Application of yeast two-hybrid screen to identify P/Q-type calcium channel-interacting proteins in rat brain
| Autor: | Chi-Ming Lee, 李啟銘 |
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| Rok vydání: | 2012 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 P/Q type voltage-gated Ca2+ channels are composed of pore-forming α1A subunit and auxiliary α2δ and β subunits. In neurons, P/Q type voltage-gated Ca2+ channels are primarily located at dendrite and axon terminal, the neurophysiological role of P/Q type voltage-gated Ca2+ channels may include the neuronal excitability and modulation of synaptic transmission. Mutations in P/Q type voltage-gated Ca2+ channels are associated with a variety of hereditary neuropathy, the detail mechanisms of which remain unclear. P/Q type voltage-gated Ca2+ channels were regulated by SNARE proteins, G proteins and calmodulin (CaM). In this study, we aim to apply the cytoplasma C-terminus of P/Q type voltage-gated Ca2+ channels as the bait for yeast two-hybrid screening to search for novel P/Q type voltage-gated Ca2+ channels interacting proteins. 156 prey clones were identified after screening a rat brain cDNA library. By DNA sequencing and eliminating the clones with incorrect reading frames, we have obtained 67 positive clones. 10 potential candidates from 67 positive clones were chosen for further characterization. X-gal assays and leucine requirement tests were performed to reconfirm the interaction between P/Q type voltage-gated Ca2+ channels and potential candidate proteins. All of the 10 candidate proteins showed blue patches on blue and/white tests and were able to grow on leucine-deficient plates, suggesting that these clones may indeed interact with P/Q type voltage-gated Ca2+ channels. We performed GST pull-down assay, and found the 7 of the 10 candidate proteins have positive reaction. We performed Co-IP assay, so far, we test 3 of these candidate proteins and found the 2 of the 3 candidate proteins have positive reaction. And then, we performed immunofluorescence microscopy imaging to observe subcelluar lococaliztion of these candidate proteins. We can find which candidate protein might colocolize with P/Q-type calcium channels. We expect to take electrophysiological experiments prove P/Q-type calcium channel properties, in addition to prove their interactions with different biochemical experiments. We hope to better understand about physiological mechanism of the P/Q-type calcium channels and the related hereditary neurodegenerative disease. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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