The Effect of Benzyl Alcohol on the Voltage-Current Characteristics of Tethered Lipid Bilayers.

In this study a lipid bilayer membrane model was used in which the bilayer is tethered to a solid substrate with molecular tethers. Voltage-current (V-I) measurements of the tethered bilayer membranes (tBLM) and tBLM with benzyl alcohol (BZA) incorporated in their structures, were measured using triangular voltage ramps of 0-500 mV. The temperature dependence of the conductance deduced from the V-I measurements are described. An evaluation of the activation energies for electrical conductance showed that BZA decreased the activation/ Born energies for ionic conduction of tethered lipid membranes. It is concluded that BZA increased the average pore radius of the tBLM.

The Effect of Cholesterol on the Voltage-Current Characteristics of Tethered Lipid Membranes.

In this study, a lipid bilayer membrane model was used, in which the bilayer is tethered to a solid substrate with molecular tethers. The V-I characteristics of the lipid bilayers were found to be non-linear which suggests the presence of pores that are voltage-dependent. At high applied voltages, the conductance reached a limiting value, presumably indicating a limit on the maximum pore size. A decrease in the spacing between tethers (increasing tether density) caused a decrease in the membrane's conductance at high applied voltage, which is consistent with the maximum pore size being determined by the spacing between the tethers. The inclusion of 10 M% cholesterol within the membrane lipid caused a decrease in the membrane conductance. However, the inclusion of higher levels of cholesterol increased the membrane conductance.

The Effect of Cholesterol on the Dielectric Structure of Lipid Bilayers.

Cholesterol plays an important role in regulating the properties of phospholipid bilayers and many mechanisms have been proposed to explain why cholesterol is so ubiquitous within biological membranes of animals. Here we present the results of studies on the effect of cholesterol on the electrical/dielectric properties of lipid membranes tethered to a solid substrate. These tethered bilayer lipid membranes tBLM were formed on a commercially available chemically modified gold substrate. These lipid bilayers are very robust. Very high-resolution electrical impedance spectroscopy (EIS) was used to determine the dielectric structure of the lipid bilayers and associated interfaces. The EIS data allowed the dielectric substructure of the lipid bilayers to be determined. The results showed that when cholesterol was present in the tethered membranes at a concentration of 10% (mol/mol); the thickness of the tBLMs increased and the membrane conductance decreased. However, when cholesterol was present in the tethered membrane at more than 30% (mol/mol) the effect of cholesterol was dramatically different; the membranes then became thinner and possessed a much larger electrical conductance. The EIS allowed a distinction to be made between a hydrophobic region in the center of the bilayer and another hydrophobic region further out towards the polar head region, in addition to the polar head region itself. Cholesterol was found to have the largest effect on the inner, hydrophobic region, although the outer hydrophobic region was also affected.

The Effect of Benzyl Alcohol on the Dielectric Structure of Lipid Bilayers.

Molecularly tethered lipid bilayer membranes were constructed on a commercially available chemically modified gold substrate. This is a new and promising product that has allowed the construction of very robust lipid bilayers. Very high resolution electrical impedance spectroscopy (EIS) was used to determine the dielectric structure of the lipid bilayers and associated interfaces. The EIS data were modelled in terms of the dielectric substructure using purpose developed software. The hydrophobic region, where the lipid tails are located, was revealed by the EIS in the frequency range of (1-100) Hz and its thickness was calculated from the capacitance of this region and found to be approximately 3-4 nm. The hydrophilic region, where the polar heads are located, was revealed at higher frequencies and its thickness was estimated to be approximately 1-2 nm. The effect of the local anaesthetic benzyl alcohol (BZA) on the tethered lipid bilayers was investigated. The effect of BZA on the membrane capacitance and conductance allowed the changes in the thickness of the polar head and hydrophobic tails regions to be determined. It was found that the addition of BZA caused a significant increase in the capacitance (corresponding to a decrease in the thickness) of the hydrophobic region and an increase in the membrane electrical conductance. The EIS allowed a distinction between a hydrophobic region in the centre of the bilayer and an outer hydrophobic region. Benzyl alcohol was found to have the largest effect on the outer, hydrophobic region, although the inner hydrophobic region was also consistently affected.

Evidence of the Key Role of H3O+ in Phospholipid Membrane Morphology.

This study explains the importance of the phosphate moiety and H3O+ in controlling the ionic flux through phospholipid membranes. We show that despite an increase in the H3O+ concentration when the pH is decreased, the level of ionic conduction through phospholipid bilayers is reduced. By modifying the lipid structure, we show the dominant determinant of membrane conduction is the hydrogen bonding between the phosphate oxygens on adjacent phospholipids. The modulation of conduction with pH is proposed to arise from the varying H3O+ concentrations altering the molecular area per lipid and modifying the geometry of conductive defects already present in the membrane. Given the geometrical constraints that control the lipid phase structure of membranes, these area changes predict that organisms evolving in environments with different pHs will select for different phospholipid chain lengths, as is found for organisms near highly acidic volcanic vents (short chains) or in highly alkaline salt lakes (long chains). The stabilizing effect of the hydration shells around phosphate groups also accounts for the prevalence of phospholipids across biology. Measurement of ion permeation through lipid bilayers was made tractable using sparsely tethered bilayer lipid membranes with swept frequency electrical impedance spectroscopy and ramped dc amperometry. Additional evidence of the effect of a change in pH on lipid packing density is obtained from neutron reflectometry data of tethered membranes containing perdeuterated lipids.

