In Silico Analysis to Study Blockade of Potassium Channels by Common Poisons and Their Relative Affinity of Binding

Send Orders for Reprints to [email protected] Neuroscience and Biomedical Engineering, 2015, 3, 11-19 11 In Silico Analysis to Study Blockade of Potassium Channels by Common Poisons and Their Relative Affinity of Binding

Sunil M. Patel1,*, Ashutosh Mishra1 and Manish Patel2

1Department of Applied Science & Biomedical Engineering, IIIT-Allahabad, India; 2Saffrony Institute of Technology, Gujrat, India

Abstract: The peptide poison binding mechanism shows how different types of potassium channels are affected or blocked by peptide poisons derived from disparate species of scorpions and snakes. The peptide poison binding mechanism to the extracellular domain of potassium channel called pore domain was examined. The binding affinity of a given poison molecule to a specific potassium channel is quantified on the basis of ZDock Score of Protein-Protein docking using Accelrys Discovery Studio. Interacting amino acid residues of peptide toxin and those of the extracellular pore domains are identified by a standalone application and mechanism behind the toxin’s binding severity to potassium channel is deduced. From ZDock score of pose with highest score obtained after docking, we found that peptide poisons have the highest binding affinity to the calcium-gated potassium channel, followed by inward rectifier potassium channel and then lastly to the voltage-gated potassium channel. Using Delphi, we calculated charges on the toxin molecule and found that Electrostatic charges on peptide poison and pore forming residues of Potassium channel are identified as key factors responsible for binding. Dendrotoxin, which is having overall and per amino acid higher positive charge, is found to be most potent toxin compared to charybdotoxin and agitoxin. Keywords: Binding analysis, MD simulation, potassium channel blocker, snake venom, scorpion venom.

1. INTRODUCTION of 38 amino acids, it binds to all state of k + channel. 1AGT 1, 2 and 3 have similar structures of triplet stranded antipar- Potassium channels are ubiquitous to the family of neu- allel β-sheet consisting of c-terminal in core and alpha-helix ron and muscle membrane protein. The genetic bases of the guarding one face [7] (Fig. 1). potassium channel is universally conserved. Potassium channels are distinct and omnipresent in membrane proteins 2.1.2. Charybdotoxin- (PDB ID - 2CRD) of not only excitable but non excitable cells as well, respon- + Found in venom of scorpion Leiurus quinquestriatus he- sible for selective conduction of K ions across the cell membrane and control a wide variety of cell functions [1-6]. braeus and is known to block calcium activated potassium channel. It consists of 37 amino acid neurotoxin with three These channels may be Voltage-gated potassium channels, di-sulfide bridges [8, 9]. Calcium-gated potassium channels or Inward rectifier potassium channels. These are diverse groups of ion channels and 2.1.3. Dendrotoxin- (PDB ID - 1DTX) play a fundamental role in the modulation of cell excitability as well as controlling the frequency and the shape of action Neurotoxin produced by mamba snake Dendroaspis potential waveform, and in the secretion of hormones and polylepispolylepis [10]. DTX has a single peptide chain of 58 neurotransmitters. Peptide toxins derived from disparate spe- amino acids, consists of several topologies, having slight cies of scorpions and snakes selectively bind to the extracel- difference in the sequence but all shows similar folding con- lular domain pore of potassium channel resulting in toxic formations [11]. A two stranded anti parallel β-sheet, con- neurological effect. Peptide toxins, which are not membrane nected by a distorted β-turn region occupies the central part permeable, may either block the channel pore or alternatively of the molecule. modify the activation of potassium channels. 2. TOOLS AND METHODS 2.1. Dataset 2.1.1. Agitoxin- (PDB ID - 1AGT) Found in the venom of East Indian red scorpion Buthus tamulus and Leiurus quinquestriatus hebraeus, AGT consists

*Address correspondence to this author at the Department of Applied Sci- Fig. (1). Structure of toxins A) Agitoxin, B) Charybdotoxin, and C) ence & Biomedical Engineering, IIIT-Allahabad, India; Dendrotoxin. E-mail: [email protected]

