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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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Fluorescent PH-PLCδ1 are expressed in adult cardiac myocytes after infection with Ad-PIP2-tool.Membrane localization of EYFP fluorescence in adult ventricular (Ve) and atrial (At) myocyte from heart of adult rat on day 4 after infection. Note that in line with previous studies, atrial myocytes show a tendency to round up within about two days in vitro, whereas ventricular myocytes under identical conditions retain their typical brick-shaped morphology for more than one week (B) Western blot from lysate of atrial myocytes 4 d after infection with Ad-PIP2-tool.
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pone-0020855-g004: Fluorescent PH-PLCδ1 are expressed in adult cardiac myocytes after infection with Ad-PIP2-tool.Membrane localization of EYFP fluorescence in adult ventricular (Ve) and atrial (At) myocyte from heart of adult rat on day 4 after infection. Note that in line with previous studies, atrial myocytes show a tendency to round up within about two days in vitro, whereas ventricular myocytes under identical conditions retain their typical brick-shaped morphology for more than one week (B) Western blot from lysate of atrial myocytes 4 d after infection with Ad-PIP2-tool.

Mentions: In order to make this molecular tool applicable to postmitotic transfection-resistant cells, the tricistronic pShuttle-CMV vector was used for recombination with the pAdEasy-1 backbone plasmid to generate a recombinant adenovirus (Ad-PIP2-tool). After infection with the virus, adult rat atrial and ventricular myocytes showed membrane-delimited fluorescence within ≥48 h (figure 4 A). In Western blots the singular fluorescent PH-domains were detected (B). Uncleaved protein was below detection limit. To demonstrate proof of principle and usefulness of this approach, we used endogenous GIRK current of atrial myocytes carried by tetrameric complexes of Kir3.1 and Kir3.4 [30]. This channel is opened by binding of Gβγ upon activation of muscarinic M2-receptors and other GPCRs that converge on pertussis toxin-sensitive Gi/o. The observation that this process requires the binding of PtdIns(4,5)P2 represents a key finding in this field [3], [31].


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)

Fluorescent PH-PLCδ1 are expressed in adult cardiac myocytes after infection with Ad-PIP2-tool.Membrane localization of EYFP fluorescence in adult ventricular (Ve) and atrial (At) myocyte from heart of adult rat on day 4 after infection. Note that in line with previous studies, atrial myocytes show a tendency to round up within about two days in vitro, whereas ventricular myocytes under identical conditions retain their typical brick-shaped morphology for more than one week (B) Western blot from lysate of atrial myocytes 4 d after infection with Ad-PIP2-tool.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111442&req=5

pone-0020855-g004: Fluorescent PH-PLCδ1 are expressed in adult cardiac myocytes after infection with Ad-PIP2-tool.Membrane localization of EYFP fluorescence in adult ventricular (Ve) and atrial (At) myocyte from heart of adult rat on day 4 after infection. Note that in line with previous studies, atrial myocytes show a tendency to round up within about two days in vitro, whereas ventricular myocytes under identical conditions retain their typical brick-shaped morphology for more than one week (B) Western blot from lysate of atrial myocytes 4 d after infection with Ad-PIP2-tool.
Mentions: In order to make this molecular tool applicable to postmitotic transfection-resistant cells, the tricistronic pShuttle-CMV vector was used for recombination with the pAdEasy-1 backbone plasmid to generate a recombinant adenovirus (Ad-PIP2-tool). After infection with the virus, adult rat atrial and ventricular myocytes showed membrane-delimited fluorescence within ≥48 h (figure 4 A). In Western blots the singular fluorescent PH-domains were detected (B). Uncleaved protein was below detection limit. To demonstrate proof of principle and usefulness of this approach, we used endogenous GIRK current of atrial myocytes carried by tetrameric complexes of Kir3.1 and Kir3.4 [30]. This channel is opened by binding of Gβγ upon activation of muscarinic M2-receptors and other GPCRs that converge on pertussis toxin-sensitive Gi/o. The observation that this process requires the binding of PtdIns(4,5)P2 represents a key finding in this field [3], [31].

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