Membrane Conduction and Ion ChannelsKey Learning Objectives: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 classyou will be asked to fabricate a tethered membrane, insert gramicidin, and measure itsconductance.6. You will be instructed to form a tethered membrane and to include in it the ion channelGramicidin.7. You will need to measure the conductivity of Gramicidin in membrane and determine hemembrane thickness.SDx tethered membranes March 2014.BioNanotechnology Practical: Ion Channels in Membranes.Background:Cell MembranesCell membrane properties control the behaviour of all plants, bacteria and animals. Cell membranes consistof self-assembled supramolecular structures formed by amphiphiles, or compounds that have polar segmentsthat strongly attract water and non-polar segments that do not. This results in the non-polar segments beingexcluded from the aqueous phase and assembling into bimolecular sheets which eventually form closedspheres which are the precursors of biological cells. The amphiphiles we are interested in here are known aslipids and the cell-like structures they form when dispersed in water are known as liposomes. Liposomes canbe 10 nm to hundreds of micrometres in diameter but all have walls that are approximately 4 nm thick, andare nearly impermeable to ions such as Na+, K+ and Cl-. The 4 nm thick lipid bilayer, that forms the wall of aliposome 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 andautonomic nervous systems.? The senses of sight, sound, taste touch and smell in animals, and related functions in plantsand bacteria.? Mitochondrial metabolism and bioenergetics.Cell membrane biochemistry is a core discipline within medical research and a core interest of thePharmaceutical 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 onreplacement antibiotics for penicillin which is becoming increasingly ineffective against methicillin resistantbacterial strains of Staphylococcus Aureus. Compounds that interact with membranes are also important inunderstanding 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 orsingle cells pieced using fragile microelectrodes. Tethered membranes provide a stable planar phospholipidbilayer over a relatively large surface area (2-3 mm2) that is a convenient alternative tool to study iontransport in membrane bound ion channels. The tethering of the membrane is achieved using sulphurchemistry to gold (gold is not totally unreactive and possesses a chemistry with sulphur). Molecular tethersare thus molecules that possess a sulphur group, polar linkers and a hydrophobic segment that embeds in thelipid bilayer. The polar linkers allow the existence of an aqueous layer, between the gold electrode and themembrane. The assembly of a tethered membrane is shown below.SDx tethered membranes March 2014.BioNanotechnology Practical: Ion Channels in Membranes.(a) Ethanol solutions containing 0.4mMdisulphides are exposed to pure fresh goldfor 30 minutes. The molecules collide withthe gold and sulphur-gold bonds form,causing the self assembly of a lipid-spacermonolayer. In todays practical class 10%of the molecules are hydrophobic lipidicanchor groups, and ninety percent arehydrophilic spacers. This ratio can bereduced to below 1% tether molecules orup to 100% tether molecules. The motivefor reducing the fraction of tethers is toprovide more space to incorporate largechannels or to increase the number oftethers to fabricate a more stable device.(b) Following the adsorption of the selfassembled monolayer at the gold surface afurther 8ul of 3mM free lipid in ethanol isallowed to assemble at the surface and thenrinsed with buffer.(c) Rinsing with buffer causes the mix oftethered and free lipids to form into atethered bilayer, 4nm thick on a 3nmhydrophilic cushion. The hydrophiliccushion mimics the inside of a cell and thelipid bilayer mimics a cell membrane.Ion Channels:SDx tethered membranes March 2014.BioNanotechnology Practical: Ion Channels in Membranes.Ion channels are molecules that create hydrophilic pathways across lipid bilayer membranes permiting ionsto cross otherwise impermeable membranes. Common bacteria such as Pneumonia, Diphtheria, GoldenStaphylococcus and Anthrax are pathogenic because the toxins they produce are ion channels that puncturethe 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 FigureBelow). Being much smaller, with molecular weight of 1882 Da, two molecules end-to-end are required tospan the lipid bilayer. Gramicidin is ion selective and is only conducive to monovalent cations (especiallyNa+).The bacterial ion channel gramicidin (gA). Monomers in the inner and outer leaflets of the bilayermembrane 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 appliedand 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 throughwhich ions pass.SDx tethered membranes March 2014.BioNanotechnology Practical: Ion Channels in Membranes.Membrane Preparation kitA 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) [SDxtethaPod™ ], that reads both the membrane conductance and capacitance. A cartridge preparation kit issupplied 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 3Cbelow)?a silicon rubber pressure pad used when attaching the electrode to the flow cell cartridge (Fig. 3Dbelow)?an aluminium pressure plate used when attaching the electrode to the flow cell cartridge (Fig. 3Ebelow)?a pressure clamp is used when attaching the electrode to the flow cell cartridge (Fig. 1B below)FIGURE 1FIGURE 2FIGURE 3SDx tethered membranes March 2014.BioNanotechnology Practical: Ion Channels in Membranes.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 forthe adhesive to seal.FIGURE 4Introduction to practical exerciseAim: 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 t
he ionselectivity.SDx tethered membranes March 2014.BioNanotechnology Practical: Ion Channels in Membranes.5. To measure the membrane capacitance.6. Use this measurement to calculate the thickness of a lipid bilayer.7.Note!Ensure all equipment, instrumentation and chemicals are available when youstart. Timing is critical for proper membrane formation. Read the entireexperiment through before commencing.Exercise 1. Prepare tethered membranes containing gramicidina. Cut open the silver foil pack, and using tweezers remove the slide.b. (Never touch the gold with fingers as this may damage the lipid coating lipid formation of themembrane.)c. The electrode is stored in ethanol and you need to stand it on a tissue to dry. This may take 1-2minutes.d. Align the dry slide over the alignment jig, ensuring electrode tracks and the SDX logo on the slideoverlay each other. Using tweezers gently push electrode into the slot.e. Remove top thin protective layer of plastic from the cartridge. (Be sure that it is only the thinprotective layer that is removed and not the entire adhesive laminate.) This will reveal a stickysurface which will then bind to the electrode upon contact.f. Position white cartridge over the top and push into position. Once the two surfaces meet do not peelthem apart or attempt to re-locate them as it will damage the electrode.g. Gently put the cartridge and electrode into the clamp and tighten. Allow to stand for at least 1minute, before loosening the pressure. The electrode is now ready for membrane formation.Membranes are formed as follows: