Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

59-POS Board 29 DESIGN AND CHARACTERIZATION OF A FLUORESCENT BIOSENSOR FOR DETERMINATION OF POTASSIUM CONCENTRATION IN LYSOSOMES Alena Shulutkova 1 ; Thomas Gensch 1 ; 1 Forschungszentrum Jülich, Institute of Biological Information Processing, Molecular and Cellular Physiology, Jülich, Germany Lysosomes serve many important functions in cells including cellular cleaning, membrane repair and material recycling. Dysfunction of lysosomes are manifested in a number of diseases like Alzheimer’s disease, lysosomal storage disorder or Parkinson’s disease. Ion concentrations of the lysosomal lumen are scarcely investigated, but crucial for understanding cellular processes such as ion homeostasis or membrane potential in lysosomes. We are developing a potassium biosensor for application in lysosomes of living cells. Nearly a decade ago, a fluorescent biosensor (GEPII 1.0 1 ) based on the native E. coli potassium-binding protein (Kbp) flanked by a pair of fluorescent proteins capable of Förster Resonance Energy Transfer (FRET). Kbp undergoes a large conformational change upon potassium binding 2 , leading to a sizeable FRET change. The largest challenge is the low pH of lysosomal lumen (4.5-5.0). A new, pH-resistant pair of fluorescent proteins has to be found preserving the large FRET change of the original sensor, which is often sacrificed. 3 We determined the efficiency of potassium binding of isolated Kbp and tested various combinations of fluorescent proteins with high pH stability and good spectral overlap to create a biosensor suitable for the low lysosomal pH. The best candidate was characterized in vitro and in vivo (expressed in HEK293T cells), underwent various adjustments and is targeted to lysosomal membrane to verify its stability and function in the lysosomal lumen. It will allow to determine lysosomal potassium concentrations with both lifetime and ratiometric fluorescence intensity change.1 H. Bischof et al. Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo. Nat Commun 8, 1422 (2017) 2 K. Ashraf et al. The potassium binding protein Kbp is a cytoplasmic potassium sensor. Structure 24, 741-749 (2016) 3 Y. Shen et al. Genetically encoded fluorescent indicators for imaging intracellular potassium ion concentration. Comm. Biol. 2:18 (2019)

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