Surface Plasmon Resonance Feasibility Experiments Neurotransmitter Interaction with Sparsely Tethered
Lipid Membranes (stBLM)
Brian Josey, Stefanie Rintoul, Siddharth Shenoy, Frank Heinrich, Mathias Lösche,
Robert Cantor
November 2013
Summary
2
Samples
2
Results
4
GABA Control
4
GABA
6
GABA Neutron Reflectometry
11
Acetylcholine Chloride Control
13
Acetylcholine Chloride
16
Serotonin Results
22
Serotonin Neutron Reflectometry
24
Discussion
26
Comparison to Wang, C. et al.
26
Further Experiments
27
Discussion Notes
28
Notes from a discussion with Robert Cantor – Summer 2013
28
Notes from a discussion with Vitalii Silin – Summer 2013
28
Notes from a discussion with Robert Cantor – Summer 2013
28
Summary
SPR measurements studying the bilayer association of GABA, acetylcholine and
serotonin with 70:30 POPC:POPG tethered lipid membranes were performed. All three
of the neurotransmitters were found to bind to the lipid bilayer. In a single-binding
process Langmuir isotherm model, GABA binds with a K D of about 0.9±0.6 µM,
acetylcholine exhibits a KD of about 10 mM and serotonin exhibits a KD of 0.08±0.2 µM.
Estimates for maximum surface densities for GABA vary between one GABA molecule
per lipid and one GABA molecule per 5 lipid molecules. Association of acetylcholine and
serotonin are linked to observable bilayer thinning. Therefore surface coverage
estimates cannot be obtained.
Samples
Tethered lipid bilayer membranes (tBLMs) completed with a 70:30 mixture of POPC and
POPG were prepared on gold-coated surfaced coated with a 70:30 β-mercaptoethanol
: HC18 SAMs. tBLMs were exposed to a series of concentrations of either GABA ,
acetylcholine or serotonin and the SPR signal was recorded for every concentration
without rinsing with pure buffer in-between. All experiments were conducted using an
instrument from SPR Biosystems (Germantown, MD).
GABA (A5835-10G),
Acetylcholine Chloride (A6625-25G) and Serotonin hydrochloride (H9523-100MG) were
purchased from Sigma-Aldrich.
Table 1. List of Samples.
Sample
Date
Buffer
stBLM
Neurotransmitter
stBLM
preparation
1
12/12/2011
100 mM NaCl
10 mM HEPES
pH 7.5
70:30
POPC:POPG
GABA
0.01µM-600mM
rapid
solvent
exchange
at
40ºC
2
12/13/2011
100 mM NaCl
10 mM HEPES
pH 7.5
70:30
POPC:POPG
GABA
0.01µM-500mM
rapid
solvent
exchange at
25ºC
3
12/14/2011
100 mM NaCl
10 mM HEPES
pH 7.5
N/A
SAM only
GABA
0.01µM-400mM
N/A
4
5/23/2012
100 mM NaCl
10 mM NaH2PO4
pH 7.5
70:30
POPC:POPG
Acetylcholine
Chloride
.01µM–5.5 mM
rapid
solvent
exchange
at
40ºC
5
6/8/2012
100 mM NaCl
10 mM NaH2PO4
pH 7.5
70:30
POPC:POPG
Acetylcholine
Chloride
0.01µM-400mM
N/A
6
6/11/2012
100 mM NaCl
10 mM NaH2PO4
pH 7.5
N/A
SAM only
Acetylcholine
Chloride
0.01µM-400mM
rapid
solvent
exchange
at
40ºC
7
6/28/2012
100 mM NaCl
10 mM NaH2PO4
pH 7.5
70:30
POPC:POPG
GABA
0.01 µM - 250
µM
rapid
solvent
exchange
at
40ºC
8
8/3/2012
100 mM NaCl
10 mM NaH2PO4
pH 7.5
70:30
POPC:POPG
Acetylcholine
Chloride
0.01µM-400mM
rapid
solvent
exchange
at
40ºC
9
2/28/2013
100 mM NaCl
10 mM HEPES
pH 7.5
70:30
POPC:POPG
GABA
0.01 µM – 500
mM
Rapid
solvent
exchange
at
22ºC
10
6/7/2013
100 mM NaCl
10 mM HEPES
pH 7.5
70:30
POPC:POPG
Serotonin
0.01 µM – 10
mM
Rapid
solvent
exchange
at
22ºC
Figure 1: GABA
Figure 2: Acetylcholine chloride
Figure 3: Serotonin hydrochloride
Results
GABA Control
A gold surface coated with a SAM from a 70:30 ethanolic solution of βmercaptoethanol and HC18 tether molecules was exposed to GABA concentrations
between 10nM and 400mM (see Figure 4). There is no observable change of the SPR
signal when adding up to 1mM of GABA. For concentrations higher than 1mM an
increase of the SPR signal that is strictly linear with GABA concentration is observed.
