| Summary: | 碩士 === 國立臺灣大學 === 口腔生物科學研究所 === 94 === As a member of the family of inwardly rectifying potassium channels, Kir1.1 (ROMK1) channel is widely distributed in different tissues and regulates many important physiological functions, including maintenance of resting potential, synaptic excitability, and renal K+ transport.
Phosphorylation and dephosphorylation are believed to regulate the activation of Kir1.1 channels. Recently, it was demonstrated that the phosphorylation of Kir1.1 channels is mediated by cyclic AMP-dependent protein kinase (PKA) through the participation of PIP2. In addition, it has been since long proven that decrease in intracellular pH (pHi) can result in the channel closure of Kir1.1. The molecular mechanism responsible for such regulations may involve the structural rearrangements of the intracellular N- and C-terminal domains in Kir1.1 channels. However, the details required for further interpretation regarding this remain unclear. Therefore, we are in attempts to solve this problem through the determination of 3-D structures of these domains in Kir1.1 channels.
To reach this goal, in this study, we have successfully expressed the N- and C-terminal intracellular domains of Kir1.1 in E. coli as fusion proteins containing GST-tag. The C-terminal domain protein has been obtained through efficient purification with affinity chromatography, whereas, however, the N-terminal part was still unavailable due to the limited expression level. Our future studies will be focused in the large-scale expression and purification of the Kir1.1 N-terminal domain protein as short-term goal with respect to the crystallization and structural determination for both N- and C-terminal parts in a long run.
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