(735.1) pHire: A Novel Genetically Encoded Red Intracellular pH Sensor Expressed in Cells and Flies

Poster Board Number: E297

Areonna Schreiber (Mayo Clinic), Adam Rossano (University of Pennsylvania), Carmen Reynolds (Mayo Clinic), Heather Holmes (Mayo Clinic), Matthew Brown (Mayo Clinic), Aleksey Matveyenko (Mayo Clinic), Michael Romero (Mayo Clinic)

Intracellular pH regulation is vital for normal cellular function and is controlled by membrane H+ and HCO3‑ transporters. Most pH reporters have green fluorescence (i.e., BCECF, pHlorin) making simultaneous Ca2+, Cl-, or other sensing (i.e., green fluorescent sensors) or GFP-tags impossible. We previously reported a red-fluorescent pH sensor (pHire) with an alkaline-shifted pKa that increases fluorescence with increasing intracellular pH. pHire was transiently transfect into TM5 cells, but these fibroblast-like cells did not address continuous expression in epithelial or excitable cells. Our objective was to evaluate pHire responses in multiple cell-types and show that it can be used for fluorescent multiplexing.

            We selected pHire-expressing rat pancreatic β-cells (INS1pHire) and porcine kidney epithelial cells (LLCPK1pHire), and then FACS sorted to keep the top 10-20% fluorescent cells. To test pHire’s usefulness for intact animals, we made transgenic, UAS-pHire Drosophila, allowing pHire expression in specific tissues via genetic crossing with promoter-GAL4 lines. By using perfusion chambers, we monitored cellular pHire-responses to acid-loading (NH4Cl pulse) and CO2/HCO3‑ addition.

Stable selection of pHire cell lines ensures single cells as well as contiguous cell layers for acid-base transporter evaluation. Addition of NH4Cl quickly alkalinizes all pHire cells. Different acid-recovery rates were found in individual cells with LLCPK1pHire cells recovering faster than INS1pHire cells. CO2/HCO3‑ addition acidified both mammalian cell-types. LLCPK1pHire cells typically show a Na+ dependent recovery (consistent with electrogenic Na+ bicarbonate cotransporter, NBCe1, activity) that is absent from INS1pHire cells. Infection of INS1pHire cells with a NBCe1A-GFP AAV allowed clear demonstration of NBCe1 activity.

To test pHire’s stability in living animals, we next generated a UAS-pHire transgenic Drosophila line, allowing pHire expression in specific tissues via genetic crossing with various promoter-GAL4 lines. We tested renal epithelial (principal cells: Uro-GAL4, CapaR-GAL4; stellate cells: c724-GAL4) and CNS (elav-GAL4) expression. As observed in mammalian cells, NH4Cl acid-loading showed robust and more uniform non-HCO3‑ recovery. The pH-calibration curves for pHire in Drosophila cells were similar those obtained in mammalian cells (pKagt;7.4). Thus, pHire characteristics are not changed by continuous expression in whole organisms.

This study shows that the pHire sensor can be constitutively expressed into multiple cell lines or whole animals (flies) with an ideal pH range of pH 6 to 9. pHire may also be multiplexed with GFP labels and biosensors to allow real-time, pH measurements with other ions of interest at a cellular scale. Finally, our data demonstrate that genetically encoded pHire is a reliable and useful tool for studying acid-base transporters in a variety of cell-types.

F32DK128987, F99DK123834, R01DK128844, R25GM075148, T32DK007013