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Sensitive red fluorescent indicators for real-time visualization of potassium ion dynamics in vivo [1]

['Lina Yang', 'School Of Life Sciences', 'Fudan University', 'Shanghai', 'Westlake University', 'Hangzhou', 'Zhejiang', 'Westlake Laboratory Of Life Sciences', 'Biomedicine', 'Institute Of Basic Medical Sciences']

Date: 2025-09 

Potassium ion (K + ) dynamics are vital for various biological processes. However, the limited availability of detection tools for tracking intracellular and extracellular K + has impeded a comprehensive understanding of the physiological roles of K + in intact biological systems. In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2, through a combination of directed evolution in Escherichia coli and subsequent optimization in mammalian cells. RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K + -specific fluorescence response with affinities of 3.55 and 14.81 mM in HEK293FT cells, respectively. We employed RGEPOs for real-time monitoring of subsecond K + dynamics in cultured neurons, astrocytes, acute brain slices, and the awake mouse in both intracellular and extracellular environments. Using RGEPOs, we were able, for the first time, to visualize intracellular and extracellular potassium transients during seizures in the brains of awake mice. Furthermore, molecular dynamics simulations provided new insights into the potassium-binding mechanisms of RGEPO1 and RGEPO2, revealing distinct K + -binding pockets and structural features. Thus, RGEPOs represent a significant advancement in potassium imaging, providing enhanced tools for real-time visualization of K + dynamics in various cell types and cellular environments.

Funding: The work was supported by start-up funding from the Foundation of Westlake University, Westlake Laboratory of Life Sciences and Biomedicine, National Natural Science Foundation of China grant 32171093, and “Pioneer” and “Leading Goose” R&D Program of Zhejiang 2024SSYS0031 to K.D.P. The authors thankfully acknowledge support from the National Science Foundation−NSF through the grant NSF CHE2338804 to A.R.W. We thank Wayne State University and Department of Chemistry for the Thomas C. Rumble University Graduate Fellowship to V. P. M.D. thanks the NIH/NINDS BRAIN Initiative grant 2U24NS107109 for supporting his Resource for Multiphoton Characterization of Genetically Encoded Probes. F.V.S. acknowledges that the work was carried out within the state assignment of NRC “Kurchatov Institute” (design part of the sensor). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All essential raw datasets, including source files for the S1 Data, supplementary figures, and raw unprocessed ND2 images of cultured neurons, have been deposited in Figshare ( https://doi.org/10.6084/m9.figshare.29917037 ). All plasmids used in this study are available from WeKwikGene ( https://wekwikgene.wllsb.edu.cn/publications/0000741 ; barcodes: 0000741, 0000742, 0000765, 0000766, 0001230, 0001231). The computational inputs for molecular dynamics simulations, including topology files, initial coordinates of all systems, the last frames of the free diffusion simulations of both RGEPO1 and RGEPO2, force field parameters for the chromophore, and a short simulation video of potassium diffusion in RGEPO1, have been deposited in Zenodo ( https://doi.org/10.5281/zenodo.13824581 ).

By integrating insights garnered from genomic mining and informed by structural guidance in potassium-binding protein architecture, we successfully developed two red fluorescent potassium indicators, designated as RGEPO1 and RGEPO2. These indicators were engineered by inserting a potassium-binding protein cloned from a hydrothermal vent bacterium into the FP mApple at β-strand 7 next to the chromophore. RGEPO1 and RGEPO2 exhibited a ΔF/F in the range of 400%–1,000% in solution and 100%–300% in HEK293FT cells in response to 150 and 200 mM K + , respectively. In HEK293FT cells, potassium affinities were determined to be 3.55 mM for RGEPO1 and 14.81 mM for RGEPO2. Additionally, we discovered the molecular mechanisms underlying the different responses of RGEPO1 and RGEPO2 to potassium ions using molecular dynamic simulation. Furthermore, RGEPOs were applied to monitor intracellular and extracellular K + dynamics in the culture neurons, astrocytes, acute brain slices, and mouse seizures in vivo. These findings substantiate the practical utility of indicators RGEPO1 and RGEPO2 as essential tools for the real-time visualization and comprehensive analysis of intracellular and extracellular potassium ion dynamics.

Genetically encoded potassium indicators (GEPOs) provide a promising alternative for K + measurement, allowing for real-time, minimally invasive monitoring with high spatial and temporal resolution [ 13 – 16 ]. Recent achievements in developing GEPOs stemmed from using a small water-soluble potassium-binding protein from Escherichia coli, known as Ec-Kbp (formerly YgaU) [ 17 ]. Among these indicators, GEPII [ 16 ] and KIRIN1s [ 15 ] are based on the Förster Resonance Energy Transfer (FRET) ( S1 Table ). The FRET-based GEPOs provide ratiometric output, which enhances the accuracy of concentration measurements. However, they require two specific emission color bands, limiting their application in multiplexed imaging experiments. Additionally, FRET-based indicators often suffer from weak signals and limited dynamic range. In contrast, single fluorescent protein (FP)-based indicators, although more challenging to engineer than FRET-based biosensors and typically intensiometric, require only single-color bands for excitation and emission. This makes them well-suited for multiparameter imaging [ 18 ], and they are generally characterized by higher dynamic range and stronger signals. The green GEPOs, GINKO1 [ 15 ], GINKO2 [ 13 ], and KRaIONs [ 14 ], based on the single GFP-like FPs, have been successfully applied to monitor intracellular K + dynamics in mammalian cells and plants ( S1 Table ). Compared to green fluorescence proteins, red fluorescence proteins provide the advantage of deeper tissue penetration, low phototoxicity, and compatibility with a large variety of existing green indicators. Thus, the development of red fluorescent GEPOs (RGEPOs) is essential for advancing K + research.