This linear increase does not show saturation and can be attributed exclusively to a
change of the reflective index of the bulk solvent rather than to surface-bound GABA.
This linear relation between the change of the bulk refractive index and the SPR signal
will be used to distinguish between surface-bound GABA and change of the bulk solvent
refractive index at high GABA concentrations in the following experiments using lipid
bilayers. To achieve this, a linear function will be fitted to the high-concentration region
of the SPR data and subtracted. In case that the high-concentration region contains
signal that arises from surface-bound GABA, the corrected signal will remain above
background. In case that the dissociation constant KD lies within the high-concentration
region, a correction of the SPR signal to a constant value is not possible, and therefore,
this situation can be identified.
Figure 4: Sample 3, no lipid bilayer - SAM only, GABA. Top: SPR raw data, pixel change
vs. time. Bottom: Binding curve extracted from equilibrium pixel changes from SPR raw data.
There is no binding to the SAM in the low concentration region up to 1 mM GABA. Above 1
mM GABA a continuous change of the refractive index of the bulk solution due to the high
concentration of GABA is observed.
GABA
Three GABA experiments were performed by exposing a tBLM composed of 70:30
POPC:POPG to GABA concentrations between 10nM and 600mM (see Figure 5),
between 10nM and 500nM (see Figure 6), and between 10nM and 250µM (see Figure
7). A summary of all three obtained dissociation constants K D and maximum SPR signal
values (pixel changes) can be found in Table .
An average KD value of 0.92±0.54 µM was observed and an average saturation of
1.82±1.68 pixel change was fitted to the binding curves. While those values show a
large variation, all measured binding curves can be fit with a single sigmoidal binding
curve and no additional or increased binding can be detected up to 100 mM GABA.
Above this concentration the correction of the SPR data for the change of the bulk
refractive index does not yield a constant signal anymore. This is either due to a failure
of the correction or due to binding of GABA to the interface above 100 mM. The
average pixel change at saturation of 1.82±1.68 corresponds to an approximated 1
GABA molecule per 2.5 lipid molecules assuming an area per lipid of 65 Å2.
Sample
kd (µM)
bmax
1
1.5±0.6
3.7±0.2
2
0.9±4.9
1.2±0.7
7
0.4±0.3
0.6±0.2
average ± sd
0.9±0.6
1.8±1.7
surface coverage estimate
(1 Pixel = 5.8 ng/cm2, area
per lipid 65Å2)
10.6
ng/cm2
1
neurotransmitter per 2.5 lipid
molecules
Table 2: Experimentally derived dissociation constants and SPR saturation values for
experiments using GABA.
A fourth SPR experiment was performed independently of the others using
neurotransmitter concentrations from 10nM to 10 mM and a buffer of 100mM NaCl and
10 mM HEPES. A negative change in the SPR pixel value was observed. Fitting this
data to a Langmuir isotherm with negative saturation gave KD value of 1.28±1.01 µM
was observed and an average saturation of 1.28±0.19 pixel change (see Figure 8).
