Preparing Ion Channel Switch Membrane-Based Biosensors.

Monitoring the changes in membrane conductance using electrical impedance spectroscopy is the platform of membrane-based biosensors in order to detect a specific target molecule. These biosensors represent the amalgamation of an electrical conductor such as gold and a chemically tethered bilayer lipid membrane with specific incorporated ion channels such as gramicidin-A that is further functionalized with detector molecules of interest.

Langmuir-Schaefer Deposition to Create an Asymmetrical Lipopolysaccharide Sparsely Tethered Lipid Bilayer.

Because they are firmly anchored to a noble metal substrate, tethered bilayer lipid membranes (tBLMs) are considerably more robust than supported lipid bilayers such as black lipid membranes (BLMs) (Cranfield et al. Biophys J 106:182-189, 2014). The challenge to rapidly create asymmetrical tBLMs that include a lipopolysaccharide outer leaflet for bacterial model membrane research can be overcome by the use of a Langmuir-Schaefer deposition protocol. Here, we describe the procedures required to assemble and test asymmetric lipopolysaccharide (LPS) tethered lipid bilayers.

Measuring Voltage-Current Characteristics of Tethered Bilayer Lipid Membranes to Determine the Electro-Insertion Properties of Analytes.

Tethered bilayer lipid membranes (tBLMs) anchored to a solid substrate can be prepared and individual triangular voltage ramps from zero to 500 mV with a period of 2-10 ms applied to give membrane voltage dependencies with and without the addition of drugs and analytes in order to measure their electro-insertion properties.

Measuring Activation Energies for Ion Transport Using Tethered Bilayer Lipid Membranes (tBLMs).

Model lipid bilayers tethered to a gold substrate with molecular tethers are constructed. The conductance versus temperature dependence curve is then obtained. Here, a method to measure the activation energy for translocation of an ion through existing transmembrane pores in a sparsely tethered bilayer lipid membranes is presented.

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes.

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

Real-time monitoring of heat transfer between gold nanoparticles and tethered bilayer lipid membranes.

Plasmon resonance frequency irradiated gold nanoparticles (GNPs) have gained interest as a laser-targeted treatment for infections, tumors and for the controlled release of drugs in situ. Questions still remain, however, as to the efficiency of heat delivery within biological tissues and how this can be reliably determined. Here, we demonstrate how a nanomaterial-electrode interface that mimics cell membranes can detect the localized heat transfer characteristics arising from plasmon resonance frequency-matched laser excitation of GNPs. We demonstrate that the lipid bilayer membrane can be affected by conjugated GNP induced hyperthermia when irradiated with a laser power output as low as 135 nW/µm2. This is four orders of magnitude lower power than previously reported. By restricting the lateral movement of the lipids in the bilayer membrane, it was shown that the change in membrane conductance as a result of the heat transfer was due to the creation of transient lipidic toroidal pores within the membrane. We further demonstrate that the heat transfer from the GNPs alters diffusion rates of monomers of the gramicidin-A peptide within the lipid leaflets. This work highlights how targeted low laser power GNP hyperthermia treatments, in vivo, could play a dual role of interfering with both cell membrane morphology and dynamics, along with membrane protein function.

A conserved GXXXG motif in the transmembrane domain of CLIC proteins is essential for their cholesterol-dependant membrane interaction.

BACKGROUND: Sterols have been reported to modulate conformation and hence the function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family of proteins. The CLIC protein family consists of six evolutionarily conserved protein members in vertebrates. These proteins exist as both monomeric soluble proteins and as membrane bound proteins. To date, the structure of their membrane-bound form remains unknown. In addition to several studies indicating cellular redox environment and pH as facilitators of CLIC1 insertion into membranes, we have also demonstrated that the spontaneous membrane insertion of CLIC1 is regulated by membrane cholesterol. METHOD: We have performed Langmuir-film, Impedance Spectroscopy and Molecular Docking Simulations to study the role of this GXXXG motif in CLIC1 interaction with cholesterol. RESULTS: Unlike CLIC1-wild-type protein, the G18A and G22A mutants, that form part of the GXXXG motif, showed much slower initial kinetics and lower ion channel activity compared to the native protein. This difference can be attributed to the significantly reduced membrane interaction and insertion rate of the mutant proteins and/or slower formation of the final membrane configuration of the mutant proteins once in the membrane. CONCLUSION: In this study, our findings uncover the identification of a GXXXG motif in CLIC1, which likely serves as the cholesterol-binding domain, that facilitates the protein's membrane interaction and insertion. Furthermore, we were able to postulate a model by which CLIC1 can autonomously insert into membranes to form functional ion channels. GENERAL SIGNIFICANCE: Members of the CLIC family of proteins demonstrate unusual structural and dual functional properties - as ion channels and enzymes. Elucidating how the CLIC proteins' interact with membranes, thus allowing them to switch between their soluble and membrane form, will provide key information as to a mechanism of moonlighting activity and a novel regulatory role for cholesterol in such a process.

Nanostructural determination of a lipid bilayer tethered to a gold substrate.

Tethered lipid bilayer membranes (tBLM) are planar membranes composed of free lipids and molecules tethered to a solid planar substrate providing a useful model of biological membranes for a wide range of biophysical studies and biotechnological applications. The properties of the tBLM depend on the free lipids and on the chemistry of the tethering molecules. We present a nanoscale characterization of a tBLM composed of deuterated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (d-DMPC) free lipids, benzyl disulfide undecaethylene glycol phytanol (DLP) tethering molecules, and benzyl disulfiide tetraethylene glycol polar spacer molecules (PSM) used to control the areal density of tethering molecules through coadsorption. The use of selected isotopic substitution provides a way to distinguish the conformation and location of the tethered lipids from the free lipids and to elucidate how the two components influence the structure of the tBLM. These findings provide useful information to optimise the insertion of transmembrane proteins into the tethered bilayer system.

Investigating Sterol and Redox Regulation of the Ion Channel Activity of CLIC1 Using Tethered Bilayer Membranes.

The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans. These are a group of enigmatic proteins, which adopt both a soluble and membrane bound form. CLIC1 was found to be a metamorphic protein, where under specific environmental triggers it adopts more than one stable reversible soluble structural conformation. CLIC1 was found to spontaneously insert into cell membranes and form chloride ion channels. However, factors that control the structural transition of CLIC1 from being an aqueous soluble protein into a membrane bound protein have yet to be adequately described. Using tethered bilayer lipid membranes and electrical impedance spectroscopy system, herein we demonstrate that CLIC1 ion channel activity is dependent on the type and concentration of sterols in bilayer membranes. These findings suggest that membrane sterols play an essential role in CLIC1's acrobatic switching from a globular soluble form to an integral membrane form, promoting greater ion channel conductance in membranes. What remains unclear is the precise nature of this regulation involving membrane sterols and ultimately determining CLIC1's membrane structure and function as an ion channel. Furthermore, our impedance spectroscopy results obtained using CLIC1 mutants, suggest that the residue Cys24 is not essential for CLIC1's ion channel function. However Cys24 does appear important for optimal ion channel activity. We also observe differences in conductance between CLIC1 reduced and oxidized forms when added to our tethered membranes. Therefore, we conclude that both membrane sterols and redox play a role in the ion channel activity of CLIC1.

Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

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.

Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol.

The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.

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.