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• W2023604813 abstract "Alternative splicing generates considerable functional diversity of large conductance calcium- and voltage-activated potassium (BK) channels. For example, inclusion of the STREX (STRess-activated EXon) insert into the unstructured cytosolic linker between the two RCK domains of the channel generates a cysteine rich domain (CRD) that confers intrinsic hypoxia sensitivity to the channel [1]. Here, we have developed YFP-mCFP fluorescent fusion proteins of the CRD and exploit these to examine conformational rearrangements in the CRD in response to hypoxia using fluorescence lifetime imaging microscopy (FLIM). The fusion protein, expressed in HEK293 cells, is targeted to the plasma membrane and responds to acute hypoxia (10 minute exposure at <5% oxygen) with a significant shift in fluorescence lifetime distribution (n=7). Pre-treatment with either oxidizing or reducing agents did not block the hypoxia induced shift in fluorescence lifetimes (n=4). In contrast, site directed mutagenesis of the cysteine residues within STREX, which we had previously shown to confer functional hypoxia sensitivity to the channel, abolished the hypoxia induced shift in lifetimes (n=6). The effects of cysteine mutagenesis and REDOX manipulation on hypoxia sensitivity were recapitulated in patch clamp assays of channel activity supporting a role for hypoxia-induced shifts in CRD conformation with changes in channel function. These findings suggest that FLIM, in conjunction with patch clamp electrophysiology, will be an important approach to monitor conformational rearrangements and investigate the structure-function relationship of the important unstructured linker region between the channel RCK domains. [1] McCartney, C.E., McClaffert, H., Huibant, J.M., Rowan, E.G., Shipston, M.J, and Rowe. I.C. (2005) A cysteine-rich motif confers hypoxia sensitivity to mammalian large conductance voltage- and Ca-activated K (BK) channel alpha-subunits. Proc. Natl. Acad. Sci. USA 102:17870-17876" @default.
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• W2023604813 date "2011-02-01" @default.
• W2023604813 modified "2023-09-27" @default.
• W2023604813 title "Investigating Structural Re-Arrangement of the BK Channel Rck1-Rck2 Linker Using Fluorescence Lifetime Imaging Microscopy" @default.
• W2023604813 doi "https://doi.org/10.1016/j.bpj.2010.12.1634" @default.
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