Figure 4: Sample 1, 70:30 POPC:POPG, GABA, bilayer preparation at 40ºC. Top: SPR raw
data, pixel change vs. time. Pixel change is related to the change of refractive index at the
interface, and, therefore the associated mass. Different colors indicate different GABA
concentration sequentially added to the stBLM. Bottom: Binding curve extracted from
equilibrium pixel changes from SPR raw data. The red data points are directly obtained from the
top panel, the blue data points are the red data corrected for the refractive index change of the
bulk solvent. An equilibrium dissociation constant of 1.4566±0.568 µM was determined and a
saturation pixel change of 3.7292±0.141 pixels.
Figure 6: Sample 2, 70:30 POPC:POPG, GABA, bilayer preparation at room
temperature. Top: SPR raw data, pixel change vs. time. Bottom: Binding curve
extracted from equilibrium pixel changes from SPR raw data. The red data points are
directly obtained from the top panel, the blue data points are the red data corrected for
the change of the refractive index of the bulk solvent. An equilibrium dissociation
constant of 0.93666±4.93 µM was determined and a saturation pixel change of
1.2±0.708 pixels.
Figure 7: Sample 7, 70:30 POPC:POPG, GABA, bilayer preparation at 40ºC. Top: SPR raw
data, pixel change vs. time. Bottom: Binding curve extracted from equilibrium pixel changes
from SPR raw data. An equilibrium dissociation constant of 0.36731±0.301 µM was determined
and a saturation pixel change of 0.55664±0.12 pixels.
Figure 5: Sample 9, 70:30 POPC:POPG, GABA, bilayer preparation at 22ºC with 100 mM
NaCl 10mM HEPES buffer. Top: SPR raw data, pixel change vs. time. Bottom: Binding curve
extracted from equilibrium pixel changes from SPR raw data. An equilibrium dissociation
constant of 1.28±1.01 µM was determined and a saturation pixel change of -1.28±0.19 pixels.
GABA Neutron Reflectometry
Neutron reflectometry experiments were performed on the MAGIK detector at NCNR
from September 19 to September 24, 2013. A tBLM with 70:30 POPC:POPG was
assembled in a flow cell with a volume of 6mL. Initially the bilayer reached 87%
completions, so a further 6mL of 70:30 POPC:POPG vesicles were flowed into the flow
cell and ruptured using osmotic shock, resulting in an approximately 100% bilayer
completion.
A baseline reflectometry measurement was performed on the neat bilayer using H2O
and D2O buffers of 100mM NaCL and 10mM NaH2PO4. Further neutron reflectometry
measurements were performed on 100nM, 1 μM and 1mM GABA concentrations in both
H2O and D2O buffers, plus an additional rinse. The data were then fitted to determine
the cross-sectional area, in 1/Å2, as a function of distance from the gold substrate in Å
(see Figure 9 to Figure 12).
GABA was observed to associate with the hydrocarbon tails of the outer leaflets with
increasing affinity as the neurotransmitter concentration increased. The depth of the
distribution of GABA into the hydrocarbon region and the thickness of the bilayers were
there calculated (see Table 3). As the neurotransmitter concentration increased, the
thickness of the bilayer decreased and the depth of the GABA penetration increased.
Rinsing the bilayer did not result in the removal of the GABA, suggesting that the
binding is irreversible.
Figure 6: Cross-sectional area 100nM GABA Data for 100nM GABA fitted to show the crosssectional area as a function of height above the substrate. GABA, red, shows a weak affinity to
the hydrocarbon chains of the outer leaflet of the bilayer, dark blue.
Figure 7: Cross-sectional area 1μM GABA Data for 1μM GABA fitted to show the crosssectional area as a function of height above the substrate. GABA, red, shows a weak affinity to
the hydrocarbon chains of the outer leaflet of the bilayer, dark blue.