2.2. Target Selection minimization, temperature equilibration, pressure equilibration and MD. Parrinello-Rahman [21] and V-rescale [22] methods Genetic screening approach reveals that the shaker gene were used for barostat and thermostat, respectively. DPPC (also known as CG33472) in Drosophila melanogaster has a lipid parameters were obtained from D. Peter Tieleman’s striking effect on the sleep quality and quantity of the fly website (http://wcm.ucalgary.ca/tieleman). Lipid parameters [12, 13]. Blastx [14] of CG33472 (Drosophila melanogaster includes 1) dppc128.pdb - the structure of a 128-lipid DPPC quiver) resulted in the identification of various domains pe- bilayer 2) dppc.itp - the molecule type definition for DPPC 3) culiar to potassium channels. Functional annotation of Volt- age-gated potassium channel of PDB ID- 1A68 was per- lipid.itp - Berger lipid parameters structures from output files formed to identify other related potassium channels, such as were used for docking purpose [23]. All potassium channels Calcium-gated Potassium channel (PDB ID - 1LNQ) and G- were prepared for docking (loop building and protonation) protein-gated inward rectifier Potassium channel in complex using Accelrys Discovery Studio Client 2.5. with the beta-gamma G protein subunits (PDB ID - 4KFM). Structures of all these Potassium channels were taken from 2.6. Binding Site Selection the RCSB database [15]. QSITEFINDER [24] was used for cavity detection in all potassium channels, in channel BK amino acid residues 2.3. Conserved Domain VGYGD & Y (60-65) in each monomer forming pocket of 3 Conserved domain analysis of all potassium channels volume 860 Å was taken as a binding site. Similarly, in GIRK2 amino acid residues GYGYR & V (156-161) of each (Kv, BK and GIRK2) were carried out with Motif Scan [16] 3 using HAMAP, PROSITE and Pfam HMMs [17]. monomer forming pocket of volume 8784.6 Å extracellularly was taken as a binding site. Kv consists of sequence SGLAFQ 2.4. Model Building of Potassium Channels & T of antiparallel β-sheet and LRD & Q of turret forms cavity of approximate volume 10630Å3, which is sufficient Naturally functional potassium channel exits as 4 TM enough to accommodate very large sized toxin peptide was symmetric subunits, protein files of Kv and GIRK2 obtained taken as a binding site. Kv and GIRK2 offer voluminous bind- from RCSB database contains only one subunit (Table 1). ing site due to the presence of turret surrounding pores. SymmDock [18] was used for forming 4th order symmetric multimer of monomer obtained from the RCSB database. 2.7. Docking Distance Constraints between units were set to auto, and the binding site of one unit to another unit was chosen on Cross docking of all potassium channels to all toxins was the basis of their X-ray structure available in the literature performed using Z-Dock in Accelrys Discovery Studio Cli- [19, 20]. ent 2.5. Z-Dock utilizes Fast Fourier Transformer [25] for rotational and translational motion and scoring was based on 2.5. Protein Preparation and Molecular Dynamics (MD) pair wise shape complementarity of proteins. Receptor Bind- Simulation ing Sites for all potassium channels were chosen using com- bined knowledge of literature review [26, 27] and pocket In model building, we used distance constrains between having highest volume extracellularly was selected on the two units as auto and we took site for assemblies formation basis of QSITEFINDER results. Bi-lipid membrane was from the literature. Therefore, in order to get near-natural form added to each potassium channel using transmembrane pro- of these assemblies we carried out MD simulations. After teins tool to avoid docking of toxins at transmembrane por- building the model (Symmetrical tetramer), Kv and GIRK2 tion of potassium channel. Clustering RMSD Cutoff was were prepared for MD run using DockPrep utility of UCSF kept 2.0. Angular step size was kept 6 (which explores 6400 Chimera. DockPrep adds hydrogens and assigns partial poses). We assume that electrostatic charges play an impor- charges to the protein. The prepared Kv and 4KFM tetramers tant role in binding of a toxin to a extracellular pore of potas- were energy minimized using GROMACS 4.5.5 using OPLS- sium channel, therefore we used Electrostatic and Desolva- AA force field in lipid bilayer of DPPC (dipalmitoyl phos- tion energy criteria in addition to original protocol for dock- phatidylcholine). Molecular dynamics run was carried out for ing [28]. All these criteria were kept same for all the 9 pairs 5 ns time period using leap-frog integrator (Fig. 2). Reading of protein-protein docking for lucid correlation of results. were taken at every 2 fs during MD. Four parameter files were Out of 6400 pores, best having highest z-dock score and used as input for MD simulations, which included energy RMSD Cutoff within limit were selected (S-2).

