Rossano Adam J, Romero Michael F
Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine;
Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine; Department of Nephrology and Hypertension, Mayo Clinic College of Medicine;
J Vis Exp. 2017 Aug 11(126):55698. doi: 10.3791/55698.
Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.
上皮离子转运对于全身离子稳态以及维持基本的细胞电化学梯度至关重要。细胞内pH值（pHi）受多种离子转运体影响，因此监测pHi是评估转运体活性的有用工具。现代基因编码pH指示剂（GEpHIs）可在细胞和亚细胞水平上对完整细胞中的pHi进行光学定量。本方案描述了通过对pHerry进行离体实时成像来实时定量黑腹果蝇马氏管（MTs）中的细胞pHi调节，pHerry是一种伪比率型GEpHI，其pKa非常适合追踪细胞质中的pH变化。提取的成年果蝇MTs由形态和功能不同的单细胞层上皮部分组成，可作为研究上皮转运的易于获取且具有遗传可操作性的模型。与传统的pH敏感荧光染料和离子选择性电极相比，GEpHIs具有多个优点。如果有合适的启动子元件，GEpHIs可以标记不同的细胞群体。这种标记在本质上异质的离体、体内和原位制剂中特别有用。GEpHIs还允许在完整组织中随时间定量pHi，而无需重复进行染料处理或组织外置。当前GEpHIs的主要缺点是在组织损伤和构建物过度表达时倾向于在胞质内含物中聚集。通过评估提取的果蝇MTs中功能不同的主细胞和星状细胞的基底外侧质子（H）转运，本方案展示了这些缺点、其解决方案以及GEpHIs的固有优势。所描述的技术和分析很容易适用于多种脊椎动物和无脊椎动物制剂，并且检测的复杂性可以从教学实验室扩展到通过特定转运体精细测定离子通量。
