In Silico  Modeling of Voltage-gated Sodium Channel Alpha Subunit to Understand Insecticide Binding Simulation in Mosquitoes

Manas Sarkar<sup>1,2</sup>; Aparajita Borkotoki<sup>1</sup>

Research Article

In Silico Modeling of Voltage-gated Sodium Channel Alpha Subunit to Understand Insecticide Binding Simulation in Mosquitoes

Manas Sarkar^1,2

, Aparajita Borkotoki¹

1. Department of Zoology, Gauhati University, Guwahati, Assam-781014, India
2. Research & Development (Advance Technology and Innovation), Godrej Consumer Products Ltd., Pirojshanagar, Mumbai-400079, India

Author

Correspondence author
Journal of Mosquito Research, 2015, Vol. 5, No. 23 doi: 10.5376/jmr.2015.05.0023
Received: 16 Sep., 2015 Accepted: 04 Nov., 2015 Published: 24 Nov., 2015

This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Preferred citation for this article:

Sarkar M., and Borkotoki A., 2015, In Silico modeling of voltage-gated sodium channel alpha subunit to understand insecticide binding simulation in mosquitoes, Journal of Mosquito Research, 5(23): 1-8 (doi: 10.5376/jmr.2015.05.0023)

Abstract

The Voltage Gated Sodium Channel (VGSC) is critical for binding of different insecticides and play key role in insecticide resistance. An important mechanism of resistance to DDT and pyrethroids is termed knockdown resistance (kdr), caused by mutations in IIS6 domain of sodium channels. To attain a better management strategy for insecticide resistance and screening of new insecticide molecules, it is important to understand the three-dimensional structure of insecticide-binding domain of VGSC and its molecular interaction with insecticides. We constructed a theoretical model of ion transport domain–II of VGSC from mosquitoes, Culex quinquefasciatus. The stereochemistry of the model shows 91.1% residues are in the most favored region. Docking studies with DDT and deltamethrin indicated that deltamethrin showed interaction with Thr⁹²⁹, Met⁹¹⁸, Ile⁹³⁶, Cys⁹³³, Leu⁹²⁵, Glu⁸⁸¹, Met⁸⁵⁷ and Gly⁸⁶⁶ and DDT showed interaction with Ile⁹³⁶, Thr⁹²⁹, Ser⁸⁷⁸, Phe⁸⁶³, Gln⁸⁶⁴, Trp⁸⁶¹ and Met⁸⁵⁷. We also predicted that mutation of Thr⁹²⁹ should confer resistance to both DDT and deltamethrin.

Keywords

Insecticide resistance; in silico modeling; Knockdown resistance; Docking; Kdr mutation

Introduction

The Voltage Gated Sodium Channel (VGSC) mediates the initiation and propagation of action potential in the nervous system and other excitable cells. The critical role of the sodium channel in membrane excitability has made it the target for a variety of neurotoxins during evolution (Wang and Wang, 2003). During an action potential, the sodium channel undergoes transactions between closed-resting, activated, and inactivated functional states and neurotoxins alter various channel properties, including ion conductance, ion selectivity, activation, or inactivation. “There are at least ten separate binding sites for ligands on the sodium channel, including those for local anesthetics and anti-convulsants” (O’Reilly et al., 2006). The site classified as ‘7’ has been recognized as binding site for the DDT [1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane] and pyrethroid insecticides (Wang and Wang, 2003).

Prior to the 1970s, DDT was used intensively in agriculture and in vector control, but following concerns over its environmental impact, its use in agriculture was discontinued or banned, but it is still recommended in vector control programs by World Health Organization (WHO). Pyrethroid, the synthetic analogues of the naturally occurring pyrethrum from the flower extracts of Chrysanthemum species, is a favored alternative of DDT. Because of the relatively low mammalian toxicity, low persistence in the environment and high excito-repellency activity, pyrethroids represent a major class of insecticide used to control many agriculturally and medically important arthropod pests.