Table 1. Conserved domain analysis of Potassium channels resulted into three specific domains.

Sr. No. Name Description

The N-terminal, cytoplasmic tetramerization domain (T1) of voltage-gated potassium channels encodes molecu- 1 pfam02214:AK_tetra lar determinants for subfamily-specific assembly of alpha-subunits into functional tetrameric channels Transmembrane ion channel family was defined in Pfam as family of tetrameric sodium, potassium, and 2 Ion_trans (PF00520) calcium ion channels, in this motif two C-terminal transmembrane helices flank a loop which determines ion selectivity of the channel pore This domain belongs to calcium activated BK potassium channel alpha sub unit. It belongs to superfamily - 3 Pfam03493/BK_channel_a BK_channel_a super In Silico Analysis to Study Blockade of Potassium Channels Neuroscience and Biomedical Engineering, 2015, Vol. 3, No. 1 13

3. RESULTS AND ANALYSIS 3.2.2. Radius of Gyration (Rg)

3.1. Conserved Domain Analysis Protein compactness is measured by using Rg. In (Fig. 3) from t=0 ps to t=1600 ps R fluctuates but in later stage of 3.2. Energy Minimization of Protein g simulation time period, it remains almost stable in range of Energy Minimization of Kv and GIRK2 was carried out 1nm - 1.2nm. In (Fig. 4A) from t=0ps to t=3675ps Rg fluctu- at ~1016.38 g/l density, ~1.00264 bar pressure and ~299.561 ates but thereafter it remains stable in range of 1.68nm- K temperature in DPPC (dipalmitoyl phosphatidylcholine). 1.7nm. Rg remains almost constant at the end of simulation for sufficient time period, this indicates the stability of 3.2.1. Root Mean Square Deviation (RMSD) Analysis tetramer assemblies (Table 2). RMSD measures the deviation or conformational changes 3.2.3. Potential Energy of the structure backbone with respect to previous structure The nativeness of structure can be hypothesized by calculat- at any time interval during the simulation. RMSD was calcu- ing potential energy of protein in the system. Potential energy is lated for c-alpha - c-alpha comparison. It is more representa- -6 in order of 10 in both the protein assemblies. This is an accept- tive for structural integrity. Both structures deviate with an able energy range for to confirm stability of such assemblies. RMSD of ~0.3nm, this is an acceptable range, which indicates that tetramer assemblies gained stability during the simulation.

A Radius of Gyration B Backbone after Isq fit to Backbone

1.6 5

4 1.4 3 Rg (nm)

RSMD (nm) RSMD 2 1.2 1

0 0 1000 2000 3000 4000 5000 0 1000 2000 3000 4000 5000 Time (ps) Time (ps) Fig. (2). Plot shows Kv tetramer assembly MD simulation (A) Radius of Gyration (B) RMSD of backbone relative to the minimized and equilibrated structure. A B Radius of Gyration Backbone after Isq fit to Backbone

1.75 0.3

0.2

1.7 Rg (nm)

RSMD (nm) RSMD 0.1

1.65 0 01000 2000 3000 4000 5000 01000 2000 3000 4000 5000

Time (ps) Time (ps)

