J Membr Biol. 2016 Dec;249(6):833-844.

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

Hadeel Alobeedallah a, Bruce Cornell b, Hans Coster a

a School of Chemical and Biomolecular Engineering, The University of Sydney, Sydney, 2006, Australia

b SDx Tethered Membranes Pty Ltd, Roseville, Sydney, 2069. Australia

 

Abstract

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 was 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 center of the bilayer and an outer hydrophobic region. BZA was found to have the largest effect on the outer, hydrophobic region, although the inner hydrophobic region was also consistently affected.

KEYWORDS: Tethered lipid membranes; Benzyl alcohol (BZA); Electrical Impedance Spectroscopy (EIS).

PMID: 27803961

 

Supplement:

The changes that occur in the structure of lipid membranes upon the addition of different substances, such as toxins, drugs and anaesthetics, or changes in physical parameters, such as temperature, have been investigated in some detail [1-6]. Moreover, the general effect of certain anaesthetics, such as benzyl alcohol (BZA, a liquid local anaesthetic) on the structure of lipid membranes has been studied [7-11]. However, the mechanisms by which anaesthetic substances induce an anaesthetised state remain unclear. In particular, the sites where anaesthetic molecules sit in the lipid membrane are still not completely clear. Where the anaesthetic molecules sit will affect the physical structure of the lipid membrane, which in turn affects the organisation and function of membrane proteins. Membrane proteins are responsible for generating action potentials in nerves, and thus blocking of the function of membrane proteins will produce an anaesthetised state. There have been previous studies on the effects of anaesthetic molecules on the electrical properties of lipid membranes but a major difficulty arises with the fragility, or lack of longevity, of artificial lipid bilayer membranes. To date, studies that have investigated the effect of anaesthetics on lipid bilayers have used mostly planar free-standing black lipid membranes (BLM) and solid supported lipid membranes (sBLM). With the new molecularly tethered lipid bilayers new avenues are available for performing experiments on the effects of incorporating molecules such as BZA into the membrane.

The molecular tethers in these tethered membranes also mimic the anchors provided by proteins within cell membranes that are cross-linked via intrinsic membrane proteins, such as spectrin. tBLM offer some degree of two-dimensional fluidity in the liquid crystalline phase and can provide exposure to aqueous reservoirs on each side of the bilayer.

Very high-resolution electrical impedance spectroscopy (EIS) was used to determine the dielectric structure, thickness and capacitance of the lipid bilayers and associated interfaces. This forms the basis [Coster et al. [12]] to determine the electrically distinct layers within the membrane. In this method, the dispersions in capacitance and conductance at ultra-low frequencies (0.001 – 1000 Hz) were used to obtain capacitance and conductivity of the individual sub-structural layers. This discovery was crucial for the determination of the effect of different chemical moieties, such as anaesthetics, on the internal component layers of lipid membranes. This technique was used in this study to elucidate the changes in the sub-structural dielectric parameters when BZA was incorporated into tethered lipid membranes.

 

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