The Role of Sarcolipin and ATP in the Transport of Phosphate Ion into the Sarcoplasmic Reticulum

In a previous study, sarcolipin (SLN) was shown to form channels selective toward chloride ion when incorporated in a mercury-supported tethered bilayer lipid membrane (tBLM). Its incorporation had only a modest permeabilizing effect on phosphate ion. In this note the resistance of a tBLM membrane incorporating sarcolipin was investigated by electrochemical impedance spectroscopy in aqueous solutions of 0.05 M sodium phosphate of pH ranging from 5.3 to 8, in the presence of ATP, adenosine monophosphate, and phenylphosphonic acid. At pH 5.3, submicromolar additions of ATP increase the conductivity of the tBLM incorporating SLN up to a maximum limiting value. The dependence of the conductivity on the ATP concentration satisfies the Michaelis-Menten equation, with an association constant of 0.1 μM. Phenylphosphonium ion and adenosine monophosphate exert an inhibitory effect on membrane permeabilization to phosphate ions by ATP if they are added before ATP, but not if they are added after it. An explanation for this behavior is provided. In conclusion, SLN acts as an ATP-induced phosphate carrier exhibiting a behavior quite similar to that of the unidentified P_i transporter described previously. No ion-channel activity is exhibited by the T18A mutant of SLN.     

Tethered lipid bilayers on electrolessly deposited gold for bioelectronic applications

This paper presents the formation of a novel biomimetic interface consisting of an electrolessly deposited gold film overlaid with a tethered bilayer lipid membrane (tBLM). Self-assembly of colloidal gold particles was used to create an electrolessly deposited gold film on a glass slide. The properties of the film were characterized using field-effect scanning electron microscopy, energy dispersive spectroscopy, and atomic force microscopy. Bilayer lipid membranes were then tethered to the gold film by first depositing an inner molecular leaflet using a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate], 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (DPGP), and cystamine in ethanol onto a freshly prepared electrolessly deposited gold surface. The outer leaflet was then formed by the fusion of liposomes made from DPGP or 1,2-dioleoyl-sn-glycero-3-phosphocholine on the inner leaflet. To provide functionality, two membrane biomolecules were also incorporated into the tBLMs: the ionophore valinomycin and a segment of neuropathy target esterase containing the esterase domain. Electrochemical impedance spectroscopy, UV/visible spectroscopy, and fluorescence recovery after pattern photobleaching were used to characterize the resulting biomimetic interfaces and confirm the biomolecule activity of the membrane. Microcontact printing was used to form arrays of electrolessly deposited gold patterns on glass slides. Subsequent deposition of lipids yielded arrays of tBLMs. This approach can be extended to form functional biomimetic interfaces on a wide range of inexpensive materials, including plastics. Potential applications include high-throughput screening of drugs and chemicals that interact with cell membranes and for probing, and possibly controlling, interactions between living cells and synthetic membranes. In addition, the gold electrode provides the possibility of electrochemical applications, including biocatalysis, bio-fuel cells, and biosensors.     

Transient Potential Gradients and Impedance Measures of Tethered Bilayer Lipid Membranes: Pore-Forming Peptide Insertion and the Effect of Electroporation

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >∼150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.     

Transient Potential Gradients and Impedance Measures of Tethered Bilayer?Lipid Membranes: Pore-Forming Peptide Insertion and the Effect of?Electroporation

In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >∼150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.     

The protein-tethered lipid bilayer: a novel mimic of the biological membrane

A new concept of solid-supported tethered bilayer lipid membrane (tBLM) for the functional incorporation of membrane proteins is introduced. The incorporated protein itself acts as the tethering molecule resulting in a versatile system in which the protein determines the characteristics of the submembraneous space. This architecture is achieved through a metal chelating surface, to which histidine-tagged (His-tagged) membrane proteins are able to bind in a reversible manner. The tethered bilayer lipid membrane is generated by substitution of protein-bound detergent molecules with lipids using in-situ dialysis or adsorption. The system is characterized by surface plasmon resonance, quartz crystal microbalance, and electrochemical impedance spectroscopy. His-tagged cytochrome c oxidase (CcO) is used as a model protein in this study. However, the new system should be applicable to all recombinant membrane proteins bearing a terminal His-tag. In particular, combination of surface immobilization and membrane reconstitution opens new prospects for the investigation of functional membrane proteins by various surface-sensitive techniques under a defined electric field.     

