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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 voltage-gated 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 highaffinity 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 sitedirected 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 penicillin-streptomycin. 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 MgSO 4, 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 Ca 2+ 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 Ca 2+. 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 Ca 2+. 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. EC 50s and Hill coefficients were
determined by fitting the data points to a standard dose–response curve (Y = 100/(1 + (X/EC50)^ − Hill)). A onetailed 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
10
5
0
WT
A477V/L480M
Figure 3. The amount of Riluzole at half maximal effective concentration (EC50) for both SK channels-mutant and
wild type. The amount of Riluzole was significantly different for the mutant versus wild type (p = 9.17 x 10-5, onetailed 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).
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.
Appendix
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 half-maximal
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 Small-Conductance Ca2+-Activated Potassium Channels”. Nature
Communications. 3:1021.
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s): Sara Ali
Title: The Interactions of Riluzole with Its Binding Pocket in SK2 Channels
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.
This experiment recently determined the binding pocket of Riluzole in SK2 channels by
using crystallography that was unclear before. Also, this experiment is investigating
whether the binding pocket of Riluzole in SK2 channels is the functional binding pocket in
which the drug can exert its modulation by using the combined techniques of site-directed
mutagenesis and electrophysiology. The data from running one-tailed t-test by StatPlus
showed a significant difference in the half-maximal effective concentrations (EC50) between
the mutant and wild type cells, which supports that the binding site identified by
crystallography is the functional binding site through which Riluzole exerts positive
modulation of SK2 channels.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our
current state of knowledge.
The strength was that the study was done in a research lab, and it was a group project,
which helped it to be more precise and accurate. Also, the topic of the study was a strong
topic. Adding some pictures that describe the steps, and the result of each step could help
the better understanding of this paper, and the paper needed some grammar changes. To
our current state of knowledge, it was a good and accurate paper.
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors,
and minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.
This paper was a final version
Recommendation
 This paper should be published as is
This paper should be published with revision
 This paper should not be published
This paper was a rough draft