Institute of Physiology, Ruhr-University Bochum, Bochum, Germany.
PLoS One. 2011;6(6):e20855. doi: 10.1371/journal.pone.0020855. Epub 2011 Jun 9.
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.
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.
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.
大多数离子通道通过多种机制在细胞膜中受磷脂酰肌醇 4,5-二磷酸（PtdIns(4,5)P(2)）调节。过去已经开发了重要的分子工具来研究活细胞中 PtdIns(4,5)P(2)对离子通道的调节。这些工具包括荧光 PH 结构域作为Förster 共振能量转移（FRET）的传感器，以监测质膜的变化。为了控制和可逆地耗尽 PtdIns(4,5)P(2)，电压感应磷酯酶（VSD）已被证明是一种优越的工具，因为它们独立于细胞信号通路。在完整细胞中结合这些方法需要多次转染。我们使用自我切割的病毒 2A 肽序列，用于腺病毒驱动的磷脂酶 Cδ1（PLCδ1）的 PH 结构域的表达，该 PH 结构域分别与 ECFP 和 EYFP 融合，并从成年大鼠心肌细胞的单个开放阅读框（ORF）中表达 Ciona intestinalis VSP（Ci-VSP）。
首先通过电压控制的 FRET 测量和 Western blot 证明，来自包含 2A 肽序列的单个三顺反子载体的 ECFP-PH-PLCδ1、EYFP-PH-PLCδ1 和 Ci-VSP 的表达和正确靶向，在 HEK293 细胞中。使用整合到腺病毒构建体中的载体，成年大鼠心肌细胞在基因转移后 4 天内表达 Ci-VSP 和两个荧光 PH 结构域。Ci-VSP 的激活导致 FRET 比迅速变化，表明质膜中 PtdIns(4,5)P(2)的耗尽。这与内源性 G 蛋白激活的 K(+)（GIRK）电流的抑制相平行。通过比较 FRET 和电流的变化，确定了肾上腺素能受体对 GIRK 抑制的一个组成部分与 PtdIns(4,5)P(2)的耗竭无关。
来自单个 ORF 的 FRET 传感器对和 Ci-VSP 的表达提供了一种有用的方法来研究细胞系和有丝分裂后细胞中磷酯酰肌醇对离子通道的调节。一般来说，包含自我切割 2A 肽序列的腺病毒构建体非常适合在成年心肌细胞中同时转移多个基因。
