| Summary: | 碩士 === 國立臺灣大學 === 生理學研究所 === 97 === ClC-2 is a member of the ClC family of chloride channels and transporters. It is mainly located on the plasma membrane and expressed in various kind of tissues, and most abundant in the brain and epithelium. Previous studies have shown that ClC-2 channel expression in neurons may modulate their membrane excitability. A disruption in the ClC-2 gene was reported to correlate with the presence of generalized epilepsy in human. In spite of the recent progress in the understanding of the physiological and pathophysiological significance of ClC-2 channels, their regulatory as well as signaling pathways remain unclear. Therefore, we applied yeast two-hybrid screen to search for ClC-2-interacting proteins, which will provide important insight on the physiological function of ClC-2 channels.
We focused on a sequence between the CBS1-CBS2 region (amino acids 581-794) at the C-terminal tail of ClC-2, which was cloned into the bait vector pGilda. After screening a rat brain cDNA library, 124 prey clones were identified. By eliminating the clones with incorrect reading frames, we have obtained 51 positive clones. 10 potential candidates from 51 positive clones were chosen for further characterization. X-gal assays and Leucine requirement tests were performed to reconfirm the interaction between ClC-2 and potential candidate proteins. All of the 10 candidate proteins showed blue patches on X-gal-containing plates and were grown on leucine-deficient plates, suggesting that these clones may indeed interact with ClC-2 channels. We performed co-immunoprecipitation and GST pull-down assay to verify the interaction between ClC-2 and potential candidates of ClC-2-interacting proteins. Despite of the fact that the signal intensity of co-immunoprecipitation was not optimal, our current data suggest that ClC-2 probably display direct interaction with with NSF (N-ethylmaleimide sensitive fusion protein), SMAD 1, and Carhsp 1 (calcium regulated heat stable phosphoprotein 1). Moreover, our preliminary GST pull-down results also support a direct interaction between NSF and ClC-2. Further experiments, hower, will be required to verify the foregoing observation. In the future, we plan to apply electrophysiological techniques to determine whether these candidate proteins may affect the biophysical properties and/or trafficking process of ClC-2 channels.
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