Abstract
Replacement of the phosphatase domain of the Ciona intestinalis voltage sensitive phosphatase (CiVSP) with fluorescence protein (FP) FRET pairs generates chimeric proteins that respond to voltage fluctuations with changes in FRET efficiency. VSFP 2.1 and Mermaid represent two different CiVSP-based voltage probes. We have developed a single FP-based, CiVSP-based probe (pKF215). These probes have different FPs and linker domains. As these constructs represent the most promising probes developed to date, we have undertaken a structure/function analysis of these constructs by: i) altering the inserted FPs and ii) replacing the VSD with species orthologs. The goal is to produce probes with a larger and faster ΔF/F response. We have created a matrix of 33 different constructs replacing the FPs of the original constructs with a range of red-shifted FPs (mCherry, mStrawberry, mTangerine, mOrange, tdTomato and TagRFP). We have also replaced the inserts with the two currently most viable FRET pairs (CFP/YFP and the Mermaid FRET pair). Finally we have chosen three other FPs which we have previously identified as ‘modulatable’ (mCerulean, PHluorin and eGFP). As the S1 to S4 domain of CiVSP has produced the most successful fluorescent voltage sensing constructs, we sought orthologs of Ciona VSD that might perform better. We identified VSPs orthologs from a variety of different species. We cloned the VSP orthologs from chicken, mouse, human, Xenopus laevis, Xenopus tropicalis, and zebra fish. To test these VSDs, we have created VSFP 2.1-like constructs from these cDNAs by inserting the CFP/YFP FRET pairs at homologous positions in the orthologous sequence. The constructs are being tested for membrane expression and voltage-dependent fluorescence changes in HEK293 cells.
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