with phospholipids, on ion transport across phosphatidylcholine-cholesterol bilayers have been investigated using three lipophilic ions and one ion-carrier complex. It was found that 3-phenylindole increased membrane electrical conductance of positively charged membrane probes and decreased electrical conductance of negatively charged probes. The enhancement of conductance detected by nonactin-K+ complex and tetraphenylarsonium+ was several orders of magnitude, whereas the suppression of conductance due to tetraphenylborate- and dipicrylamine- was less than a factor of ten. Presence of 3-phenylindole in aqueous phase slightly decreased adsorption of tetraphenylborate- and dipicrylamine- at the membrane surface. From the voltage dependence of the steady-state conductance it was shown that 3-phenylindole induced kinetic limitation of membrane transport of potassium mediated by nonactin. No such limitation was found in the case of tetraphenylarsonium+ transport. These results are shown to be consistent with the present concept of ion diffusion in membranes and the assumption that 3-phenylindole decreases the electric potential in the membrane interior. The asymmetry of the effect of 3-phenylindole on the magnitude of conductance changes for positively and negatively charged membrane permeable ions is also discussed as a reflection of the discreteness of both the absorbed 3-phenylindole and lipid dipoles.     

Electrochemical study of ion channel behavior in incorporated poly L-glutamate bilayer lipid membranes

The lipid layer membranes were fabricated on the glassy carbon electrode (GC) and demonstrated to be bilayer lipid membranes by impedance spectroscopy. The formation of incorporated poly L-glutamate bilayer lipid membrane was achieved. The ion channel behavior of the incorporated poly L-glutamate membrane was determined. When the stimulus calcium cations were added into the electrolyte, the ion channel was opened immediately and exhibited distinct channel current. Otherwise, the ion channel was closed. The cyclic voltammogram at the GC electrode coated with incorporated poly L-glutamate DMPC film response to calcium ion is very fast compared with that at the GC electrode coated only with DMPC film. Ion channel current is not dependent on the time but on the concentration of calcium. The mechanism of the ion channel formation was investigated.     

Permeation of ammonia across bilayer lipid membranes studied by ammonium ion selective microelectrodes.

Ammonium ion and proton concentration profiles near the surface of a planar bilayer lipid membrane (BLM) generated by an ammonium ion gradient across the BLM are studied by means of microelectrodes. If the concentration of the weak base is small compared with the buffer capacity of the medium, the experimental results are well described by the standard physiological model in which the transmembrane transport is assumed to be limited by diffusion across unstirred layers (USLs) adjacent to the membrane at basic pH values (pH > pKa) and by the permeation across the membrane itself at acidic pH values. In a poorly buffered medium, however, these predictions are not fulfilled. A pH gradient that develops within the USL must be taken into account under these conditions. From the concentration distribution of ammonium ions recorded at both sides of the BLM, the membrane permeability for ammonia is determined for BLMs of different lipid composition (48 x 10(-3) cm/s in the case of diphytanoyl phosphatidylcholine). A theoretical model of weak electrolyte transport that is based on the knowledge of reaction and diffusion rates is found to describe well the experimental profiles under any conditions. The microelectrode technique can be applied for the study of the membrane permeability of other weak acids or bases, even if no microsensor for the substance under study is available, because with the help of the theoretical model the membrane permeability values can be estimated from pH profiles alone. The accuracy of such measurements is limited, however, because small changes in the equilibrium constants, diffusion coefficients, or concentrations used for computations create a systematic error.     

Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides.

We made use of a planar lipid bilayer system to examine the action of synthetic basic peptides which model the prepiece moiety of mitochondrial protein precursors and have antibacterial activity against Gram-positive bacteria. The sequences of the peptides used were as follows: Ac-(Ala-Arg-Leu)3-NHCH3 (3(3], Ac-(Leu-Ala-Arg-Leu)2-NHCH3 (4(2], Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], Ac-(Leu-Leu-Ala-Arg-Leu)2-NHCH3 (5(2]. These peptides interacted differently with planar lipid bilayer membranes and membrane conductance increased by the formation of ion channels. The effects of the peptides on the macroscopic current-increase and on the probability of channel formation, at the single channel level were in the order of 4(3) greater than 4(2) approximately 5(2) much greater than 3(3), a finding which correlates with the antibacterial activity of these peptides. The micromolar (microM) order concentration at which the channel was formed resembles that causing antibacterial activity. Thus, the peptide antibacterial activity may occur through an increase in ion permeability of the bacterial membrane. The single-channel properties were investigated in detail using 4(3), the peptide with the highest ion channel-forming activity. Many types of channels were observed with respect to conductance (2-750 pS) and voltage dependency of gating. However, the channels were all cation-selective. These results suggest that the ion channels formed by peptide 4(3) may be able to take on a variety of conformations and/or assembly.     

Structure and stability of the Boc(D-Pro-L-Pro)2OCH3 Na+ complex: a model of poly(D,L-proline), an alkali ion channel across membranes

In order to elucidate the molecular mechanisms of alkali ions conduction of poly(D,L-proline), which shows the typical behavior of an ion channel across membranes, conformations of the tetrameric derivative Boc(D-Pro-L-Pro)2OCH3 in solution, in the presence of the Na+ ion, were investigated by CD and nmr spectroscopy. Both theoretical and experimental data indicate the formation in solution of a monomeric complex between the tetramer, in the all-trans conformation, and the cation. The results allow to confirm the channel model, previously proposed on the phenomenological behavior of poly(D,L-proline) in membranes.     

