The Interactions of Riluzole with Its Binding Pocket in SK2 Channels
Sara Ali
Department of Biological Science
Saddleback College
Mission Viejo, CA 92692
Riluzole, the only FDA-approved drug for Amyotrophic Lateral Sclerosis (ALS),
works by modulating multiple drug targets including small conductance Ca2+-activated
potassium (SK) channels. However, the functional binding site of Riluzole is still unclear.
We recently determined the binding pocket of Riluzole in SK2 channels through
crystallography. This study investigates whether this binding pocket of Riluzole in SK2
channels is the functional binding pocket in which the drug exerts its modulation. With
combined techniques of site-directed mutagenesis and electrophysiology, results supported
that the binding site identified by crystallography is the functional binding pocket.
Mutations A477V/L480M, which mimic the corresponding residues in SK4 channels, were
introduced into SK2 channels to test the hypothesis. The mutant channel was then coexpressed with Calmodulin in HEK293 cells. Inside out macro-patch recordings were
performed to measure the SK channel current as a function of Riluzole concentration
(μM). Dose response curves for the potentiation of SK2 channel activity were constructed.
The half maximal effective concentrations (EC50), were significantly different (p = 9.17 x
10-5, one tailed unpaired t-test) between the mutant and wild type cells. The data supports
that the binding site identified by crystallography is the functional binding site through
which Riluzole exerts positive modulation of SK2 channels.
Introduction
SK2 channels belong to a family of channels
named small conductance Ca2+-activated potassium
channels or, SK channels (Faber and Sah, 2005). The
family consists of three main channels, SK1-3 which is
widely expressed in neurons and is found numerously
throughout the central nervous system. When an action
potential is initiated, calcium influx through voltagegated calcium channels triggers the opening of SK
channels, resulting in hyperpolarization (Faber et al.,
2007). Calmodulin (CaM) is tethered to the SK
channels and serves as a high-affinity Ca2+ sensor.
Once calcium binds to CaM, the conformation of CaM
changes and subsequently, opens the SK Channels. SK
channels play a direct role in the medium duration
after-hyperpolarization and when these channels are
blocked, the firing rate increases (Seutin and Liégeois,
2007).
Activation of SK channels decreases the firing
rate of action potentials, which then contributes to the
regulation of Ca2+ of neuronal excitability, dendritic
integration, synaptic transmission, and plasticity in the
central nervous system (Zhang et al., 2012). These
factors that have to do with regulation of Ca2+ goes
hang in hand with Amyotrophic Lateral Sclerosis
(ALS).
ALS is a neurodegenerative disease in which
upper and lower motor neurons both degenerate. Some
research has pointed to mutations as being the possible
cause of ALS however the pathology of ALS is still
largely unknown (NIH, 2015). Due to this lack of
information there is only one FDA approved drug in
the market for the treatment of ALS: Riluzole.
Recently, SK channels were identified as a critical
target for the neuro-protective effect of Riluzole
(Dimitriadi et al., 2013).
This study investigates whether putting the
mutations (A477V/L480M) that mimic residues in SK4
channels responsible for Riluzole binding, in SK2
channels, will help increase drug potentiation. Drug
potentiation is measured by normalized SK2 current
(%) as a function of drug concentration (μM). The
hypothesis of this study is that introducing mutations
A477V/L480M, there will be more binding between
the drug and receptors, which will increase the overall
potency of Riluzole, making for a more efficient
treatment.
Figure 1. The chemical structure of Riluzole and a
molecular dock model of Riluzole and mutation A477V
with its binding pocket shown. Adapted from Dr.
Zhang.
Materials and Methods
Mutagenesis
In order to perform mutagenesis SK2 (WT)
needed to be sub-cloned into an expression vector
(Invitrogen) along with Calmodulin (CaM) since CaM
is used as a calcium sensor and a signal transducer
which both are key in electrophysiology. Mutations
(A477V/L480M) were then introduced into SK2 using
the QuickChange XL site-directed mutagenesis kit
(Stratagene-Agilent) and subsequently confirmed by
DNA sequencing. Riluzole was provided by Tocris.
