Hi, I have 7 physiological systems questions that I need desperate help with. I have attached the document containing the 7 questions thank you. If i'm pleases with the answers I also have another 13 potential questions. Please include all working out for the question. 

Attached is also the notes for the experiment it is based on.

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BioNanotechnology Practical: Ion Channels in Membranes.

Membrane Conduction and Ion Channels
Key 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 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.

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BioNanotechnology Practical: Ion Channels in Membranes.

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 important 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.

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BioNanotechnology Practical: Ion Channels in Membranes.

(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 stable 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:
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BioNanotechnology Practical: Ion Channels in Membranes.

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.

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BioNanotechnology Practical: Ion Channels in Membranes.

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

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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 for

the adhesive to seal.

FIGURE 4

Introduction to practical exercise
Aim:
1.
2.
3.
4.

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.

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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 you
start. Timing is critical for proper membrane formation. Read the entire
experiment through before commencing.
Exercise 1. Prepare tethered membranes containing gramicidin
a. 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 the
membrane.)
c. The electrode is stored in ethanol and you need to stand it on a tissue to dry. This may take 1-2
minutes.
d. Align the dry slide over the alignment jig, ensuring electrode tracks and the SDX logo on the slide
overlay 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 thin
protective layer that is removed and not the entire adhesive laminate.) This will reveal a sticky
surface 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 peel
them 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 1
minute, before loosening the pressure. The electrode is now ready for membrane formation.
Membranes are formed as follows:
Chambe
r
1
2
3
4
5
6

Constituent in Phospholipid
0nM gA
40nM gA
60nM gA
80nM gA
100nM gA
120nM gA

a. Start stop watch.
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BioNanotechnology Practical: Ion Channels in Membranes.

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).

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BioNanotechnology Practical: Ion Channels in Membranes.

Exercise 2. Testing the bilayer using AC impedance spectroscopy
The 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 (µS) and capacitance (nF)
Typical conduction values for a freshly formed membrane using the proprietary SDx TM AM199 in PBS are
0.35 ±0.15 µS 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 inversely
proportional to the membrane thickness. A significant additional measure using a tethaPod™ is the
Goodness 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 Spectroscopy
A sine wave excitation of 20mV is applied across the tethered membrane between the tethering gold
electrode 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 the
modelled 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.
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BioNanotechnology Practical: Ion Channels in Membranes.

FIGURE 6

The DVM display on the conductance reader .

FIGURE 7

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BioNanotechnology Practical: Ion Channels in Membranes.

The 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. (The
instrument is actually fitting a complex impedance function from the sample at a range of
frequencies from 1kHz to 0.1Hz. To avoid the user having to deal with complex impedances the
instrument 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.
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BioNanotechnology Practical: Ion Channels in Membranes.

h. Wait until reader stabilises. Repeat measurement. Save data into an Excel Spreadsheet (do not save
the 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:
Channel
1
2
3
4
5
6

gA (nM)
0
40
60
80
100
120

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Gm (0mV bias)
0.271
1.042
1.905
4.098
0.271
8.947

Gm (-100mV bias)
0.307
1.172
3.126
4.204
0.307
5.637

Gm (+100mV)
0.359
0.605
1.347
2.322
0.359
12.04

BioNanotechnology Practical: Ion Channels in Membranes.

Questions:
Describe the effect of applying a positive or negative bias potential from the outer to inner
surface of the membrane. Note! positive here is taken from the reader configuration and means
negative 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 = 3mM
Volume added = 8?L
Number of Molecules = Molarity (mol/L) x Volume (L) x Avogadro’s number (molecules/mol)
= ? molecules

3. Calculate the number of molecules of lipid in tethered monolayer film on gold.

Area per tethered molecule = 1nm2
Area 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
= ~? Molecules
7. 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/molecule
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BioNanotechnology Practical: Ion Channels in Membranes.
(iii) Calculate membrane thickness from the relationship between area plate separation and

permittivity 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 and
area A = 3mm2
d = ? nm

(iv) What changes occur to the membrane thickness when more gA is added? Why might these

changes be occurring?

SDx tethered membranes March 2014

Physiological Systems questions.docx  Download Attachment

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1.

 Calculate the number of lipid molecules we added to each cell to 
make the tethered membrane.  (5 marks)
Molarity of lipid = 3mM 
Volume added = 8m
L

2.

Calculate the number of molecules of lipid in the tethered 
monolayer film on gold. (5 marks)
Area per tethered molecule = 1nm2
Area of gold electrode = 2mm2   

3.

What is the fraction of added phospholipid that is incorporated into
the membrane? (3 marks)
           

4.

5.

6.

            
Calculate the number of molecules of gramicidin in 8m
l of a 50nM 
solution. (5 marks)
                     
                       
Assume the same fraction of gramicidin remains as part of the  
membrane as the fraction of lipids (they are very similar molecular
weights), then how many molecules of gramicidin are there in the 
membrane (3 marks)
                        
Calculate the approximate conductance generated per gramicidin 
(in pS) in the membrane. (4 marks)
Total Siemens generated = 5µS

 7. One can calculate membrane thickness from the relationship below 
between area plate separation and permittivity of a capacitor.

The following results were obtained from the biosensor 
experiment. 
gA (nM)
Capacitance (nF)
0

17.6

40

21.9

80

26.4

For a capacitor of area, A (m2) and thickness, d (m) the 
capacitance, Cm (F) is given by: 

Cm = e 0 x e r x A /d
where e 0 is the permittivity of free space = 8.854 x 10­12 F/m and 
e r is the relative permittivity of membrane lipid ~ 2.3 and 

area A = 3mm2
(a) Calculate the membrane thickness at the three different 
gramicidin concentrations. (9 marks)
    (b) What changes occur to the membrane thickness when more gA 
is added? Why might these changes be occurring? (4 marks)