Lymphatic or bancroftian filariasis is considered as the predominant infection in the continental Asia (Gyapong et al., 2005). The mosquitoes, Culex quinquefasciatus is the principal vector of the parasitic worm Wuchereria bancrofti the agent of bancroftian filariasis throughout the continental Asia. Due to indiscriminate and intensive use of insecticides, many insects including mosquitoes have developed resistance to these compounds. An important mechanism of resistance to DDT and pyrethroids is termed as knockdown resistance (kdr) was first discovered in houseflies (Milani, 1954) and subsequently in many other insect and arachnid species (Soderlund and Bloomquist, 1990; Soderlund and Knipple, 2003). Extensive research has shown that kdr or kdr-like mechanisms are resulted by mutations in sodium channels (Dong, 2002; Soderlund and Knipple, 2003; Vais et al., 2001). Like mammalian sodium channel alpha subunits, the primary structure of insect sodium channel proteins consists of four homologous domains, each containing six transmembrane segments (S1 – S6) (Loughney et al., 1989). Mutations in the domain-II region of the channel protein are commonly responsible for insecticide resistance. The most common mutation is leucine to phenylalanine at IIS6 domain and is referred to as the kdr mutation.

To attain a better management strategy for insecticide resistance, development of effective interference mechanisms and screening of new insecticide molecules, it is essential to understand the three-dimensional structure of voltage-gated sodium channel and the molecular interaction between the target site and its ligands. Therefore, we designed a three-dimensional theoretical model of the insecticide-binding domain (i.e., IIS6 domain) of voltage-gated sodium channel alpha subunit from Culex quinquefasciatus and studied the molecular interactions between channel protein and insecticides. We computed this molecular interaction using two insecticides, DDT, and deltamethrin. Automated docking studies were used to perform this insecticide binding simulation analysis.

1 Material and Methods

1.1 In Silico modeling

The amino acid sequence of Voltage gated sodium channel alpha subunit of Culex quinquefasciatus (Taxonomic Identifier: 7176, NCBI) was retrieved from the Uniprot database (http://www.uniprot.org/uniprot/A5I9E7; accession number: A519E7). In this sequence, the site of kdr mutation that occurred due to substitution of leucine to phenylalanine is at position 1016, whereas in general this mutation found at position 1014 in other referral sequences (Martinez-Torres et al., 1998, 1999; Chandre et al., 1998; Wondji et al., 2008). However, we did not make any manual adjustment to the retrieved sequence during modeling. But during discussion of ligand binding simulation, this error had been taken into consideration and based on the available sequence in literatures, the position of all amino acid residues were accepted as –2 (minus two) from their original position in the three-dimensional model.

The sequence was submitted to the Pfam to search for its families and domains (http://pfam.sanger.ac.uk/search). BLAST search was used to find the homology of the query sequence with other known sequences of the database. Initially full-length protein sequence (accession number: A519E7) was submitted to the server 3D-JIGSAW (version 3.0) POPULUS (http://bmm.cancerresearchuk.org/~populus/populus_submit.html) in search of suitable templates for homology modeling of VGSC protein. The server identified templates using HMM (Soding, 2005) and returned alignments were used to build the model. All models were preselected using POPULUS ENERGY, gaps and missing residues were closed and filled using POPULUS REPAIR, finally all models were recombined using basic POPULUS approach (Offman et al., 2006), where Genetic Algorithm (GA) was used for conformational space search engine. All models were ranked using a fine and coarse energy function weighted according to the highest sequence identity. The server constructed several models on split site basis using different templates rather than building a single model covering the whole sequence.

After analyzing all the initial models, we did not found any suitable template for construction of the whole VGSC protein model. Therefore, depending on the sequence id and energy function, only domain-II of VGSC (residues 833 to 1047) was modeled. We selected the X-ray crystal structure of the rat brain Kv 1.2–Kv 2.1 Paddle Chimera Channel (PDB Code: 2R9R, chain ‘B’), a member of the Shaker family of voltage depended potassium channel as a structural template to generate a homology model of VGSC domain-II. The model was then recombined using basic POPULUS to other 29 models (Table 1), which were selected according to their coverage, sequence id and domains, for the construction of the final three-dimensional structure of VGSC domain-II (residue coverage: 833 to 1047).

Table 1 Templates used for POPULUS recombination to generate the VGSC domain-II model

The backbone conformation and stereo-chemical properties of the constructed model was evaluated by Psi/Phi Ramachandran plot

Journal of Mosquito Research

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