Ryanodine receptors (RyRs) are calcium channels located on the endo(sarco)plasmic reticulum of muscle cells and neurons. They are the largest ion channels known made up of four monomers, each 565kDa in size. Mammals have 3 different RyR isoforms, encoded by different genes, while insects express only one isoform of the receptor, which is only 46% similar (at the amino acid level) to its mammalian counterpart(s). RyRs function to regulate the release of luminal Ca2+ stores into the cell cytoplasm and play a key role in muscle excitation-contraction coupling (ECC). The plant alkaloid ryanodine, from which the receptor derives its name, has been investigated extensively as a potential pest control agent, but to date no commercial products have been identified. Recently two synthetic insecticides selectively targeting pest RyRs were introduced to the market. These compounds belong to the novel group of insecticides called diamides. In this study two insect ryanodine receptors were isolated sequenced and cloned into suitable expression vectors from economically important pests M. persicae and P. xylostella to identify protein site of interaction for the novel compounds. Both proteins were expressed in HEK 293 cells and Sf9 cells and analysed for evidence of function using ryanodine binding assays and calcium release imaging. In the case of M. persicae RyR the expression level was not sufficient to obtain any functional data. However the expression of P. xylostella RyR showed evidence of function in both HEK and Sf9 cells. Functional studies showed that expressed P. xylostella RyR can bind [3H] ryanodine and respond to various caffeine concentrations; the protein was also sensitive to both diamide compounds. DNA sequencing of RyR from field evolved diamide resistant strains of P. xylostella identified a mutation causing amino acid change G4946E. Functional analysis of modified RyR construct in Sf9 cells showed significantly reduced sensitivity to to both diamide compounds while retaining caffeine and ryanodine sensitivity comparable to the expressed WT form.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:611003 |
| Date | January 2013 |
| Creators | Troczka, Bartlomiej Jakub |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/60069/ |
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