Bruce Cornell

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.

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.

EPR and NMR measurements on high-temperature superconductors

Some EPR and NMR experiments at the Cu sites in the mixed-phase materials Ba0.6Y0.4CuOy and Ba0.2Y0.8CuOy, and the pure-phase compounds YBa2Cu3Oy (1:2:3), BaCuO2 (0:1:1), Y2Cu2O5 (2:0:2), Y2BaCuO8 (2:1:1), YBa3Cu2Oy (1:3:2) and CuO are presented and discussed. In particular, it is shown that any EPR signal in nominally pure 1:2:3 superconducting material is probably due to a small amount of impurity 2:1:1 (greenphase) compound. For the 2:1:1 and 1:3:2 compounds the authors find g/sub /// approximately=2.23 and gperpendicular to approximately=2.09 with a linewidth of approximately=0.025 T, whereas for the 2:0:2 compound g=2.12(2) with a linewidth of 0.047(3)T. Some suggestions are also made concerning the absence of both NMR and EPR signals in the 1:2:3 compound, in terms of (i) Andersons resonating bond model and (ii) recent FLAPW band-structure calculations. In particular, it is argued that the delocalised nature of the Cu d electrons probably gives rise to exceptional spin-spin dipolar broadening of both the NMR and EPR transitions.

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.

Ion-Channel Biosensors—Part I: Construction, Operation, and Clinical Studies

This paper deals with the construction and operation of a novel biosensor that exploits the molecular switching mechanisms of biological ion channels. The biosensor comprises gramicidin A channels embedded in a synthetic tethered lipid bilayer. It provides a highly sensitive and rapid detection method for a wide variety of analytes. In this paper, we outline the fabrication and principle of operation of the ion-channel switch (ICS) biosensor. The results of a clinical study, in which the ion-channel biosensor is used to detect influenza A in untreated clinical samples, is presented to demonstrate the utility of the technology. Fabrication of biochip arrays using silicon chips decorated with ¿ink jet¿ printing is discussed. We also describe how such biochip arrays can be used for multianalyte sensing. Finally, reproducibility/stability issues of the biosensor are addressed.

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.

Bacterial Mechanosensitive Channels: Models for Studying Mechanosensory Transduction

SIGNIFICANCE Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. RECENT ADVANCES As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. CRITICAL ISSUES In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. FUTURE DIRECTIONS A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology.

Ion Channel Biosensors—Part II: Dynamic Modeling, Analysis, and Statistical Signal Processing

This paper deals with the dynamic modeling, analysis, and statistical signal processing of the ion channel switch biosensor. The electrical dynamics are described by a second-order linear system. The chemical kinetics of the biosensor response to analyte concentration in the reaction-rate-limited regime are modeled by a two-timescale nonlinear system of differential equations. Also, the analyte concentration in the mass-transport-influenced regime is modeled by a partial differential equation subject to a mixture of Neumann and Dirichlet boundary conditions. By using the theory of singular perturbation, we analyze the model so as to predict the performance of the biosensor in transient and steady-state regimes. Finally, we outline the use of statistical signal processing algorithms that exploit the biosensor dynamics to classify analyte concentration.

Orientation dependence of NMR relaxation time, T1ρ, in lipid bilayers

The orientation dependence of the low frequency NMR relaxation time, T(1rho), of protons in aligned phospholipid bilayers was measured using 13C cross polarisation and direct proton experiments. The contribution of intra- and inter-molecular interactions to proton T(1rho) was determined by using dimyristoyl phosphatidylcholine (DMPC) with one hydrocarbon chain deuterated and dispersed in perdeuterated DMPC. The results indicated that intramolecular motions on the kHz timescale were the major cause of T(1rho) relaxation in phospholipid bilayers.

A molecular machine biosensor: Construction, predictive models and experimental studies

This paper describes the construction, operation and predictive modeling of a molecular machine, functioning as a high sensitivity biosensor. Embedded gramicidin A (gA) ionchannels in a self-assembled tethered lipid bilayer act as biological switches in response to target molecules and provide a signal amplification mechanism that results in high sensitivity molecular detection. The biosensor can be used as a rapid and sensitive point of care diagnostic device in different media such as human serum, plasma and whole blood without the need for pre and post processing steps required in an enzyme-linked immunosorbent assay. The electrical reader of the device provides the added advantage of objective measurement. Novel ideas in the construction of the molecular machine, including fabrication of biochip arrays, and experimental studies of its ability to detect analyte molecules over a wide range of concentrations are presented. Remarkably, despite the complexity of the device, it is shown that the response can be predicted by modeling the analyte fluid flow and surface chemical reactions. The derived predictive models for the sensing dynamics also facilitate determining important variables in the design of a molecular machine such as the ion channel lifetime and diffusion dynamics within the bilayer lipid membrane as well as the bio-molecular interaction rate constants.