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.
Highlights
Phosphoinositides represent key molecules in numerous aspects of cellular signaling, including regulation of vesicular trafficking, cytoskeletal organization, cell cycle progression, and function of membrane proteins [1]
Adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes
In a first step we verified functionality of the two fluorescent PH-domains expressed from the bicistronic vector pShuttle-EYFPPH-PLCd1P2A-ECFP-PH-PLCd1 in HEK293 cells
Summary
Phosphoinositides represent key molecules in numerous aspects of cellular signaling, including regulation of vesicular trafficking, cytoskeletal organization, cell cycle progression, and function of membrane proteins [1]. 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 Forster resonance energy transfer (FRET), to monitor changes in plasma membrane. For controlled and reversible depletion of PtdIns(4,5)P2, voltagesensing 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-Cd1 (PLCd1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes
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