WT and mutant channels, along with CaM and green
fluorescent protein, were expressed in human cell line
cells (TsA201 cells), which was cultured in a formula
to sustain the cells. This formula used was DMEM,
with 10% fetal bovine serum, and penicillinstreptomycin. After the cells were cultured, a calcium–
phosphate method was used for transfection of SK2
cDNA (WT or mutants), together with CaM and green
fluorescent protein at a ratio of 5/2.5/1 (weight). For
the calcium—phosphate method, we prepared a 2M
CaCl2 solution and a phosphate buffer solution that
included the SK2 cDNA, CaM, and the green
fluorescent protein. After mixing the two solutions,
DNA-calcium phosphate co-precipitated. This
precipitate is what was up taken by the cell, after
having been introduced to the cell wells. After the cells
took up the cDNA, CaM, and the green fluorescent
protein, transfection was complete as was mutagenesis.
Inside-out Macro Patch Readings
Channel activities were recorded 1–2 days
after transfection, with a Multiclamp 700B or an
Axon200B amplifier (Molecular Devices) at room
temperature. pClamp 10.2 (Molecular Devices) was
used for data acquisition and analysis. The resistance of
the patch electrodes ranged from 3–7 MΩ. The pipette
solution (with the electrode inside the pipette)
contained 140 mM KCl, 10 mM HEPES, 1 mM
MgSO4, at pH 7.4. The bath solution contained 140
mM KCl and 10 mM HEPES, at pH 7.2. EGTA (1
mM) and HEDTA (1 mM) were mixed with Ca2+ to
obtain 0.2 μM free Ca2+, calculated using the software
of Stanford University. Currents were recorded using
an inside-out patch configuration. For SK2 and its
mutants, the intracellular face was initially exposed to a
zero-Ca2+ bath solution, and subsequently to bath
solutions with 0.2 μM Ca2+. Currents were recorded by
repetitive 1-s-voltage ramps from −100 mV to +100
mV from a holding potential of 0 mV. One minute after
switching of bath solutions, ten sweeps with a 1-s
interval were recorded at concentrations for Riluzole in
the presence of 0.2 μM Ca2+. The integrity of the patch
was examined by switching the bath solution back to
the zero-Ca2+ buffer. Data from patches, which did not
show significant changes in the seal resistance after
solution changes, were used for further analysis.
Data Analysis
To construct the dose-dependent potentiation
of channel activities, the current amplitudes at −90 mV
in response to various concentrations of Riluzole were
normalized to that obtained at maximal concentration.
The normalized currents were plotted as a function of
the concentrations of Riluzole. EC50s and Hill
coefficients were determined by fitting the data points
to a standard dose–response curve (Y = 100/(1 +
(X/EC50)^ − Hill)). A one-tailed t-test performed by
StatPlus was used to determine whether the data points
were consistent with the hypothesis.
Results
A dose response curve was constructed to
reflects the amount of concentrated Riluzole needed for
a certain response (Figure 2). Response was measured
by electrophysiology; the machine captures the current
produced by the activated SK2 channels. The mean
EC50 value for wild type cells was 11.43 ± 0.59 μM (±
SEM) and for the mutant cells it was 1.81 ± 0.12 μM (±
SEM), which are shown in Figure 3. The mean half
maximal effective concentration required for both
mutant and wild type cells are evaluated in Figure 3.
Normalized SK2 current (%)
100
SK2 WT
SK2 (A477V/L480M)
80
60
40
20
0
0.1
1
10
100
Riluzole (M)
Figure 2. Dose response curve for potentiation by
mutations of the SK2 channel activities.“Normalized”
refers to the fact that the maximal activation was set as
denominator when calculating % SK2 current.
Riluzole EC50 (M)
15
The concentration needed in order for the
population to show response, is significantly lower
(p = 9.17 x 10-5, one-tailed unpaired t-test) for the
mutant in comparison to the wild type SK2 channels.
Ultimately what this means is that there is a
significantly lower amount of Riluzole needed to
produce the same response for the mutant SK2
channels in comparison to the wild type SK2 channels.