Figure 8: Cross-sectional area 1mM GABA Data for 1mM GABA fitted to show the crosssectional area as a function of height above the substrate. GABA, red, shows a weak affinity to
the hydrocarbon chains of the outer leaflet of the bilayer, dark blue.
Figure 9: Cross-sectional area post-rinse GABA Data for GABA after rinsing with buffer fitted
to show the cross-sectional area as a function of height above the substrate. GABA, red, shows
an association to the head groups of the lipids, light blue, suggesting that adsorption is
irreversible.
GABA
Concentration
Neurotransmitter
volume (Å3/Å2)
Neurotransmitter
Penetration (Å)
Thickness
Change (Å)
1 μM
100 nM
1 mM
Rinse
0.82-0.31 +0.52
0.56-0.24 +0.42
0.91-0.36 +0.46
0.94-0.41 +0.50
-0.51-9.95 +4.47
-1.52-5.6 +2.9
-4.41-8.48 +0.06
-2.23-8.56 +2.86
-0.12-0.30 +0.19
-0.14-0.15 +0.15
-0.58-0.19 +0.17
-0.96-0.26 +0.16
Table 3: Parameter values obtained from neutron data analysis for GABA. The error bars were
obtained using Monte-Carlo resampling technique and give 68% confidence on the parameters.
The neurotransmitter location represents the distance from the surface of the hydrocarbon
region that the center of mass of the neurotransmitter peak is located; a negative value
represents a penetration into the hydrocarbon region while a positive value represents
association outside of the hydrocarbons.
Acetylcholine Chloride Control
A gold surface coated with a SAM from a 70:30 ethanolic solution of βmercaptoethanol and HC18 tether molecules was exposed to acetylcholine chloride
concentrations between 10nM and 400mM (see Figure 13). As for GABA, there is no
observable change of the SPR signal when adding up to 1mM of acetylcholine. For
concentrations higher than 1mM an increase of the SPR signal that is strictly linear with
acetylcholine concentration is observed. This linear increase does not show saturation
and can be attributed exclusively to a change of the reflective index of the bulk solvent
rather than to surface-bound acetylcholine.
As for GABA, this linear relation between the change of the bulk refractive index and the
SPR signal will be used to distinguish between surface-bound GABA and change of the
bulk solvent refractive index at high acetylcholine concentrations in the following
experiments using lipid bilayers. For this correction, a linear function was subtracted
that had been determined by fitting the data points between 5.5mM and 100mM. The
correction is valid up to 100mM and above this concentration, the control curve is nonlinear with respect to the concentration of AcCh (see Figure 14).
Figure 13: Sample 6, no lipid bilayer - SAM only, Acetylcholine Chloride. Top: SPR raw
data, pixel change vs. time. Bottom: Binding curve extracted from equilibrium pixel changes
from SPR raw data. There is no binding to the SAM in the low concentration region up to 1 mM
Acetylcholine Chloride. Above 1 mM Acetylcholine Chloride a continuous change of the
refractive index of the bulk solution due to the high concentration of Acetylcholine Chloride is
observed. This signal is used as a correction for samples 4 and 5.
Figure 14: Sample 6, no lipid bilayer - SAM only, Acetylcholine Chloride. Red: SPR raw
signal (pixel change), blue: corrected signal by subtracting the high-concentration slope
determined from the data point from 5.5 103 µM to 105 µM. The correction is valid up to 105 µM
and the corrected curve deviates less than 1 pixel from zero in this range.
Acetylcholine Chloride
Three experiments with acetylcholine were performed by exposing a tBLM composed of
70:30 POPC:POPG to neurotransmitter concentrations between 10nM and 5.5mM (see
Figure 15), and between 10nM and 400mM (see Figure 16 and Figure 17).