Fig. (3). Plot shows GIRK2 tetramer assembly MD simulation (A) Radius of gyration (B) RMSD of backbone relative to the minimized and equilibrated structure. A Gromacs EnergiesB Gromacs Energies

Potential Potential -1.4x105 0.5x105 -1.6x105 0 -1.8x105 (kl/mol) (kl/mol) -0.5x106 -2x105 -1x105

0 50 100 150 200 0 500 1000

Time (ps) Time (ps)

Fig. (4). Potential energies of (A) Kv tetramer assembly (B) GIRK2 tetramer assembly. During simulation structures were almost stable and potential energy was in order of 10-6 kj/mol. 14 Neuroscience and Biomedical Engineering, 2015, Vol. 3, No. 1 Sunil M Patel

3.3. Docking From (Fig. 4) it is quite evident that 1DEM is having the highest affinity to block all types of potassium channels under To validate the ligand orientation and position in receptor study. After 1DEM, 2CRD is considered more potent as com- pocket, method of docking and re-docking was used. All pared to 1AGT based on Z Dock score. It is also clear form ligands were cross-docked to all potassium channels, gener- the plot that BK is the most vulnerable to poisonous attack, in ating total 9 combinations. The output is sorted according to comparison to GIRK2 and KV. Amongst all channel under ZDock score. Docked pose having highest ZDock scores is consideration, KV remains least affected to toxin attack. selected.

ZDock score showing comaprative Affinity of Toxin to bind pore of Potassium channel 25

19.54 20.22 20 18.66 16.54 16.84 16.96 15.04 15.18 15 11.58

10 ZDock Score

0 1AGT 2CRD 1DEM 1AGT 2CRD 1DEM 1AGT 2CRD 1DEM Kv G1RK2 BK Toxin-Potassium channel pair

Fig. (5). A comparison using Z dock score showing comparative affinity of Toxin to bind potassium channel. 3.4. Binding Analysis Table 2. Binding residue of Kv to 1AGT 1. 3.4.1. Kv Approximate Bond Turrets are protruding ~10Å extracellularly [29]. Extracel- Residues of Kv Residues of 1AGT lular exposed two antiparallel β-sheet (of six TM) and turret of Length(Å) each subunit of the channel constitute a framework for binding of toxin molecule to the pore domain [30]. Pore is located at ARG-76:NH1|A 2.4 ASN-4:O|A the center of 4 turrets at the base of bowl like structure. ~25Å ARG-76:NH2|A 2.7 SER-6:2HB|A of cross turret distance is sufficient enough to accommodate small toxin peptide. All toxin peptides under study were found ARG-106:NE|A 1.4 SER-6:1HB|A to occlude the ion passage by occupying pore (Table 3). ASN-107:N|A 1.6 ASN-30:1HD2|A

ASN-107:O|A 1.8 LYS-31:2HZ|A

GLY-74:O|B 2.1 ASN-4:1HD2|A Toxin Moleeule ARG-76:NH1|B 2.5 ASN-4:2HD2|A

Pore LEU-105:N|B 2.9 ARG-19:2HH1|A

ARG-106:NH2|B 1.8 ARG-19:2HB|A

PRO-104:O|C 2.2 ASN-22:1HB|A Interacting Base Interacting Turret Residues Residues LEU-105:O|C 1.7 ASN-22:2HB|A

ARG-106:O|C 0.9 HIS-21:HD1|A

Fig. (6). Shows binding of Toxin molecule to Kv (a) Side view ARG-106:NE|C 2.6 PCA-1:1HG|A shows space between two red lines which indicates TM region. (b) GLU-108:OE1|C 1.5 HIS-21:HE1|A Top view of bound state of toxin molecule to the cavity of Kv toxin molecule is shown as tube, represented in violet color, Turret forming ARG-76:NH1|D 1.4 LYS-32:3HZ|A residue (105-107 of all four fragment) in extracellular region are shown by green color in th space filling region. Pore forming residue (73-77 of all four fragments) in are shown by orange space filled 1Binding site residues for all cross docking combinations are identified by a region. Kv is shown as tan ribbon. Image was generated using VMD standalone Perl script. and processed with Persistence of Vision Ray-Tracer™ (POV-Ray). In Silico Analysis to Study Blockade of Potassium Channels Neuroscience and Biomedical Engineering, 2015, Vol. 3, No. 1 15

