Molecular biology and pharmacology of insect sodium channels

Voltage-gated sodium channels are essential for action potential initiation and propagation in the nervous system and other excitable cells. In the past two decades, ten different mammalian sodium channel α-subunit genes were identified. It appears that the structural, functional and pharmacological diversity of mammal sodium channels is achieved primarily through expression of distinct sodium channel genes. In Drosophila melanogaster, however, para is the only gene that has been demonstrated to encode functional sodium channels. How do insects achieve sodium channel diversity? Work in my laboratory has been focused on this question. We cloned a total of 69 full-length German cockroach sodium channel clones (BgNav, formerly para^CSMA) by direct reverse transcription polymerase chain reaction using RNA from heads and thoraces (Tan et al., 2002a,b). Functional characterization of these channels in Xenopus oocytes showed that alternative splicing and RNA editing are two major posttranscriptional mechanisms in the production of tissue/cell type-specific and functional distinct variants of sodium channels in insects (Tan et al, 2002b; Song et al. 2004; Liu et al., 2004). Currently we are using the powerful genetic resources available for Drosophila to generate D. melanogaster mutants that lack specific splicing or RNA edited variants and investigating the roles of alternative splicing and RNA editing in the regulation of neuronal excitability in insects.

We are also examining responses of sodium channel variants to various neurotoxins, with emphasis on insecticides and insect-specific scorpion toxins (in collaboration with Michael Gurevitz and Dalia Gordon, Tel-Aviv University, Israel). This project is supported by a National Science Foundation grant (IBN-0224877) and a BARD grant with M.Gurevitz and D. Gordon.

Knockdown resistance (kdr) of insects to pyrethroid insecticides

Voltage-gated sodium channels are targets of various neurotoxins including pyrethroid insecticides. Pyrethroid insecticides are widely used to control many agriculturally and medically important insect pests. Due to intensive use of pyrethroids, however, many pest populations have developed resistance to these compounds. One major mechanism of pyrethroid resistance, conferred by the knockdown resistance gene (kdr), is reduced target-site (sodium channel) sensitivity to DDT and pyrethroids. In the past several years we have been characterizing the molecular mechanism of kdr in the German cockroach (Dong, 1997; Dong et al., 1998; Liu et al., 2000; Tan et al., 2002a; Liu et al., 2002). We and others demonstrated that multiple point mutations in the sodium channel gene reduced the sodium channel sensitivity to pyrethroids, and that these mutations are likely responsible for kdr and kdr-type resistance to pyrethroids in insects.

Our current research is focused on understanding how kdr mutations reduce the sodium channel sensitivity to pyrethroids and how pyrethroids interact with sodium channels at the molecular level. This research project is supported by National Institutes of Health. In addition, in collaboration with Dr. Zachary Huang (Michigan State University) we are characterizing the mechanism(s) of pyrethroid resistance in the Varroa mite (an ecto-parasite of honeybee) (Wang et al., 2002). This project is supported by USDA-NRI. We published the full-length cDNA sequence of the varroa mite sodium channel gene (Wang et al., 2003). Currently, we are conducting functional expression of this varroa mite sodium channel in Xenopus oocytes.

Functional characterization of BSC1, a novel voltage-gated calcium-selective channel

Eighteen years ago, a putative sodium channel gene, DSC1, was reported. This gene was isolated from a D. melanogaster genomic DNA library using a cDNA probe encoding the eel sodium channel (Salkoff et al., 1987). The DSC1 protein shares a high sequence similarity with Para and mammalian sodium channel α-subunits. However, the functional identity of the DSC1 channel has never been determined. We cloned an ortholog of DSC1, called BSC1, from Blattella germanica (Liu et al., 2001). In collaboration with Al Goldin’s laboratory at UC, Irvine, we conducted molecular and functional characterization of the BSC1 channel, revealing that BSC1 encodes a voltage-gated Ca²⁺-permeable cation channel with gating properties distinct from those of classic sodium or calcium channels (Zhou et al., 2004). The physiological role of DSC1/BSC1 in neuronal signaling in vivo is the focus of current investigation.

Recent Lab Publications:

Tan, J, Liu, Z, Wang, R, Huang, ZY, Chen, AC, Gurevitz, M, and Dong, K. (2004) Identification of amino acid residues in the insect sodium channel critical for pyrethroid binding. Mol Pharmacol. 67:513-522.

Song, W., Liu, Z., Tan, J., Nomura, Y. and Dong, K. (2004) RNA editing generates tissue-specific sodium channels with distinct gating properties. J. Biol. Chem. 279:32554-32561.

Liu, Z., Song, W. and Dong, K. (2004) Persistent tetrodotoxin-sensitive sodium current resulting from U-to-C RNA editing of an insect sodium channel. Proc. Natl. Acad. Sci. USA 101:11862-11867.

Zhou, W., Chung, I., Liu Z. Goldin, A. L. and Dong, K. (2004) A voltage-gated calcium-selective channel encoded by a sodium channel-like gene. Neuron 42: 101-112.

Wang R., Huang Z. Y. and Dong K. (2003) Molecular characterization of an archnid sodium channel gene from the varroa mite (Varroa destructor). Insect Biochem. Molec. Biol. 33:733-739.

Tan, J. Liu, Z. Nomura, Y. Goldin, A.L. Dong, K. (2002) Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels. J. Neurosci. 22:5300-5309.

Wang R., Liu Z., Dong K., Elzen P., Pettis J. and Huang Z. Y. (2002) Novel mutations in a sodium channel gene are associated with fluvalinate resistance in the varroa mite, Varroa destroctor. J Apicultural Research 40:17-25.

Liu, Z., Tan, J., Valles, S. M. and Dong, K. (2002) Synergistic interaction between two cockroach sodium channel mutations and a tobacco budworm sodium channel mutation in reducing channel sensitivity to a pyrethroid insecticide. Insect Biochem. Molec. Biol. 32:397-404.

Tan, J., Liu, Z., Tsai, T., Valles, S. M., Goldin, A. L. and Dong, K. (2002) Novel sodium channel gene mutations in Blattella germanica reduce the sensitivity of expressed channels to deltamethrin. Insect Biochem. Molec. Biol. 32:445-454.

Liu, Z., Chung, I. and Dong, K. (2001) Alternative splicing of the putative BSC1 sodium channel gene generates tissue-specific isoforms in the German Cockroach. Insect Biochem. Molec. Biol. 31, 703-713.

Liu, Z. Valles, S. M. and Dong, K. (2000) Novel point mutations in the German cockroach para sodium channel gene are associated with knockdown resistance (kdr) to pyrethroid insecticides. Insect Biochem. Molec. Biol. 30, 991-997.

Links of Interest

Michigan State University Department of Entomology
Michigan State University Neuroscience Program