None of these experiments showed any neurotransmitter binding between 10nM and
5.5mM. However, all experiments showed a decrease of the SPR signal by less than 1
pixel over this range. The second and the third experiment, which were extended to
higher acetylcholine concentrations, did show the characteristic increase of the SPR
signal at high concentrations due to changes of the bulk refractive index. However, this
region could not be fit to a linear function.
Therefore in the second experiment, the whole binding curve was fit to a single
sigmoidal absorption isotherm with a KD in the high-concentration region plus a linear
function that models the high-concentration refractive index increase. This model
function is able to fit the data only when a negative saturation pixel change is used. The
fit values are KD=15.6±6.2mM and Bmax=-8.8±2.2 pixel. The negative saturation value is
consistent with the decrease of the SPR signal at lower concentrations observed in both
experiments. It might be interpreted as an adsorption of acetylcholine to the bilayer that
results in a substantial bilayer thinning.
In order to study the region of about 10 mM in more detail, a higher measurement point
density was used in that region during the third experiment. Figure 17 displays a
difference plot of the binding curve shown in Figure 18 and a linear function. Before and
after the feature that deviates from a linear function, the difference is a constant. The
feature itself is composed of an initial increase in the SPR signal and a later decrease
that levels out at a net decrease of the signal. Therefore, the second and the third
acetylcholine experiments are in agreement with each other. Further, the increase of the
SPR signal due to the associated mass of acetylcholine at the bilayer and the decrease
of the SPR signal due to the (presumed) membrane thinning are offset. A first attempt of
fitting the difference signal also showed that both processes probably cannot be
described by single-adsorption Langmuir isotherms.
Figure 15: Sample 4, 70:30 POPC:POPG, Acetylcholine Chloride, bilayer preparation at
40ºC. Top: SPR raw data, pixel change vs. time. Bottom: Binding curve extracted from
equilibrium pixel changes from SPR raw data.
Coefficient values ± one standard deviation
offset
=0.7 ± 0.127
kd
=15576 ± 6.19e+03
bmax
=-8.8088 ± 2.11
slope
=0.00056992 ± 1.67e-05
concoffset =1 ± 0
SPR pixel change
100
10
1
0.1
10
-2
-1
10
10
0
1
10
2
10
concentration / µM
10
3
4
10
10
5
Figure 16: Sample 5, 70:30 POPC:POPG, Acetylcholine Chloride, bilayer preparation at
40ºC. Top: SPR raw data, pixel change vs. time. Bottom: Binding curve extracted from
equilibrium pixel changes from SPR raw data. The binding curve was fitted to a single sigmoidal
function plus a linear function of the concentration. Obtained fit results are KD=15.6±6.2mM and
Bmax=-8.8±2.2 pixel.
Figure 17: Sample 8, 70:30 POPC:POPG, Acetylcholine Chloride, bilayer preparation at
40ºC. Top: SPR raw data, pixel change vs. time. Bottom: Binding curve extracted from
equilibrium pixel changes from SPR raw data. The binding curve was fitted to a linear function of
the concentration. Clearly visible are deviations from the linear function at around 104 µM.
Figure 18: Subtraction of a linear function from the binding curve of sample 8. Before and after
the feature around 104 µM the difference is a constant. The two enveloping red curves are
sigmoidal functions in analogy to a binding curve with parameters KD=8000 µM, and Bmax values
of 5 and -2.6.
Serotonin Results
A single experiment with serotonin was performed by exposing a tBLM composed of
70:30 POPC:POPG to neurotransmitter concentration between 10nM and 10mM (see
Figure 19). This experiment did not show any neurotransmitter binding between 10nM
and 100uM, showing a decrease of the SPR signal of 1.9 pixels over this range. Above
100μM, the SPR pixel value increased as the bulk concentration of the neurotransmitter
increased.