Table 3. Binding residue of Kv to 2CRD. 3.4.2. BK Eight RCK domains provided by the four channel Approximate Bond Residues of Kv Residues of 2CRD subunits form a gating ring in BK (Table 4). The pore is ~5Å Length(Å) in diameter and made up of about 400 amino acids (~22.2% + ARG-76:NH1|A 1.4 LYS-32:3HZ|A of total), providing a path for the movement of K ions be- ARG-76:NH1|B 2.3 ASN-4:O|A tween the cytoplasm and the pore [31, 32] (Fig. 5). Amino acid from 61 to 65 of the extracellular domain interacts with ARG-76:NH2|B 2.6 SER-6:2HB|A toxins. Unlike Kv and GIRK2, BK has no turret like struc- ARG-106:NE|B 1.5 SER-6:1HB|A ture present in the extracellular region (Fig. 6). ASN-107:N|B 1.7 ASN-30:1HD2|A ASN-107:O|B 2.0 LYS-31:2HZ|A GLY-74:O|C 2.8 ASN-4:ND2|A ARG-76:NH1|C 2.6 ASN-4:2HD2|A LEU-105:N|C 2.6 ARG-19:2HH1|A LEU-105:O|C 2.5 ARG-19:2HH1|A ARG-106:NH2|C 1.8 ARG-19:2HB|A PRO-104:O|D 2.4 ASN-22:1HB|A LEU-105:O|D 1.9 ASN-22:2HB|A ARG-106:O|D 2.1 HIS-21:HA|A ARG-106:NE|D 2.7 PCA-1:1HG|A GLU-108:OE1|D 1.6 HIS-21:HE1|A Fig. (7). Shows binding of Toxin molecule to BK (a) Side view Table 4. Binding residue of Kv to 1DEM. shows space between two red lines which indicates TM region. (b) Perspective view of bound state of toxin molecule to the cavity of BK toxin molecule is shown as tube representation in green color, Approximate Bond Residues of Kv Residues of 1DEM Length(Å) interacting residues (105-107 of all four fragment) in extracellular region are shown by orange color space filling region. Pore forming ARG-106:NH2|A 2.0 THR-36:HB|A residues (73-77 of all four fragment) are shown by red color space filled region. BK is shown as silver ribbon. Image is generated us- ARG-76:NE|B 2.4 ARG-11:NH1|A ing VMD and processed with Persistence of Vision Ray-Tracer™ PRO-104:O|B 1.5 GLY-42:N|A (POV-Ray). LEU-105:N|B 2.9 GLY-42:H|A LEU-105:O|B 3.1 ASN-12:ND2|A ARG-106:N|B 2.6 ASN-12:HA|A Table 5. Binding residues of BK to 1AGT.

Table 6. Binding residues of BK to 2CRD.

Residues of Approximate Bond Residues of 1DEM BK Length(Å)

GLY-63:O|A 2.2 THR-9:HG1|A

GLY-63:O|A 1.8 THR-9:1HG2|A

ASP-64:N|A 1.6 THR-9:3HG2|A

GLY-63:O|B 2.7 LYS-11:HA|A

ASP-64:O|B 1.0 TRP-14:HZ3|A Fig. (8). Shows binding of Toxin molecule to GIRK2 (a) Side view GLY-63:N|C 1.9 LYS-27:2HE|A shows space between two red lines which indicates TM region. (b) GLY-63:O|C 1.7 LYS-27:HA|A Perspective view of bound state of toxin molecule to the cavity of GIRK2. Toxin molecule is shown as cartoon representation in or- ASP-64:N|C 2.0 LYS-27:1HG|A ange color, Turret residues (105-107 of all four fragments) in ex- ASP-64:N|C 1.5 LYS-27:1HD|A tracellular region are shown by green color space filled region. Pore forming residues (73-77 of all four fragment) are shown by purple ASP-64:N|C 1.9 LYS-27:1HE|A color space filled region. GIRK2 is shown as blue ribbon. Image is ASP-64:N|C 1.4 LYS-27:2HE|A generated using VMD and processed with Persistence of Vision Ray-Tracer™ (POV-Ray). ASP-64:O|C 2.3 LYS-27:1HD|A

