Purification and Application of Genetically Encoded Potassium Ion Indicators for Quantification of Potassium Ion Concentrations within Biological Samples

Abstract

Vital cells maintain a steep potassium ion (K+) gradient across the plasma membrane. Intracellular potassium ion concentrations ([K+]) and especially the [K+] within the extracellular matrix are strictly regulated, the latter within a narrow range of ~3.5 to 5.0 mM. Alterations of the extracellular K+ homeostasis are associated with severe pathological alterations and systemic diseases including hypo- or hypertension, heart rate alterations, heart failure, neuronal damage or abnormal skeleton muscle function. In higher eukaryotic organisms, the maintenance of the extracellular [K+] is mainly achieved by the kidney, responsible for K+ excretion and reabsorption. Thus, renal dysfunctions are typically associated with alterations in serum- or plasma [K+]. Generally, [K+] quantifications within bodily fluids are performed using ion selective electrodes. However, tracking such alterations in experimental models such as mice features several difficulties, mainly due to the small blood volume of these animals, hampering the repetitive collection of sample volumes required for measurements using ion selective electrodes. We have recently developed highly sensitive, genetically encoded potassium ion indicators, the GEPIIs, applicable for in vitro determinations of [K+]. In addition to the determination of [K+] within bodily fluids, GEPIIs proved suitable for the real-time visualization of cell viability over time and the exact determination of the number of dead cells.

Immobilization of Recombinant Fluorescent Biosensors Permits Imaging of Extracellular Ion Signals

“…Such an approach might be also used to co-detect and correct for pH fluctuations, which have the potency to perturb the performance of most FP-based biosensors. 31 ■ CONCLUSIONS AND OUTLOOK While purified biosensors have mainly been used for sensor characterization purposes, 3,10,32,33 we describe a novel biotinylation-based procedure to use genetically encoded sensors for the recording of extracellular ions locally on cell surfaces and glass slides upon their immobilization using three workflows (Figure S8): first, purification of the recombinant TAv biosensor, second, expression of the biotinylated cell surface tag or using a biotinylated glass slide, and third, coupling of the TAv biosensor to the biotinylated surface.…”

Probing Intracellular Potassium Dynamics in Neurons with the Genetically Encoded Sensor lc-LysM GEPII 1.0 in vitro and in vivo

“…At an OD value between 0.4 and 0.6 protein expression was induced using IPTG with a nal concentration of 0.5 mM and further incubated for 16 hours at 18°C. Puri cation of the 6x histidine-tagged biosensors was performed using gravity-based Ni-NTA a nity chromatography method as described elsewhere 24 . Puri ed proteins were concentrated using Ultracel® Regenerated Cellulose (30kDa MWCO) Amicon tubes and kept at -80°C for further usage.…”