As with the acetylcholine experiment, the absorption curve for the serotonin was fitted to
a sigmoidal absorption isotherm and a negative saturation index increase. The values
for 1mM and 10mM concentration of serotonin were excluded from the fit due to their
being a higher concentration of bulk serotonin at these two concentrations. The fit
values are KD=0.08±0.02μM and Bmax=-2.1±0.1 pixel. The negative saturation value is
consistent with the decrease of the SPR signal at lower concentrations observed in the
experiment, and may be interpreted as the adsorption of serotonin to the bilayer
resulting in bilayer thinning.
Figure 19: Sample 10, 70:30 POPC:POPG, Serotonin hydrochloride, bilayer preparation at
22ºC. Top: SPR raw data, pixel change vs. time. Bottom: Binding curve extracted from
equilibrium pixel changes from SPR raw data. The binding curve was fitted with a Langmuir
isotherm with a negative saturation value. Clearly visible are deviations from the curve around
100μM.
Serotonin Neutron Reflectometry
After rinsing the flow cell used for the GABA neutron reflectometry experiment above
with 100mM NaCL and 10mM NaH2PO4 in H2O and D2O buffers, serotonin experiments
were performed in the same flow cell. Three concentrations of serotonin, 10 nM, 100nM
and 10μM, were each prepared in 6mL of H2O and D2O buffers of 100mM NaCL and
10mM NaH2PO4. Reflectometry measurements were then performed on each of these
concentrations of serotonin. The results of which were then fitted to determine the
cross-sectional area, in 1/Å2, as a function of distance from the gold substrate in Å (see
Figure 19 to Figure 22). Serotonin showed a greater association with the bilayer than
GABA.
The thickness of the bilayer and location of the serotonin distribution was then
calculated from this data (see Table 4). Serotonin showed a greater affinity to the
bilayer than GABA as demonstrated by the higher values of protein volume per bilayer
area. Furthermore, the distribution of serotonin moved towards the center of the bilayer
as the concentration of neurotransmitter increased, resulting in a broad distribution at
the highest concentration.
Figure 20: Cross-sectional area 10 nM Serotonin Data for 10nM serotonin fitted to show the
cross-sectional area as a function of height above the substrate. Serotonin, red, shows a weak
affinity to the hydrocarbon core of the bilayer, dark blue.
Figure 21: Cross-sectional area 100nM Serotonin Data for 100nM serotonin fitted to show the
cross-sectional area as a function of height above the substrate. Serotonin, red, shows a weak
affinity to the hydrocarbon core of the bilayer, dark blue.
Figure 10: Cross-sectional area 100μM Serotonin Data for 100μM serotonin fitted to show the
cross-sectional area as a function of height above the substrate. Serotonin, red, shows an
affinity to the hydrocarbon core of the bilayer, dark blue.
Serotonin Concentration
Neurotransmitter volume (Å3/Å2)
Neurotransmitter Penetration (Å)
Thickness Change (Å)
10 nM
0.40-0.22 +0.40
-8.15-5.63+4.23
-0.09-0.13+0.16
100 nM
0.52-0.21 +0.33
-11.52-3.55 +5.08
-0.34-0.15+0.15
100 μM
0.86-0.20 +0.25
-18.80-1.44 +5.94
-0.35-0.19+0.17
Table 4: Parameter values obtained from neutron data analysis for serotonin. The error bars
were obtained using Monte-Carlo resampling technique and give 68% confidence on the
parameters. The neurotransmitter location represents the distance from the surface of the
hydrocarbon region that the center of mass of the neurotransmitter peak is located; a negative
value represents a penetration into the hydrocarbon region while a positive value represents
association outside of the hydrocarbons.
Discussion
Comparison to Wang, C. et al.
In comparison to the publication “Wang, C. et al., 2011. Journal Of Physical Chemistry
B, 115(1), pp.196–203”, which uses membranes composed of 90:10 DMPC/DMPG, the
present work uses a membrane composition of 70:30 POPC/POPG. See Figure 11 for a
reproduction of figure 3 from Wang et al. that contains the measured concentrationdependent binding affinities.