ASP-64:O|B 0.9 TRP-37:HH2|A Table 9. Binding residues2 of GIRK2 to 2CRD. TYR-65:N|B 2.8 TRP-37:HH2|A

GLY-63:O|D 2.5 GLU-52:HG2|A Approximate Bond Residues of GIRK2 Residues of 2CRD Length(Å)

3.4.3. GIRK2 GLY-158:O|A 1.5 ASN-4:1HD2|A Pore contains four polar amino acids (one from each TYR-159:OH|A 1.7 HIS-21:ND1|A subunit) projecting towards the ion pathway (Table 5). These TYR-159:OH|A 2.2 HIS-21:HD1|A polar amino acids are ASP-173 except in Kir1.1 and Kir6.1 where ASN is present [33]. A beta sheet and alpha-helix TYR-159:OH|A 0.8 HIS-21:HE1|A (pore helix) aligned to M2 (TM region) containing amino acid residue from 132-138 and 139-149 respectively of each TYR-159:OH|A 1.8 HIS-21:NE2|A subunit form ion pathway (Fig. 7). A bowl like cavity 8784.6 ASN-137:ND2|D 2.6 LYS-32:2HD|A Å3 is so formed by M1, M2 and selectivity filter residue (Table 6). All toxins under study occlude the pore by resid- ASN-137:ND2|D 2.3 LYS-32:NZ|A ing onto cavity. Even highest volume toxin under study, ASN-137:ND2|D 1.3 LYS-32:2HZ|A 2CRD of volume 5926 Å3 easily occupies the cavity. Pore guarding residues are present deeply into the bowl like cav- GLY-158:O|D 1.7 ASN-4:2HB|A ity made by turret residues (Fig. 8). In Silico Analysis to Study Blockade of Potassium Channels Neuroscience and Biomedical Engineering, 2015, Vol. 3, No. 1 17

(Table 9) contd…. (Table 10) contd….

Approximate Bond Approximate Bond Residues of GIRK2 Residues of 2CRD Residues of GIRK2 Residues of 1DEM Length(Å) Length(Å)

