Romero, Michael F.

Maintaining pH is critical for proper cellular and organ function. To measure intracellular pH, we made a novel genetically encoded protein, pHire, that increases red fluorescence with increasing pH. Thus, pHire allows for the coupling of multiple fluorescent tags without spectral overlap. We have expressed pHire in mammalian epithelial cells, Drosophila, and mice. In this study, our goal was to characterize pHire’s spectral properties by expression in human cells (HEK293) and in E.coli. To biophysically characterize pHire, we made a maltose binding protein (MBP)-pHire fusion protein to purify from E.coli. Pelleting individual colony growths revealed pink bacterial pellets which fluoresced with mCherry filters. Rather than purifying the MBP-pHire protein, equal volumes of bacteria were permeabilized with nigericin and high K+ solution for pH 5 to 9. These aliquots were then used to gather the fluorescence excitation and emission spectra of pHire, allowing us to determine the excitation and emission peaks of pHire as a function of pH. Our results showed peaks at Ex579 nm and Em611 nm, or roughly mCherry. Pelleted bacteria could also be fixed in perfusion chambers, revealing the same calibration curve as in mammalian cells and flies. Next, stable HEK293-pHire cells were selected to assay acid-base transport using NH4Cl or CO2/HCO3− in perfusion experiments. These HEK-pHire cells have less fluorescence than MBP-pHire bacteria but show the same Ex/Em peaks. However, an in-frame ATG 5’ to pHire in a mouse (adding 23 amino acids), removed pHire pH-dependence and decreased bacterial fluorescent output. Together these results indicate that pHire is biophysically similar in cells tested but that unstructured N-terminal additions block pH-responses. Funding: R25-DK101405, R21-DK129897. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Key Points Drosophila can be a model for Dent Disease type 1. Drosophila Clc-C mutations function similar to human CLC-5 Dent 1 mutations. Background Drosophila serve as exceptional alternative models for in vivo and ex vivo research and may provide an avenue for in-depth investigation for human ClC-5 and Dent disease type 1 (DD1). The Drosophila ClC-c (CG5284) has sequence homology with human ClC-5 and is hypothesized to encompass similar functional and phenotypical roles with ClC-5 and variants that cause DD1. Methods Ion transport function and activity of Drosophila ClC-c and homologous DD1 variants were assessed by voltage clamp electrophysiology. Membrane localization was demonstrated in Drosophila expressing a GFP-labeled construct of ClC-c. Genetic expression of an RNAi against ClC-c mRNA was used to generate a knockdown fly that serves as a DD1 disease model. Tubule secretion of cations and protein were assessed, as well as the crystal formation in the Malpighian tubules. Results Voltage clamp experiments demonstrate that ClC-c is voltage-gated with Cl − -dependent and pH-sensitive currents. Inclusion of homologous DD1 mutations pathogenic variants (S393L, R494W, and Q777X) impairs ClC-c ion transport activity. In vivo expression of ClC-c-eGFP in Malpighian tubules reveals that the membrane transporter localizes to the apical membrane and nearby cytosolic regions. RNAi knockdown of ClC-c (48% decreased mRNA expression) causes increased secretion of both urinary protein and Ca 2+ and increased occurrence of spontaneous tubule crystals. Conclusions Drosophila ClC-c shows orthologous function and localization to human ClC-5. Thus, Drosophila and ClC-c regulation may be useful for future investigations of Cl − transport, Ca 2+ homeostasis, and urinary protein loss in DD1.

Abstract Aquaporin (Aqp) 10 is a member of the aquaglyceroporin subfamily of water channels, and human Aqp10 is permeable to solutes such as glycerol, urea, and boric acid. Tetrapods have a single aqp10 gene, whereas ray-finned fishes have paralogs of this gene through tandem duplication, whole-genome duplication, and subsequent deletion. A previous study on Aqps in the Japanese pufferfish Takifugu rubripes showed that one pufferfish paralog, Aqp10.2b, was permeable to water and glycerol, but not to urea and boric acid. To understand the functional differences of Aqp10s between humans and pufferfish from an evolutionary perspective, we analyzed Aqp10s from an amphibian (Xenopus laevis) and a lobe-finned fish (Protopterus annectens) and Aqp10.1 and Aqp10.2 from several ray-finned fishes (Polypterus senegalus, Lepisosteus oculatus, Danio rerio, and Clupea pallasii). The expression of tetrapod and lobe-finned fish Aqp10s and Aqp10.1-derived Aqps in ray-finned fishes in Xenopus oocytes increased the membrane permeabilities to water, glycerol, urea, and boric acid. In contrast, Aqp10.2-derived Aqps in ray-finned fishes increased water and glycerol permeabilities, whereas those of urea and boric acid were much weaker than those of Aqp10.1-derived Aqps. These results indicate that water, glycerol, urea, and boric acid permeabilities are plesiomorphic activities of Aqp10s and that the ray-finned fish-specific Aqp10.2 paralogs have secondarily reduced or lost urea and boric acid permeability.