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

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.

研究背景
绝大多数离子通道可通过多种机制，受细胞膜上磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P2）的调控。既往已开发出多种用于在活细胞中研究PtdIns(4,5)P2对离子通道调控作用的分子工具，其中包括用作福斯特共振能量转移（FRET）传感器的荧光PH结构域（PH-domain），用于监测细胞膜的动态变化。针对PtdIns(4,5)P2的可控、可逆耗竭，电压敏感型磷酸肌醇磷酸酶（VSD）已被证实是优异的工具，因其不依赖细胞信号通路。但在完整细胞中联合使用上述两类方法需要进行多次转染操作。本研究利用自剪切病毒2A肽序列，在成年大鼠心肌细胞中，通过单个开放阅读框（ORF）实现腺病毒驱动的、分别与增强型青色荧光蛋白（ECFP）、增强型黄色荧光蛋白（EYFP）融合的磷脂酶Cδ1（PLCδ1）PH结构域，以及肠海鞘源电压敏感磷酸酶（Ci-VSP）的共表达。

方法与结果
本研究首先在HEK293细胞中，通过电压控制的FRET检测与蛋白质免疫印迹实验，验证了携带2A肽序列的三顺反子载体可成功表达ECFP-PH-PLCδ1、EYFP-PH-PLCδ1及Ci-VSP，并实现其正确靶向。将该载体整合至腺病毒构建体后进行基因转染，成年大鼠心肌细胞可在转染后4天内表达Ci-VSP与两种荧光PH结构域。通过去极化激活Ci-VSP后，FRET比值快速变化，提示细胞膜上的PtdIns(4,5)P2发生耗竭，该现象同时伴随内源性G蛋白激活型钾电流（GIRK）的抑制。通过对比FRET信号与电流的变化，本研究发现肾上腺素能受体对GIRK的抑制存在一类不依赖PtdIns(4,5)P2耗竭的通路组分。

研究结论
从单个开放阅读框中同时表达FRET传感器对与Ci-VSP的策略，可为在细胞系及转染抗性的有丝分裂后细胞中研究磷酸肌醇对离子通道的调控作用提供可行方案。总体而言，携带自剪切2A肽序列的腺病毒构建体，非常适合在成年大鼠心肌细胞中同时转染多个外源基因。