Question;BioNanotechnology Practical: Ion Channels in Membranes.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 morestable 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 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 2014Background:Cell MembranesCell 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 plantsand 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 FigureBelow). 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 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 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 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) [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 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 3In 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 (8?l) and rinse with buffer(100?l)(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: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 ionselectivity.5. To measure the membrane capacitance.6. Use this measurement to calculate the thickness of a lipid bilayer.7.b. Add 8?L phospholipid solution (0nMgA) to chamber 1.c. At 15 seconds, add 8?L 40nM gA solution to chamber 2.d. At 30 seconds, add 8?L 60nM gA solution tochamber 3.e. At 45 seconds, add 8?L 80nM gA solution to chamber 4.f. At 60 seconds, add 8?L 100nM gA solution to chamber 5.g. At 75 seconds, add 8?L 120nM gA solution to chamber 6.h. At 120 seconds, to chamber 1 add 100?L PBS.i. At 135 seconds, to chamber 2 add 100?L PBS.j. At 150 seconds, to chamber 3 add 100?L PBS.k. At 165 seconds, to chamber 4 add 100?L PBS.l. At 180 seconds, to chamber 5 add 100?L PBS.m. At 195 seconds, to chamber 6 add 100?L PBS. (Total 3 minutes, 15 seconds elapsed).Exercise 2. Testing the bilayer using AC impedance spectroscopyThe conductance and capacitance of the tethered membrane may be measured by inserting the assembled electrode within the flow cell cartridge into a tethaPod? reader.The reader simplifies the interpretation of the AC impedance spectrum and provides a measure of membrane conductance (?S) and capacitance (nF)Typical conduction values for a freshly formed membrane using the proprietary SDx TM AM199 in PBS are 0.35 ?0.15 ?S and capacitance values of 18?2 nF at room temperature.The conductance is proportional to the ion flux through the membrane and the capacitance is inverselyproportional to the membrane thickness. A significant additional measure using a tethaPod? is theGoodness of Fit (GOF). This indicates the quality of match between the experimental data and a model of the tethered membrane. GOF values of less than 0.2 indicate a good match of the data to this simple model, and suggest that the membrane is uniform.Alternating Current (a.c.) Impedance SpectroscopyA sine wave excitation of 20mV is applied across the tethered membrane between the tethering goldelectrode and the gold counter electrode. The TethaPod device used here fits a three capacitor, one conductor model to the experimental data and provides a readout of Gm (membrane conductance) and Cm (membrane capacitance), thus avoiding the need to perform the more complex calculations.Software The ?Setup? menu, provides the ability to choose the communuication port to your computer. This is usually the highest numbered port displayed. Also ?Setup? permits setting a bias voltage (d.c. potential) across the tethered membrane circuit, (+100mV to -100mV).. Note that this d.c. potential only charges the coupling capacitors at the tethering gold surface and at the counter electrode. No potential is applied across the membrane elements Gm and Cm.The ?Chart? menu, permits a choice of variables to display as a function of time on the graphical trace or a numerical DVM (digital voltmeter) display. Also, The ?Table? menu, permits a choice of variables to show in the tabulation at the bottom of the display. Once the display method is chosen click ?Start? and a display will appear in 1-2 minutes.Note the GoF, GoF is an acronym for ?goodness of fit? which is a measure of the match between themodelled spectrum for the displayed Gm and Cm and the experimental data. A high GoF means a bad fit. A low GoF means a good fit. GoF values should be less than 0.2. Should the GoF be greater than 0.2 it means the tethered membrane is not capable of being modelled by this equivalent circuit and the readings of Gm and Cm values should be disregarded, e.g. should the conducting channels aggregate into rafts that are farther apart than the distance ions can flow in the time of the excitation frequency of approximately one hundred millisecond then a superposition will be seen of some membrane patches that are conductive and other patches that are sealed. This will result in two impedance spectra being