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Fabian Hertel, Agathe Switalski, Elisa Mintert-Jancke, Katharina Karavassilidou, Kirsten Bender, Lutz Pott, Marie-Cécile Wellner-Kienitz

• \(\it {Background:}\) Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate \((PtdIns(4,5)P_{2})\) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by \(PtdIns(4,5)P_{2}\) in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane. For controlled and reversible depletion of \(PtdIns(4,5)P_{2}\), voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-C\(\delta\)1 (PLC\(\delta\)1) fused to ECFP and EYFP respectively and \(\textit {Ciona intestinalis}\) VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes. \(\textit {Methods and Results:}\) Expression and correct targeting of ECFP-PH-PLCδ1, EYFP-PH-PLC\(\delta\)1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of \(PtdIns(4,5)P_{2}\) in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated \(K^{+}\) (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P2 was identified. \(\it {Conclusions:}\) Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.