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- W2016076342 abstract "Ci-VSP, a voltage-activated phosphatase, which dephosphorylates PI-(4,5)-bisphosphate (PIP2) to PI(4)P combined with fluorescent (FRET) probes for optical monitoring of changes in plasma membrane PIP2 is an emerging tool to study regulation of ion channels and transporters by PIP2 (Murata et al., Nature 43, 2004). In the present study we used HEK239 cells to express GIRK1/GIRK4 channels by means of a conventional bi-cistronic vector containing an IRES sequence. The channel could be activated by a co-expressed A1 receptor. For expression of Ci-VSP and a pair of FRET-generating PIP2 binding probes (PH-PLCδ1-CFP and PH-PLCδ1-YFP) a multicistronic vector was constructed. This vector contained the cDNAs for the phosphatase and the fluorescent PH-domains separated by viral 2A-peptide sequences in a single ORF. The 2A-sequences result in cotranslational dissociation of the polyprotein while allowing translation to continue (de Felipe et al. JBC 278, 2003). Depolarizations to + 60 mV of variable duration (1 to 10 s) resulted in reductions in FRET ratio, indicating depletion of PIP2. Concomitantly, adenosine-activated GIRK current was reduced. The onset of current inhibition was faster than the onset of reduction in FRET, whereas upon repolarization the latter recovered faster (t1/2 ∼ 10 s) as compared to recovery of the current. This suggests the PIP2 affinity of the regulatory binding sites on the channel subunit(s) to be lower than the affinity of the PLCδ1 PH-domain. Our data demonstrate versatility of 2A-peptide based expression vectors for manipulation and quantifying membrane phosphoinositides in cell lines and primary cells." @default.
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- W2016076342 date "2010-01-01" @default.
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- W2016076342 title "A Multicistronic 2a-Peptide-Based Vector Encoding for Ci-VSP and a Pair of FRET Sensors to Study Effects of PIP2-Depletion on Receptor-Activated GIRK Current" @default.
- W2016076342 doi "https://doi.org/10.1016/j.bpj.2009.12.3829" @default.
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