The potentiation of Riluzole increases significantly
with mutant SK2 channels. Overall the data collected
allows for the acceptance of the original hypothesis.
The results are consistent to the Dimitriadi et
al. (2013) study where they found Riluzole improved
motor neuron function in Drosophilia and C. elegans
by directly acting on SK channels. What would make
this study better however would be to redo the
experiment with a larger population of cells. Overall,
the results will help patients with ALS. Some future
directions include experimentation with a larger
population of cells, perhaps more experimentation with
SK channel mutations applied to other
neurodegenerative diseases, and possibly clinical trials
for patients with existing ALS.
10
Appendix
5
0
WT
A477V/L480M
Figure 3. The amount of Riluzole at half maximal
effective concentration (EC50) for both SK channelsmutant and wild type. The amount of Riluzole was
significantly different for the mutant versus wild type
(p = 9.17 x 10-5, one-tailed unpaired t-test). Error bars
are ± SEM.
Discussion
The hypothesis of this investigation was that
introducing mutations A477V/L480M into SK2
channels would increase the potency of Riluzole and
further show that this mutation serves as the functional
binding pocket in which Riluzole exerts its modulation.
SK2 channels with the mutation show a lesser amount
of drug concentration is needed to exert higher
activation of channels in comparison to the wild type
channels (Figure 2). Higher activation reflects the
relative amount of binding of the drug to SK2
channels. The dose response curve for the mutant SK2
channels lies well above the wild type’s curve showing
that SK2 mutant channels are much more favorable
when the drug is administered (Figure 2). SK2 mutant
channels show more binding to the drug where as the
wild type SK2 channels do not show nearly as much
binding (Figure 3).
CaM: Calmodulin, used as a calcium sensor and signal
transducer and characterized as an
intermediate messenger protein
TsA201 Cells: a specific kind of cell from the human
embryonic kidney cell (HEK293) line, must
be stored in liquid nitrogen
DMEM: Dulbecco’s Modified Eagle Medium. A
growth medium that contains typical amino
acids, glucose, pH indicator, salts, and
vitamins, used to sustain cells.
cDNA: complementary DNA (introduced to the cell so
that when the cell replicates, it will
incorporate the new foreign DNA in the
cDNA)
HEPES: an organic chemical buffering agent
commonly used in cell culturing due to its
ability to maintain a physiological pH.
EGTA: a reagent used to chelate Ca2+ in the presence of
Mg2+, helps protect micronutrients
HEDTA: another chelating reagent used to protect
micronutrients
EC50: the half maximal effective concentration, it is the
concentration of a drug that gives halfmaximal response…when half the population
gives the desired response.
Literature Cited
Dimitriadi, M., Kye, M.J., Kalloo, G., Yersak, J.M.,
Hart, A. 2013. “The Neuroprotective Drug Riluzole
Acts Via Small Conductance Ca2+-Activated K+
Channels to Ameliorate Defects in Spinal Muscular
Atrophy Models”. The Journal of Neuroscience.
33(15):6557-6562.
Faber, L.E., Delaney, A.J., Sah, P. 2005. “SK Channels
Regulate Excitatory Synaptic Transmission and
Platicity in The Lateral Amygdala”. Nature
Neuroscience 8, 635-641.
Faber, L.E., Sah, P. 2007. “Functions of SK Channels
in Central Neurons”. Clinical & Experimental
Pharmacology & Physiology. 34(10): 1077-1083.
NIH. 2015. “Amyotrophic Lateral Sclerosis (ALS) Fact
Sheet”. NINDS. No. 12-916.
Seutin, V., Liégeois, J. 2007. “SK Channels Are On
The Move” British Journal of Pharmacology. 151(5):
568-870.
Zhang, M., Pascal, J.M., Schumann, M., Armen, R.S.,
Zhang, J. 2012. “Identification of The Functional
Binding Pocket for Compounds Targeting SmallConductance Ca2+-Activated Potassium Channels”.
Nature Communications. 3:1021.