Lysenin forms a voltage-dependent channel in artificial lipid bilayer membranes

Lysenin, a hemolytic protein derived from the body fluid of earthworm, was incorporated into artificial bilayer membranes. Upon insertion, it formed a voltage-dependent large conductance channel in asolectin bilayers in a sphingomyelin-dependent manner. The channel had low ion-selectivity. Single-channel conductance was calculated as approximately 550 pS in 100 mM KCl. The channel in asolectin bilayers closed when the membrane was held at a positive potential. In contrast, the channel showed no voltage dependency in membranes made of pure phosphatidylcholine and sphingomyelin, suggesting some lipid contents included in the asolectin membranes affected channel gating.     

Charge transfer across a single lipid-water interface causes ion pumping across the bilayer.

The photoformation of magnesium-porphyrin cations (P+) at a single lipid bilayer-water interface can pump lipophilic borate anions completely across the lipid bilayer and causes an actual reversal of the photovoltage. The system consists of a lipid bilayer containing magnesium octaethylporphyrin, an aqueous or interfacial electron acceptor on one side, and chloro- or fluoro-substituted tetraphenylborate in both aqueous electrolyte solutions. With 1-micros pulsed illumination, an immediate positive photovoltage is observed, which decreases on the microsecond and millisecond time scales. On the time scale of seconds, as the P+ cation concentration decays in reverse electron transfer, the voltage swings negative to a value almost equal to its initial value and finally decays with a half-time (approximately 20 s) longer than the time constant of the system (approximately 5 s). Thus, an ion gradient across the membrane is formed, trapped by the nonlinear relation between ion mobility and ion concentration. Continuous light illumination confirms that negative charge moves in the direction opposite that of the initial photoinduced electron transfer. Steady-state measurements indicate an ion pumping efficiency of approximately 30%. This simple mechanism may be a progenitor of photobiological ion pumps.     

The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes

Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the organization of cholesterol and phospholipids in membranes formed from Chol/POPS (cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine) mixtures, with mixing ratios from 0 to 3. It was confirmed using the discrimination by oxygen transport and polar relaxation agent accessibility methods that the immiscible cholesterol bilayer domain (CBD) was present in all of the suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST) in the POPS membrane. The behavior of phospholipid molecules was monitored with phospholipid analogue spin labels (n-PCs), and the behavior of cholesterol was monitored with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio of ~2. Additionally, EPR spectra for n-PC, ASL, and CSL indicated that both phospholipids and cholesterol exist in these suspensions in the lipid-bilayer-like structures. EPR spectral characteristics of n-PCs (spin labels located in the phospholipid cholesterol bilayer, outside the CBD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CBD forms an integral part of the phospholipid bilayer when formed from a Chol/POPS mixture up to a mixing ratio of ~2. Interestingly, CSL in cholesterol alone (without phospholipids) when suspended in buffer does not detect formation of bilayer-like structures. A broad, single-line EPR signal is given, similar to that obtained for the dry film of cholesterol before addition of the buffer. This broad, single-line signal is also observed in suspensions formed for Chol/POPS mixtures (as a background signal) when the Chol/POPS ratio is much greater than 3. It is suggested that the EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the phospholipid bilayer.     

Molecular dynamics simulation of a synthetic ion channel.

A molecular dynamics simulation has been performed on a synthetic membrane-spanning ion channel, consisting of four alpha-helical peptides, each of which is composed of the amino acids leucine (L) and serine (S), with the sequence Ac-(LSLLLSL)3-CONH2. This four-helix bundle has been shown experimentally to act as a proton-conducting channel in a membrane environment. In the present simulation, the channel was initially assembled as a parallel bundle in the octane portion of a phase-separated water/octane system, which provided a membrane-mimetic environment. An explicit reversible multiple-time-step integrator was used to generate a dynamical trajectory, a few nanoseconds in duration for this composite system on a parallel computer, under ambient conditions. After more than 1 ns, the four helices were found to adopt an associated dimer state with twofold symmetry, which evolved into a coiled-coil tetrameric structure with a left-handed twist. In the coiled-coil state, the polar serine side chains interact to form a layered structure with the core of the bundle filled with H2O. The dipoles of these H2O molecules tended to align opposite the net dipole of the peptide bundle. The calculated dipole relaxation function of the pore H2O molecules exhibits two reorientation times. One is approximately 3.2 ps, and the other is approximately 100 times longer. The diffusion coefficient of the pore H2O is about one-third of the bulk H2O value. The total dipole moment and the inertia tensor of the peptide bundle have been calculated and reveal slow (300 ps) collective oscillatory motions. Our results, which are based on a simple united atom force-field model, suggest that the function of this synthetic ion channel is likely inextricably coupled to its dynamical behavior.     

Voltammetric study of charge transfer across supported bilayer lipid membranes (s-BLMs).

Stable bilayer lipid membranes on a metal support (s-BLMs) and on an agar gel salt bridge (sb-BLMs) have been formed and their potential usefulness as practical sensors demonstrated. This paper presents the preparation method of s-BLMs and sb-BLMs, and the application of cyclic voltammetry (CV) to their investigation. The instrument and data analysis of the CV are described. The application of CV to the C60-doped BLM system is presented. This technique is a basis for biosensor development, and a useful tool for membrane research.