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.

Discovery of "punch-through" or membrane electrical breakdown and electroporation.

As part of a detailed study in Alex Hope's laboratory of the V-I characteristics of the membrane of the giant cells of Chara corallina, it was discovered that at a well defined potential difference of around 500 mV (depending on the temperature), the cell membrane undergoes a reversible electrical breakdown. The author coined the word "punchthrough" to describe this electrical breakdown phenomenon. Detailed studies followed on the nature of this electrical breakdown phenomenon, in various cells, aimed at elucidating the physical mechanism(s) involved. The applications and the significance of the phenomenon that were subsequently developed in later years were not foreseen at that time. Electrical breakdown/electroporation is now a commonplace procedure and has entered into the mainstream biological vocabulary. Here we trace its humble beginnings to experiments carried out in Alex Hope's laboratory and review briefly some of the aspects of this phenomenon and its applications that were developed much later by others as well as the author. The discovery of membrane electrical breakdown described below took whilst the author was his student of Alex Hope but whilst Alex was away on sabbatical leave in the UK. Because this occurred in his absence, Alex Hope elected to not put his name on the paper that described the discovery in 1965.

Biographical memoir: Alexander Beaumont Hope, Australian biophysicist, 1928-2008.

This introductory article is the first of four short articles from the Tribute to Alex Hope Symposium held at the 2008 Australian Society for Biophysics meeting in Canberra, Australia, as a tribute to Professor Alex Hope, who died in July last year. As well as briefly introducing the other three articles by three former PhD students, it will also be a biographical memoir of Alex Hope.

Electric field effects in proteins in membranes.

Both the organization and function of protein nanostructures in membranes are related to the substructural properties of the lipid portion of the membrane. Potential differences that are established across the membrane and generate electric fields in these very thin portions are shown to modulate the organizational and functional properties of the protein modules. Many protein modules also have nonisotropic distributions of charged sites, including configurations in which there are regions containing predominantly positive fixed charges, juxtaposed with adjacent regions containing predominantly negative fixed charges. In these double fixed charge regions, very large electric fields can manifest in the ionic depletion layer at the junction of the two fixed charge regions. Consideration is also given to the manner in which the intense electric fields that are established in protein modules, such as proton ATPases, can modulate the chemical reactions that are associated with proton transport and dehydration reactions.

Electrical breakdown of human erythrocytes: a technique for the study of electro-haemolysis.

This paper describes a technique suitable for investigating the electromechanical breakdown properties of erythrocyte cells. The cells were exposed to square wave electric pulses of precise duration and voltage. The erythrocytes were suspended in normal isotonic saline between two opposing platinum electrodes. A red LED light source and photodiode detector system were positioned orthogonally to the electrodes to record changes in the light transmission that occur immediately after applying an electric pulse. The light transmitted through the electrically treated erythrocyte suspension could be monitored continuously. Experiments were conducted to explore the inter-relationship between the critical voltage and pulse length for haemolysis. Human blood taken from "healthy" donors underwent haemolysis at a critical field strength of 304 kV/m for a 5 micros pulse and 292 kV/m for a 50 micros pulse. The relationship of critical pulse length and critical voltage for the blood samples was found to be inversely linear.

Dielectrophoretic spectra of translational velocity and critical frequency for a spheroid in traveling electric field.

An analysis has been made of the dielectrophoretic (DEP) forces acting on a spheroidal particle in a traveling alternating electric field. The traveling field can be generated by application of alternating current signals to an octapair electrode array arranged in phase quadrature sequence. The frequency dependent force can be resolved into two orthogonal forces that are determined by the real and the imaginary parts of the Clausius-Mossotti factor. The former is determined by the gradient in the electric field and directs the particle either toward or away from the tip of the electrodes in the electrode array. The force determined by the imaginary component is in a direction along the track of the octapair interdigitated electrode array. The DEP forces are related to the dielectric properties of the particle. Experiments were conducted to determine the DEP forces in such an electrode arrangement using yeast cells (Saccharomyces cervisiate TISTR 5088) with media of various conductivities. Experimental data are presented for both viable and nonviable cells. The dielectric properties so obtained were similar to those previously reported in literature using other DEP techniques.

Structural characterization of organic multilayers on silicon(111) formed by immobilization of molecular films on functionalized Si-C linked monolayers.

Silicon(111)-H surfaces were derivatized with omega-functionalized alkenes in UV-mediated and thermal hydrosilylation reactions to give Si-C linked monolayers. Additional molecular layers of organic compounds were coupled either directly or via linker molecules to the functionalized alkyl monolayers. In the first instance, amino-terminated monolayers were prepared from a tert-butoxycarbonyl-protected omega-aminoalkene followed by removal of the protecting group. Various thiols were coupled to the monolayer using a heterobifunctional linker, which introduced maleimide groups onto the surface. In the second system, N-hydroxysuccinimide (NHS) ester-terminated monolayers were formed by reaction of Si-H with N-succinimidyl undecenoate. The reactivity of the NHS ester groups was confirmed by further modification of the monolayer. The stepwise assembly of these multilayer structures was characterized by X-ray reflectometry and X-ray photoelectron spectroscopy.