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A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells.

Hertel F, Switalski A, Mintert-Jancke E, Karavassilidou K, Bender K, Pott L, Kienitz MC - PLoS ONE (2011)

Bottom Line: 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.This was paralleled by inhibition of endogenous G protein activated K(+) (GIRK) current.Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology, Ruhr-University Bochum, Bochum, Germany.

ABSTRACT

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-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 results: Expression 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)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)P(2) was identified.

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.

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Continuous recording of ECFP and EYFP fluorescence from a single HEK293 cell transfected with pShuttle- PIP2-tool.The cell was voltage-clamped in whole cell mode. Fluorescence changes (A) were evoked by step depolarizations of 30 s in duration from −90 mV holding potential to the levels indicated. The resulting corrected fluorescence signals (FEYFP- yellow trace and FECFP- blue trace) were normalized to values before agonist application (F/Fo) or expressed as FRET ratio (FEYFP/FECFP, red trace). (B) Superimposed changes in FRET ratio from A on expanded timescale. (C) Changes in FRET ratio evoked by voltage-steps to −20 mV (blue) and +80 mV (black) were superimposed and scaled to match the steady-state levels. (D) Plot of t1/2 for rise time (depletion) and recovery of FRET signals against membrane potential. (E) Mean values±SEM of half times of FRET signal indicating PtdIns(4,5)P2-depletion and recovery by voltage steps to +80 mV (n = 6).
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pone-0020855-g002: Continuous recording of ECFP and EYFP fluorescence from a single HEK293 cell transfected with pShuttle- PIP2-tool.The cell was voltage-clamped in whole cell mode. Fluorescence changes (A) were evoked by step depolarizations of 30 s in duration from −90 mV holding potential to the levels indicated. The resulting corrected fluorescence signals (FEYFP- yellow trace and FECFP- blue trace) were normalized to values before agonist application (F/Fo) or expressed as FRET ratio (FEYFP/FECFP, red trace). (B) Superimposed changes in FRET ratio from A on expanded timescale. (C) Changes in FRET ratio evoked by voltage-steps to −20 mV (blue) and +80 mV (black) were superimposed and scaled to match the steady-state levels. (D) Plot of t1/2 for rise time (depletion) and recovery of FRET signals against membrane potential. (E) Mean values±SEM of half times of FRET signal indicating PtdIns(4,5)P2-depletion and recovery by voltage steps to +80 mV (n = 6).

Mentions: The positive detection of the ECFP/EYFP-fused PH-domains, which are downstream of the Ci-VSP-T2A sequence, predicts that Ci-VSP was expressed, C-terminally fused with 20 residues from the T2A-peptide [28]. In the majority of cases, fusion of a protein with a 2A-peptide moiety does not compromise its function. However, examples of proteins that are functionally disturbed by the peptide residues have been published [24], [29]. As illustrated in figure 2 (A–D), in HEK293 cells transfected with pShuttle-PIP2-tool, graded changes in FRET ratio, indicating depletion of PtdIns(4,5)P2, were induced by depolarizing voltage steps. Both, the amplitude and the on-kinetics of the FRET-response were voltage-dependent and, in line with previous studies, saturated at membrane potentials positive to +60 mV [18]. The kinetics of recovery upon repolarization, which should be governed by replenishment of PtdIns(4,5)P2 via phosphatidylinositol-4-phosphate 5-kinase, was independent of the preceding depolarization (C, D). Both the kinetics of depletion at saturating voltage and the recovery showed little variation in different cells (E). In conclusion, these data demonstrate expression and correct processing of the three proteins encoded by the single ORF in a standard mammalian cell line.


A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells.

Hertel F, Switalski A, Mintert-Jancke E, Karavassilidou K, Bender K, Pott L, Kienitz MC - PLoS ONE (2011)

Continuous recording of ECFP and EYFP fluorescence from a single HEK293 cell transfected with pShuttle- PIP2-tool.The cell was voltage-clamped in whole cell mode. Fluorescence changes (A) were evoked by step depolarizations of 30 s in duration from −90 mV holding potential to the levels indicated. The resulting corrected fluorescence signals (FEYFP- yellow trace and FECFP- blue trace) were normalized to values before agonist application (F/Fo) or expressed as FRET ratio (FEYFP/FECFP, red trace). (B) Superimposed changes in FRET ratio from A on expanded timescale. (C) Changes in FRET ratio evoked by voltage-steps to −20 mV (blue) and +80 mV (black) were superimposed and scaled to match the steady-state levels. (D) Plot of t1/2 for rise time (depletion) and recovery of FRET signals against membrane potential. (E) Mean values±SEM of half times of FRET signal indicating PtdIns(4,5)P2-depletion and recovery by voltage steps to +80 mV (n = 6).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111442&req=5

pone-0020855-g002: Continuous recording of ECFP and EYFP fluorescence from a single HEK293 cell transfected with pShuttle- PIP2-tool.The cell was voltage-clamped in whole cell mode. Fluorescence changes (A) were evoked by step depolarizations of 30 s in duration from −90 mV holding potential to the levels indicated. The resulting corrected fluorescence signals (FEYFP- yellow trace and FECFP- blue trace) were normalized to values before agonist application (F/Fo) or expressed as FRET ratio (FEYFP/FECFP, red trace). (B) Superimposed changes in FRET ratio from A on expanded timescale. (C) Changes in FRET ratio evoked by voltage-steps to −20 mV (blue) and +80 mV (black) were superimposed and scaled to match the steady-state levels. (D) Plot of t1/2 for rise time (depletion) and recovery of FRET signals against membrane potential. (E) Mean values±SEM of half times of FRET signal indicating PtdIns(4,5)P2-depletion and recovery by voltage steps to +80 mV (n = 6).
Mentions: The positive detection of the ECFP/EYFP-fused PH-domains, which are downstream of the Ci-VSP-T2A sequence, predicts that Ci-VSP was expressed, C-terminally fused with 20 residues from the T2A-peptide [28]. In the majority of cases, fusion of a protein with a 2A-peptide moiety does not compromise its function. However, examples of proteins that are functionally disturbed by the peptide residues have been published [24], [29]. As illustrated in figure 2 (A–D), in HEK293 cells transfected with pShuttle-PIP2-tool, graded changes in FRET ratio, indicating depletion of PtdIns(4,5)P2, were induced by depolarizing voltage steps. Both, the amplitude and the on-kinetics of the FRET-response were voltage-dependent and, in line with previous studies, saturated at membrane potentials positive to +60 mV [18]. The kinetics of recovery upon repolarization, which should be governed by replenishment of PtdIns(4,5)P2 via phosphatidylinositol-4-phosphate 5-kinase, was independent of the preceding depolarization (C, D). Both the kinetics of depletion at saturating voltage and the recovery showed little variation in different cells (E). In conclusion, these data demonstrate expression and correct processing of the three proteins encoded by the single ORF in a standard mammalian cell line.

Bottom Line: 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.This was paralleled by inhibition of endogenous G protein activated K(+) (GIRK) current.Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology, Ruhr-University Bochum, Bochum, Germany.

ABSTRACT

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-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 results: Expression 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)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)P(2) was identified.

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.

Show MeSH
Related in: MedlinePlus