Probing membrane permeabilization by the antimicrobial peptide distinctin in mercury-supported biomimetic membranes

The mechanism of membrane permeabilization by the antimicrobial peptide distinctin was investigated by using two different mercury-supported biomimetic membranes, namely a lipid self-assembled monolayer and a lipid bilayer tethered to the mercury surface through a hydrophilic spacer (tethered bilayer lipid membrane: tBLM). Incorporation of distinctin into a lipid monolayer from its aqueous solution yields rapidly ion channels selective toward inorganic cations, such as Tl(+) and Cd(2+). Conversely, its incorporation in a tBLM allows the formation of ion channels permeable to potassium ions only at non-physiological transmembrane potentials, more negative than -340mV. These channels, once formed, are unstable at less negative transmembrane potentials. The kinetics of their formation is consistent with the disruption of distinctin clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth. Comparing the behavior of distinctin in tBLMs with that in conventional black lipid membranes strongly suggests that distinctin channel formation in lipid bilayer requires the partitioning of distinctin molecules between the two sides of the lipid bilayer. We can tentatively hypothesize that an ion channel is formed when one distinctin cluster on one side of the lipid bilayer matches another one on the opposite side.     

A tethered bilayer sensor containing alamethicin channels and its detection of amiloride based inhibitors

Alamethicin, a small transmembrane peptide, inserts into a tethered bilayer membrane (tBLM) to form ion channels, which we have investigated using electrical impedance spectroscopy. The number of channels formed is dependent on the incubation time, concentration of the alamethicin and the application of DC voltage. The properties of the ion channels when formed in tethered bilayers are similar to those for such channels assembled into black lipid membranes (BLMs). Furthermore, amiloride and certain analogs can inhibit the channel pores, formed in the tBLMs. The potency and concentration of the inhibitors can be determined by measuring the change of impedance. Our work illustrates the possibility of using a synthetic tBLM for the study of small peptide voltage dependent ion channels. A potential application of such a device is as a screening tool in drug discovery processes.     

Channel-forming activity of syringomycin E in two mercury-supported biomimetic membranes

The lipodepsipeptide syringomycin E (SR-E) interacts with two mercury-supported biomimetic membranes, which consist of a self-assembled phospholipid monolayer (SAM) and of a tethered bilayer lipid membrane (tBLM) separated from the mercury surface by a hydrophilic tetraethyleneoxy (TEO) spacer that acts as an ionic reservoir. SR-E interacts more rapidly and effectively with a SAM of dioleoylphosphatidylserine (DOPS) than with one of dioleoylphosphatidylcholine (DOPC). The proximal lipid monolayer of the tBLM has no polar head region, being linked to the TEO spacer via an ether bond, while the distal monolayer consists of either a DOPC or a DOPS leaflet. The ion flow into or out of the spacer through the lipid bilayer moiety of the tBLM was monitored by potential step chronocoulometry and cyclic voltammetry. With the distal monolayer bathed by aqueous 0.1 M KCl and 0.8 μM SR-E, an ion flow in two stages was monitored with DOPC at pH 3 and 5.4 and with DOPS at pH 3, while a single stage was observed with DOPS at pH 5.4. This behavior was compared with that already described at conventional bilayer lipid membranes. The sigmoidal shape of the chronocoulometric charge transients points to an aggregation of SR-E monomers forming an ion channel via a mechanism of nucleation and growth. The ion flow is mainly determined by potassium ions, and is inhibited by calcium ions. The contribution to the transmembrane potential from the distal leaflet depends more on the nature of the lipid than that of the ion channel.     