TYR-159:OH|D 2.1 LYS-32:3HZ|A ASN-137:OD1|J 1.7 LYS-28:HA|A

ASN-137:OD1|G 2.6 CYS-7:HA|A ASN-137:OD1|J 1.7 LYS-28:HG2|A

ASN-137:ND2|G 2.2 SER-6:O|A ASN-137:OD1|J 1.9 LYS-28:HG3|A

ASN-139:OD1|G 2.6 THR-8:2HG2|A ASN-137:ND2|J 2.2 LYS-28:O|A

TYR-159:OH|G 1.3 SER-6:1HB|A ASN-137:ND2|J 2.1 LYS-28:HG2|A

TYR-159:OH|G 1.6 CYS-7:H|A ASN-137:ND2|J 1.9 LYS-29:N|A

ASN-137:OD1|J 2.3 ARG-19:1HB|A GLY-158:O|J 2.1 LYS-29:HZ3|A

ASN-137:OD1|J 1.9 ARG-19:2HB|A TYR-159:OH|J 1.3 ASN-26:HD22|A

ASN-137:OD1|J 2.0 ARG-19:1HG|A TYR-159:OH|J 2.2 LYS-28:HB2|A

ASN-137:OD1|J 1.6 ARG-19:1HD|A TYR-159:OH|J 2.0 LYS-28:HB3|A

ASN-137:OD1|J 0.9 ARG-19:2HD|A

ASN-137:OD1|J 2.5 ARG-19:NE|A 3.5. Charge Calculation of Toxins

ASN-137:ND2|J 2.2 ARG-19:2HB|A Charge of toxins was calculated using Delphi [33]. Del- phi uses a two dielectric implicit solvent model and a finite TYR-159:OH|J 2.4 SER-15:HG|A difference method to solve the Poisson-Boltzmann Equation. Amber force field was used to type charges on atoms of tox- Table 10. Binding residues2 of GIRK2 to 1DEM. ins (Table 7). Sum of total charges and per amino acid charge on toxins are reported. Refer (Fig. S-3) for visualiza- tion of charge distribution around pores of potassium chan- Approximate Bond Residues of GIRK2 Residues of 1DEM nel (Tables 8, 9). Length(Å)

ASN-137:OD1|A 2.2 ARG-54:HH22|A Table 11. Total and per amino acid charge on peptide toxins.

TYR-159:O|D 2.4 PHE-23:HZ|A CONCLUSION

TYR-159:OH|D 2.4 GLU-51:HG3|A Potassium channels are primarily responsible for hyper- polarization of the cell and hence responsible for the tran- ASN-137:OD1|G 1.8 THR-36:H|A sient refractory period. In this work, we studied mode and relative affinity of different peptide toxins which bind to ASN-137:ND2|G 1.6 PHE-35:HA|A different potassium channels. Toxins are showing highest ASN-139:ND2|G 2.2 GLN-18:HG2|A affinity of binding to the calcium gated potassium channel, subsequently to G-protein-gated inward rectifier potassium ASN-139:ND2|G 2.4 THR-36:HG23|A channel and lastly to the voltage gated potassium channel TYR-159:OH|G 2.2 PHE-35:N|A (Table 10). In voltage gated potassium channel, turrets guard pore openings. In G-protein gated inward rectifier potassium TYR-159:OH|G 0.4 PHE-35:O|A channel, pore opening is located at the base of deep bowl like structure. Due to the absence of turrets and a peculiar shape guarding the cavity, calcium gated channels remain more sensitive to toxin blockage. Toxins bind to both the 24KFM tetramer obtained from SymmDock contains some chain name in repetition. For avoiding ambiguity all chain are renamed by using open and the closed states of calcium gated potassium chan- chain_changer.pl perl script (S-5) therefore, all residues reported above have nel. GLY74, ARG76, PRO104, LEU105, ARG106, ASN107 changed chain id accordingly. 18 Neuroscience and Biomedical Engineering, 2015, Vol. 3, No. 1 Sunil M Patel and GLU108 are identified as key residues where toxin binds [4] Davies AG, Pierce-Shimomura JT, Kim H, et al. A Central Role of the to the voltage gated potassium channel. GLY63, ASP64 and BK Potassium Channel in Behavioral Responses to Ethanol in C. ele- gans. Cell 2003; 115: 655-66. TYR65 are found to be key residues in calcium gated potas- [5] Billman GE. Role of ATP sensitive potassium channel in extracel- sium channel, where toxin binds. ASN139, ASN137, lular potassium accumulation and cardiac arrhythmias during myo- GLY158 and TYR159 are reported to be key residues in G- cardial ischaemia. Cardiovasc Res 1994; 28: 762-69. protein gated inward rectifier potassium channel where toxin [6] Jentsch TJ. Neuronal KCNQ potassium channels: physiology and binds. For all types of potassium channels Dendrotoxin role in disease. Nat Rev Neurosci 2000; 1: 21-30. [7] Krezel AM, Kasibhatla C, Hidalgo P, MacKinnon R, Wagner G. shows highest affinity among all toxins under study followed Solution structure of the potassium channel inhibitor agitoxin 2: by Charybdotoxin then Agitoxin. Potassium channel’s pore caliper for probing channel geometry. Protein Sci 1995; 4: 1478- guarding residues are mostly found to be negatively charged. 89. 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Received: January 05, 2015 Revised: June 29, 2015 Accepted: June 30, 2015