BioNanotechnology Practical: Ion Channels in Membranes.

1. Bilayer lipid membranes (BLM) are the major constituent of cell membranes.
2. BLMs block the passage of ions such as Na+, K+, Cl- and Ca++.
3. Ion channels penetrate cell membranes permitting the passage of ions across the membrane.
4. A convenient tool for studying the ion transport properties is a tethered membrane that is more
stable and easier to study than many other model systems.
5. Gramicidin is bacterial polypeptide and is an example of an ion channel. In this practical class
you will be asked to fabricate a tethered membrane, insert gramicidin, and measure its conductance.

6. You will be instructed to form a tethered membrane and to include in it the ion channel Gramicidin.
7. You will need to measure the conductivity of Gramicidin in membrane and determine he membrane thickness.

SDx tethered membranes March 2014

Background:
Cell Membranes
Cell membrane properties control the behaviour of all plants, bacteria and animals. Cell membranes consist of self-assembled supramolecular structures formed by amphiphiles, or compounds that have polar segments that strongly attract water and non-polar segments that do not. This results in the non-polar segments being excluded from the aqueous phase and assembling into bimolecular sheets which eventually form closed spheres which are the precursors of biological cells. The amphiphiles we are interested in here are known as lipids and the cell-like structures they form when dispersed in water are known as liposomes. Liposomes can be 10 nm to hundreds of micrometres in diameter but all have walls that are approximately 4 nm thick, and are nearly impermeable to ions such as Na+, K+ and Cl-. The 4 nm thick lipid bilayer, that forms the wall of a liposome is similar to that found in all cell membranes, whether they are from bacteria, plants or animals.

Alterations in membrane ionic permeability are the basis of:
 Signalling between neurones in the brain, and between neurones in the sympathetic and autonomic nervous systems.
 The senses of sight, sound, taste touch and smell in animals, and related functions in plants
and bacteria.

 Mitochondrial metabolism and bioenergetics.
Cell membrane biochemistry is a core discipline within medical research and a core interest of the Pharmaceutical Industry when searching for drug targets to address a wide range of medical conditions.
Membrane research is a significant component of a current major international research effort focussed on replacement antibiotics for penicillin which is becoming increasingly ineffective against methicillin resistant bacterial strains of Staphylococcus Aureus. Compounds that interact with membranes are also importan t in understanding the effects of many types of venom, toxins, and some chemical warfare agents..
Tethered membranes:
Traditional techniques used to study transmembrane ion transport require the use very small liposomes or single cells pieced using fragile microelectrodes. Tethered membranes provide a stable planar phospholipid bilayer over a relatively large surface area (2-3 mm2) that is a convenient alternative tool to study ion transport in membrane bound ion channels. The tethering of the membrane is achieved using sulphur chemistry to gold (gold is not totally unreactive and possesses a chemistry with sulphur). Molecular tethers are thus molecules that possess a sulphur group, polar linkers and a hydrophobic segment that embeds in the lipid bilayer. The polar linkers allow the existence of an aqueous layer, between the gold electrode and the membrane. The assembly of a tethered membrane is shown below.

(a) Ethanol solutions containing 0.4mM disulphides are exposed to pure fresh gold for 30 minutes. The molecules collide with the gold and sulphur-gold bonds form, causing the self assembly of a lipid-spacer monolayer. In todays practical class 10% of the molecules are hydrophobic lipidic anchor groups, and ninety percent are hydrophilic spacers. This ratio can be reduced to below 1% tether molecules or up to 100% tether molecules. The motive for reducing the fraction of tethers is to provide more space to incorporate large channels or to increase the number of tethers to fabricate a more table device.
(b) Following the adsorption of the self assembled monolayer at the gold surface a further 8ul of 3mM free lipid in ethanol is allowed to assemble at the surface and then rinsed with buffer.

(c) Rinsing with buffer causes the mix of tethered and free lipids to form into a tethered bilayer, 4nm thick on a 3nm hydrophilic cushion. The hydrophilic cushion mimics the inside of a cell and the lipid bilayer mimics a cell membrane.

Ion Channels:

Ion channels are molecules that create hydrophilic pathways across lipid bilayer membranes permiting ions to cross otherwise impermeable membranes. Common bacteria such as Pneumonia, Diphtheria, Golden Staphylococcus and Anthrax are pathogenic because the toxins they produce are ion channels that puncture the cells of target organisms and collapse their transmembrane potentials.
Gramicidin (gA): Another ion channel, found in the soil bacteria, B. brevis is gramicidin A (See Figure
Below). Being much smaller, with molecular weight of 1882 Da, two molecules end-to-end are required to span the lipid bilayer. Gramicidin is ion selective and is only conducive to monovalent cations (especially Na+).
The bacterial ion channel gramicidin (gA). Monomers in the inner and outer leaflets of the bilayer
membrane need to align to form a continuous channel to permit ions to cross the membrane.

(a) Schematic figure of gramicidin A in a tethered membrane. An excitation potential of 20mV a.c. is applied and the current due to ions being driven back and forth across the membrane is measured.
(b) More detail of gramcidin A showing two gramicidin monmers aligning and forming a conductive dimer. Beneath the image of the dimer is an end view showing the pore through the centre of gramicidin through which ions pass.

Membrane Preparation kit
A six-channel electrode is provided in this practical class that is to be assembled into a flow cell cartridge (Fig 1A and 2A below). The assembled cartridge plugs into a conductance reader (Fig 2B below) [SDx tethaPod™ ], that reads both the membrane conductance and capacitance. A cartridge preparation kit is supplied by which consists of:
 individually packaged electrodes pre-coated with tethering chemistry (Fig. 3A below)
a flow cell cartridge top containing the gold counter electrode (Fig.2A and 3B below)
an alignment jig for use when attaching the electrode to the flow-cell cartridge (Fig. 1A and 3C
below)
a silicon rubber pressure pad used when attaching the electrode to the flow cell cartridge (Fig. 3D
below)
an aluminium pressure plate used when attaching the electrode to the flow cell cartridge (Fig. 3E
below)
a pressure clamp is used when attaching the electrode to the flow cell cartridge (Fig. 1B below)

FIGURE 1

FIGURE 2

FIGURE 3

In addition to the supplied membrane preparation kit you will need:
(i) Pair of scissors to open the slide pack
(ii) A 10ul and 100ul pipette and tips to deliver the phospholipid (8µl) and rinse with buffer(100µl)
(iii) Tweezers to remove the slide from the sealed pack.
(iv) Waste bin to collect used tips.
(v) Phosphate buffered saline (100ml).
(vi) Timer to measure 2 minute incubation times for forming the membrane and a one minute delay for
the adhesive to seal.

FIGURE 4
Introduction to practical exercise
Aim:
To prepare tethered membranes containing gramicidin A (gA).
To measure the conductance dependence of the membrane on gramicidin concentration.
Use this measurement to calculate the conduction of a dimeric gramicidin channel.
To determine the dependence of conductivity on the bias potential and from this determine the ion
selectivity.
5. To measure the membrane capacitance.
6. Use this measurement to calculate the thickness of a lipid bilayer.

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