Probing membrane permeabilization by the antibiotic lipopeptaibol trichogin GA IV in a tethered bilayer lipid membrane

The lipopeptaibol trichogin GA IV (TCG) can be incorporated in the lipid bilayer moiety of a mercury-supported tethered bilayer lipid membrane (tBLM) at a non-physiological transmembrane potential of about -240mV, negative on the trans side of the bilayer. Once incorporated in the tBLM, TCG is stable over the range of physiological transmembrane potentials and permeabilizes the membrane at transmembrane potentials negative of -80÷-90mV. The chronocoulometric behavior is consistent with a kinetics of nucleation and growth of bundles of TCG building blocks with ion-channel properties. The TCG building blocks also permeabilize the lipid bilayer, albeit at more negative transmembrane potentials, and can be tentatively regarded as dimers of aligned TCG helical monomers. The cyclic voltammograms of tBLMs incorporating TCG point to a voltage-gated behavior of the TCG channel, similar to that exhibited by the peptaibol alamethicin.     

Antigenic topology of chlamydial PorB protein and identification of targets for immune neutralization of infectivity

The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines.     

Concentration-dependent behavior of nisin interaction with supported bilayer lipid membrane

Nisin is a positively charged antibacterial peptide that binds to the negatively charged membranes of gram-positive bacteria. The initial interaction of the peptide with the model membrane of negatively charged DPPG (dipalmitoylphosphatidylglycerol) was studied by cyclic voltammetry and a.c. impedance spectroscopy. Nisin could induce pores in the supported bilayer lipid membrane, thus, it led to the marker ions Fe(CN)(6)(3-/4-) crossing the lipid membrane and giving the redox reaction on the glassy carbon electrode (GCE). Experimental results suggested that the pore formation on supported bilayer lipid membrane was dependent on the concentration of nisin and it included three main concentration stages: low, middling, high concentration.     

Construction of P-glycoprotein incorporated tethered lipid bilayer membranes

To investigate drug–membrane protein interactions, an artificial tethered lipid bilayer system was constructed for the functional integration of membrane proteins with large extra-membrane domains such as multi-drug resistance protein 1 (MDR1). In this study, a modified lipid (i.e., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG)) was utilized as a spacer molecule to elevate lipid membrane from the sensor surface and generate a reservoir underneath. Concentration of DSPE-PEG molecule significantly affected the liposome binding/spreading and lipid bilayer formation, and 0.03 mg/mL of DSPE-PEG provided optimum conditions for membrane protein integration. Further, the incorporation of MDR1 increased the local rigidity on the platform. Antibody binding studies showed the functional integration of MDR1 protein into lipid bilayer platform. The platform allowed to follow MDR!-statin-based drug interactions in vitro. Each binding event and lipid bilayer formation was monitored in real-time using Surface Plasmon Resonance and Quartz Crystal Microbalance–Dissipation systems, and Atomic Force Microscopy was used for visualization experiments.     

Characterization and functional analysis of PorB, a Chlamydia porin and neutralizing target

A predicted protein (CT713) with weak sequence similarity to the major outer membrane protein (20.4% identity) in Chlamydia trachomatis was identified by Chlamydia genome analysis. We show that this protein is expressed, surface accessible, localized to the chlamydial outer membrane complex and functions as a porin. This protein, PorB, was highly conserved among different serovars, with nearly identical sequences between serovars D, B, C and L2. Sequence comparison between C. trachomatis and Chlamydia pneumoniae showed less conservation between species with 59.3% identity. Immunofluorescence staining with monospecific antisera to purified PorB revealed antigen localized within chlamydial inclusions and found throughout the developmental cycle. Antibodies to PorB neutralized infectivity of C. trachomatis in an in vitro neutralization assay confirming that PorB is surface exposed. As PorB was found to be in the outer membrane, as well as having weak structural characteristics similar to major outer membrane protein (MOMP) and other porins, a liposome-swelling assay was used to determine whether this protein had pore-forming capabilities. PorB had pore-forming activity and was shown to be different from MOMP porin activity.     

