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 co-expressed 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 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 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 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.
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.
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, 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).
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.