recorded each with different characteristics but superimposed into a single spectrum. The reader will reject such recordings as not fitting the single membrane Gm and Cm model. This filter is useful in determining the presence of such channel aggregation.Note the state, The ?state? indicates the stage to which the model has been fitted. Wait until at least state 3 is reached before interpreting the data. State 4 will be a more accurate refinement but states 1&2 are meaningless intermediates.FIGURE 6The DVM display on the conductance reader.FIGURE 7The Chart display on the conductance reader.Measure the conductance and capacitance.a. Insert the tethaPlate cartridge into the TethaPod.b. Open ?TethaPod? Software. Select the highest communication port under ?Setup?.c. A green LED lights on the front panel of the tethaPod when the instrument is working properly.Examine the menus Table, Setup, Graphs.(i) Set: ?GoF? (goodness of fit) to 0.20 (Table/Set GoF Threshold)(ii) Set the potential bias to 100mV. (Setup/Set Bias)(iii) Set instrument to show Gm. (Table/?Gm).(iv) Press ?Start?.d. The instrument will measure the membrane conductance from each chamber sequentially. (Theinstrument is actually fitting a complex impedance function from the sample at a range offrequencies from 1kHz to 0.1Hz. To avoid the user having to deal with complex impedances theinstrument fits capacitance and conductance values to the data.)e. Once all channels read ?Yes? (Ready column) stop recording.f. Save data into an Excel Spreadsheet. [Edit/copy selection/paste into Excel/save spreadsheet].g. Set new bias at -100mV.h. Wait until reader stabilises. Repeat measurement. Save data into an Excel Spreadsheet (do not savethe file when asked at step e).i. Set new bias at +100mV. Repeat measurement. Save data into an Excel Spreadsheet (do not save the file when asked at step e).Exercise 3. Writing Report:Tabulation:From the data you recorded, generate a Table of conduction (Gm in?S) versus gramicidin concentration ([gA] in nM), for 0, 100mV and -100 mV bias. An example is given as Table 1 below.Table1:Channel123456gA (nM)0406080100120Gm (0mV bias)0.2711.0421.9054.0980.2718.947Gm (-100mV bias)0.3071.1723.1264.2040.3075.637Gm (+100mV)0.3590.6051.3472.3220.35912.04BioNanotechnology Practical: Ion Channels in Membranes.Questions:Describe the effect of applying a positive or negative bias potential from the outer to innersurface of the membrane. Note! positive here is taken from the reader configuration and meansnegative on the tethering gold relative to counter electrode.What is an explanation for this effect?(i) Calculate Conduction per gA Channel(ii) 1. Estimate the maximum slope obtained for graph of conduction vs gramicidin concentration. Mark on your graph how this was obtained. i.e. 5?S for 50nM gA (+100mV bias).2. Calculate the number of lipid molecules we added to each cell to make the tethered membrane.Molarity of lipid = 3mMVolume added = 8?LNumber of Molecules = Molarity (mol/L) x Volume (L) x Avogadro?s number (molecules/mol)=? molecules3. Calculate the number of molecules of lipid in tethered monolayer film on gold.Area per tethered molecule = 1nm2Area of gold electrode = 2mm2 =? tethered molecules.4. Fraction of added plipid incorporated into membrane. Remember it is a bilayer.=? tethered molecules/? molecules~? % of the added lipid.Note: this tells us that most of the added material is flushed away and only??% remains trapped as a membrane.5. Calculate the number of molecules of gramicidin in 8?l of 50nM.Molecules moles = Molarity (M) x Volume (L) x Avogadro?s number (molecules/mole).=? molecules. Remember it is a bilayer.6. Assume the same fraction of gramicidin remains as part of the membrane as the fraction of lipids(they are very similar molecular weights), then the number of gramicidin in the membrane= ~? Molecules7. Calculate the approximate conductance generated per gramicidin (in pS) in the membrane..Siemens per ion channel = Total Siemens generated/number of gramicidin molecules=? S /? molecules~? S/moleculeSDx tethered membranes March 2014.BioNanotechnology Practical: Ion Channels in Membranes.(iii) Calculate membrane thickness from the relationship between area plate separation andpermittivity of a capacitor:From the Chart menu select Cm and DVM. Read value for each chamber.For a capacitor of area, A (m2) and thickness, d (m) and capacitance, Cm (F) is given by:Cm =?0 x?r x A /d,where?0 is the permittivity of free space = 8.854 x 10-12 F/m and?r is the relative permittivity of membrane lipid ~ 2.3 andarea A = 3mm2d =? nm(iv) What changes occur to the membrane thickness when more gA is added? Why might thesechanges be occurring?
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