Characterization and property of DNA incorporated bilayer lipid membranes

Calf-thymus DNA-incorporated bilayer lipid membranes supported on a glassy carbon (GC) electrode was prepared by making layers of phosphatidylcholine dimyristoyl (DMPC) on GC electrode. DNA in the BLM was characterized by cyclic voltammetry, IR and AFM, and lipid layers formed on the GC electrode were demonstrated to be a bilayer lipid membrane by electrochemical impedance experiment. In IR and AFM experiments the findings indicated that DNA was incorporated into BLM. The ion channel of bilayer lipid membranes incorporated was studied. The result showed that the ion channel was opened in the presence of the stimulus quinacrine. In the absence of quinacrine the channel was switched. The process can repeat itself many times. The impedance spectroscopy measurements demonstrate that the stimulus quinacrine opens the channel for permeation of marker ion. The mechanism of forming an ion channel was investigated.     

Interaction of K7Fe3+P2W17O62H2 with supported bilayer lipid membranes on platinum electrode

The influence of K(7)Fe(3+)P(2)W(17)O(62)H(2) on l-alpha-phosphatidylcholine/cholesterol bilayer lipid membrane on Pt electrode was studied by voltammetry and AC impedance spectroscopy. The interaction of the polyoxometalates with the BLM can promote the access of Ru(NH(3))(6)(3+) and [Fe(CN)(6)](3-/4-) to the electrode surface. It was found that some kind of pores had been formed on the BLM by AFM. The phenomenon is attributed to the interaction of K(7)Fe(3+)P(2)W(17)O(62)H(2) with phosphatidylcholine phosphate groups located in its outer leaflet. Experimental results are helpful to understand the biological activity of the polyoxometalates in vivo.     

1,2-diacyl-phosphatidylcholine flip-flop measured directly by sum-frequency vibrational spectroscopy

Sum-frequency vibrational spectroscopy (SFVS) is used to measure the intrinsic rate of lipid flip-flop for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in planar-supported lipid bilayers (PSs). Asymmetric PSLBs were prepared using the Langmuir-Blodgett/Langmuir-Schaefer method by placing a perdeuterated lipid analog in one leaflet of the PSLB. SFVS was used to directly measure the asymmetric distribution of the native lipid within the membrane by measuring the decay in the CH3 v(s) intensity at 2875 cm(-1) with time and as a function of temperature. An average activation energy of 220 kJ/mol for the translocation of DMPC, DPPC, and DSPC was determined. A decrease in alkyl chain length resulted in a substantial increase in the rate of flip-flop manifested as an increase in the Arrhenius preexponential factor. The effect of lipid labeling was investigated by measuring the exchange of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n,n-Dimethyl-n-(2',2',6',6'-tetramethyl-4'-piperidyl) (TEMPO-DPPC). The rate of TEMPO-DPPC flip-flop was an order-of-magnitude slower compared to DPPC. An activation energy of 79 kJ/mol was measured which is comparable to that previously measured by electron spin resonance. The results of this study illustrate how SFVS can be used to directly measure lipid flip-flop without the need for a fluorescent or spin-labeled lipid probe, which can significantly alter the rate of lipid translocation.     

Modulation of concentration fluctuations in phase-separated lipid membranes by polypeptide insertion

The lateral membrane organization and phase behavior of the binary lipid mixture DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) - DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine) without and with incorporated gramicidin D (GD) as a model biomembrane polypeptide was studied by small-angle neutron scattering, Fourier-transform infrared spectroscopy, and by two-photon excitation fluorescence microscopy on giant unilamellar vesicles. The small-angle neutron scattering method allows the detection of concentration fluctuations in the range from 1 to 200 nm. Fluorescence microscopy was used for direct visualization of the lateral lipid organization and domain shapes on a micrometer length scale including information of the lipid phase state. In the fluid-gel coexistence region of the pure binary lipid system, large-scale concentration fluctuations appear. Infrared spectral parameters were used to determine the peptide conformation adopted in the different lipid phases. The data show that the structure of the temperature-dependent lipid phases is significantly altered by the insertion of 2 to 5 mol% GD. At temperatures corresponding to the gel-fluid phase coexistence region the concentration fluctuations drastically decrease, and we observe domains in the giant unilamellar vesicles, which mainly disappear by the incorporation of 2 to 5 mol% GD. Further, the lipid matrix has the ability to modulate the conformation of the inserted polypeptide. The balance between double-helical and helical dimer structures of GD depends on the phospholipid chain length and phase state. A large hydrophobic mismatch, such as in gel phase one-component DSPC bilayers, leads to an increase in population of double-helical structures. Using an effective molecular sorting mechanism, a large hydrophobic mismatch can be avoided in the DMPC-DSPC lipid mixture, which leads to significant changes in the heterogeneous lipid structure and in polypeptide conformation.     

