A Genetically Encoded Tool Kit for Manipulating and Monitoring Membrane Phosphatidylinositol 4,5-Bisphosphate in Intact Cells

Abstract

BackgroundMost ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P2 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)P2, 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-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes.Methods and ResultsExpression and correct targeting of ECFP-PH-PLCδ1, EYFP-PH-PLCδ1, 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)P2 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.ConclusionsExpression 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.

Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

“…However, the VSP enzymes offer several further advantages over the rapamycin-inducible changes: they allow induction of extremely rapid, quantitatively precise, and reversible changes in PM PtdIns(4,5)P 2 . Therefore, VSP is becoming an important experimental tool in phosphoinositide research for the controlled manipulation of PM phosphoinositides (422,609,953). C) PLIP/PTPMT1.…”

Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors

“…Chimeric ␣ 1A -AR displayed marked basal and agonistinduced phosphorylation (in contrast to the wild-type ␣ 1A -AR), whereas the opposite was observed in chimeric ␣ 1B -AR (34). The rapid decline of the ␣ 1B /␣ 1A -CT-induced DAG signal upon overexpression of WT PKC␦ supports the idea that the C ter-that is based on CFP-and YFP-labeled PH domains of PLC␦1 (22). To monitor the production of the second messenger DAG, we used receptor species as indicated (0.5) and the biosensor DAGR (0.5), which reports conformational changes of a CFP/YFP-labeled DAG-binding domain of protein kinase C (PKC␤2) (18).…”

“…Both cell lines were transfected using either polyethyleneimine as described in Ref. 22 or Lipofectamine (Invitrogen) according to the manufacturer's instructions. Prior to experiments, cells were seeded on sterile, poly-L-lysine-coated glass coverslips and analyzed 24 h (cells expressing DAGR) or 48 h after transfections.…”

“…Details on optical filters and beam splitters of the setup are given in Ref. 22. The individual FRET traces for obtaining concentration-response curves were normalized to the maximal response of the G protein biosensor at saturating agonist concentrations (FRET/ FRET 10 M ), denoted as FRET/FRET max ).…”

“…Presence of Phenylephrine-We investigated the depletion of membrane PIP 2 following ␣ 1B or M 1 receptor stimulation in CHO and HEK cells by utilizing a biosensor that directly reports the depletion of membrane PIP 2 during G q PCR/PLC activation with a decrease in FRET ratio (21,22). As illustrated in a representative FRET recording in Fig.…”