Figure 11: Reproduction of Figure 3 from: Wang, C. et al., 2011. Affinity of four polar
neurotransmitters for lipid bilayer membranes. Journal Of Physical Chemistry B, 115(1),
pp.196–203.
Wang et al. find membrane affinities for GABA and acetylcholine that depend linearly on
their concentration in the bulk medium. Affinities Γ3 approximate number of
neurotransmitters per lipid and range between 0.01 and 0.08 over a concentration range
of 5mM to 40mM for both, GABA and acetylcholine. These affinity values are smaller
than the estimates of the maximum surface coverage obtained with GABA in this work
which ranges between 1 and 5 lipid molecules per neurotransmitter or Γ3 values
between 0.2 and 1.
In complete disagreement with Wang et al. is the obtained dissociation constant K D of
GABA binding to POPC/POPG. We found a KD of about 1 µM and did not see any
further adsorption of GABA up to a concentration of 100 mM. The estimate of K D≈10mM
for acetylcholine that was obtained in the present work, does agree with the observed
binding of Wang et al. in the region between 5mM and 40mM. However, in the present
work we observe saturation at about 40mM, for which there is no indication in the data
from Wang et al.
Further Experiments
With the present data, the bilayer thinning observed with acetylcholine is still a
hypothesis. Neutron reflectivity experiment should be able to verify bilayer thinning. One
would have to measure three subsequent conditions: the neat bilayer, the bilayer in
contact with NT, and rinse.
We certainly want to extend the experiments with PG lipids to PS lipids.
Discussion Notes
Notes from a discussion with Robert Cantor – Summer 2013
1. The low KD values for GABA and serotonin are still surprising and ideally need
confirmation by another technique. In principal NR is such a technique, but Fred
Lanni’s experiments on the influence of NT on the bilayer undulations of red
blood cells might another such experiment.
2. The second biggest issue is the lack of reversibility seen in the NR experiments
upon rinsing of the system. One should check on this with SPR and consider
alternate model systems, like solid supported membranes. A general bias of the
tBLMs towards insertion would have far-reaching consequences.
3. The SPR data in Figure 4 of GABA at a 70:30 POPC:POPG membrane, appears
to have another binding at 1 mM. It might be interesting to experimentally follow
up on this.
4. The magnetic reference layer technique should prove useful to confirm and
repeat the NR data.
5. There were GUV micro-pipetting experiments with NTs. They might give insight
into binding affinities - check literature.
6. Consider measuring NT partitioning into bilayers using the Nagle’s equipment.
7. Do we have access to fluorine NMR experiments and could we get kinetic
information from them.
Notes from a discussion with Vitalii Silin – Summer 2013
In general the results are convincing, but there are a number of things that would
strengthen the work:
(1) The linear response of the SPR signal with respect to NT concentration for both
control experiments should be backed up with refractive index measurements of
a series of NT concentrations. One should find the same limit to linearity in those
measurements than in the SPR measurements.
(2) The SPR measurements miss a rinsing step at the end of each concentration
series. This would be necessary to check for reversibility of the NT binding. One
could also find arguments to perform a rinsing step after each incubation with
NT.
(3) If one would like to discern bilayer thinning and NT association, as it might be
important for acetylcholine, a stopped flow setup might be helpful. The
dissociation step in this setup might give extra information.
Notes from a discussion with Robert Cantor – Summer 2013
Robert remains skeptical towards the measured low KD of GABA and he looks for a
potential explanation of that result. He states that such a low K D would not be consistent
with his kinetic model of GABA-membrane interaction. A few future experiments were
identified:
1. Repeat the GABA experiment with 10% POPG instead of 30%. This repeat
would match the bilayer condition that Peter West was using.
2. Glycine is expected to behave similar to GABA because it is also zwitterionic.
The downside is that Glycine is even smaller than GABA.
3. Neutron experiments for acetylcholine and GABA.
4. Experiments with serotonin.