Evaluation of Magainin I interactions with lipid membranes: An optical and electrochemical study

Most antimicrobial peptides (AMPs) have shown clear activity related to the disruption of lipid bilayers. In order to improve knowledge of this subject, the interaction of Magainin I (MagI) with phospholipid layers (PLs), uncoated or coated with synperonic (Synp), was studied using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) techniques. MagI peptide was immobilized on gold electrode via a self-assembling monolayer obtained from liposomes and liposomes covered by Synp. MagI induces pores in the supported lipid membranes, which are reflected in an increased amperometric-response and also a decreased electron-transfer resistance (R(CT)). In addition, MagI showed a significant interaction with the PL-Synp-modified gold electrode, but MagI showed a reliable contact with the PL-modified gold electrode, leading to a decrease in the relative resistance charge transfer value of -17.38%. Our results demonstrated that Synp acts as a membrane sealant after exposure of the lipid membrane to MagI. A parallel reaction model was proposed for the interaction of MagI and a hybrid layer that result in a complex bimolecular interaction. In short, the importance of triblock copolymer to stabilize liposomes for future applications as drug delivery systems for MagI was demonstrated.     

Impedance analysis and single-channel recordings on nano-black lipid membranes based on porous alumina

Ordered porous alumina substrates with pore diameters of 55 and 280 nm, respectively, were produced and utilized as a support to prepare membranes suspending the pores of the material. Highly ordered porous alumina was prepared by an anodization process followed by dissolution of the remaining aluminum and alumina at the backside of the pores. The dissolution process of Al(2)O(3) at the backside of the pores was monitored by electrical impedance spectroscopy ensuring the desired sieve-like structure of the porous alumina. One side of the porous material with an area of 7 mm(2) was coated with a thin gold layer followed by chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol. The hydrophobic monolayer on top of the upper surface was a prerequisite for the formation of suspending membranes, termed nano-black lipid membranes (nano-BLMs). The formation process, and long-term and mechanical stability of the nano-BLMs were followed by electrical impedance spectroscopy indicating the formation of lipid bilayers with typical specific membrane capacitances of (0.65 +/- 0.2) micro F/cm(2) and membrane resistances of up to 1.6 x 10(8) Omega cm(2). These high membrane resistances allowed for single-channel recordings. Gramicidin as well as alamethicin was successfully inserted into the nano-BLMs exhibiting characteristic conductance states.     

High-pressure infrared study of phosphatidylserine bilayers and their interactions with the local anesthetic tetracaine

High-pressure Fourier-transform infrared (FT-IR) spectroscopy was used to study the barotropic behavior of phosphatidylserine bilayers and their interactions with the local anesthetic tetracaine. The model membrane systems studied were multilamellar aqueous dispersions of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) in the absence and the presence of tetracaine at pH 5.5 and 9.5. The infrared spectra were measured at 28 degrees C in a diamond anvil cell as a function of pressure up to 25 kbar. The results show that the barotropic behavior of the negatively charged phosphatidylserine bilayers is very similar to that observed for zwitterionic phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, with corresponding acyl chains. The results also indicate that the local anesthetic partitions into phosphatidylserine bilayers in an environment close to the membrane-water interface and interacts electrostatically with the lipid head group. Application of high hydrostatic pressure on the lipid-anesthetic systems results in the pressure-induced expulsion of the anesthetic from a membrane to an aqueous environment. The pressures required for expulsion of anesthetic from bilayers are much higher for the unsaturated lipid (DOPS) than for the saturated lipid (DMPS) (approximately 6 kbar vs approximately 2 kbar, respectively). Whereas incorporation of the anesthetic into DOPS bilayers does not affect significantly the structural and dynamic properties of the disordered acyl chains in the liquid-crystalline phase, it orders the DMPS acyl chains in the gel phase.