Traditionally, K + levels have been measured using ion-sensitive electrodes [ 8 , 9 ] or fluorescent dyes [ 10 – 12 ]. While ion-sensitive electrodes provide direct and precise measurements of absolute K + concentrations [ 9 ], they are highly invasive and typically offer limited spatial resolution and throughput of measurements, making them unsuitable for studying fine-scale cellular processes in intact cells and tissues. Fluorescent dyes, such as potassium-sensitive fluorophores, offer improved spatial resolution compared to electrodes and allow for noninvasive imaging of K + levels in live cells [ 10 , 12 ]. However, they suffer from drawbacks such as poor ion selectivity, labeling specificity, limited dynamic range, cytotoxicity, and lack of targetability to specific cell types or subcellular compartments.

Potassium ion (K + ) homeostasis and dynamics play critical roles in a multitude of biological activities in all domains of life [ 1 , 2 ]. For example, in neurons, K + is vital for generating action potentials and maintaining the resting membrane potential, which is critical for proper signal transmission [ 3 , 4 ]. Astrocytes, on the other hand, regulate extracellular K + levels through a process known as potassium buffering, which is essential for preventing neuronal hyperexcitability and maintaining overall ion homeostasis [ 5 ]. Under resting conditions in the mammalian cells, K + has an intracellular concentration of 142–175 mM, while extracellular concentration is in the range of 3.5–5 mM, maintaining the resting membrane potential at −70 to −80 mV [ 6 , 7 ]. Therefore, sensitive ion probes with a high dynamic range at physiological concentrations are essential for accurately analyzing K + homeostasis and its functional transients.

Results

Molecular mechanism of potassium sensitivity To explore the molecular mechanism underlying potassium binding, we performed classical molecular dynamics (MD) simulations for both the RGEPO1 and RGEPO2 systems in solution at low (20 mM) and high (300 mM) potassium concentrations. Low concentration was selected to match the affinity of the sensors in the solution, and high concentration corresponds to the upper limit of the sensor dynamic range. The initial protein structures were generated using AlphaFold2, revealing two key structural differences in the linker regions of RGEPO1 and RGEPO2 that were maintained and stable in the MD (S15 Fig). Specifically, in RGEPO2, residues N298 and P299 exhibit a tendency towards an open conformation, which results in the loss of beta-sheet character below residue P299 (S16 Fig). This structural variation renders the phenolate end of the chromophore in RGEPO2 more accessible to the solvent as compared to RGEPO1. This could explain some of the differences in molecular brightness in K+-bound states between them, since the fluorescence mechanism likely requires a structural shift upon K+ binding to stabilize the phenolate and emissive form. RGEPO2, therefore, would require a larger conformational change as compared to RGEPO1, though they have similar recognition mechanisms, as discussed further below. The MD simulations of RGEPO1 revealed two distinct binding pockets for K+ ions (Fig 4A). The first, referred to as the main binding pocket, was observed in both 20 and 300 mM ionic concentrations and exhibited significant K+ binding duration (~140 ns; Fig 4A upper inset). The second pocket, termed the transient binding pocket, showed a shorter binding time (~100 ns) and was observed only at 20 mM (Fig 4A lower inset). The main binding pocket was formed by 13 amino acids, including K240/E241/P242/V243/D294/L295/S297/A298/C299/V300/S301/V349/D350/R374. Among them, D294 and D350 directly coordinate potassium ions via ionic interactions. R374 is highly mobile prior to K+ binding, and subsequently forms a key hydrogen bond with D294 that stabilizes K+ in the pocket for a significant amount of time. The transient pocket consisted of only 5 amino acids, namely D156/V158/K159/S228/K229. Among them, only V158 and K159 were shared with the binding pocket identified in the Ec-Kbp domain of the KRaION1 biosensor [14]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Molecular mechanism of potassium sensitivity. (A) The predicted 3D structure of RGEPO1 is shown in red, with K+ ions in green. The residues around the binding pockets are depicted as blue sticks. The binding regions are enlarged on the right with labels for the respective binding residues (upper main binding site and lower transient site). (B) The predicted 3D structure of RGEPO1 is shown in red, with K+ ions in green. The residues around the binding pockets are depicted as blue sticks. The binding region is enlarged on the right, and labels are placed for the respective binding residues. (C) Charge distribution of the surface of RGEPO1 (i and ii), RGEPO2 (iii). Surface color is assigned such that red is more electronegative and bluer is more electropositive. The green spheres represent the K+ ions. (D) Sensitivity mechanism of (i) RGEPO1 and (ii) RGEPO2. Left, the prominently observed hydrogen bonding network with O− of phenolate ring of RGEPO1 upon K+ binding. Right, the prominently observed hydrogen bonding interactions with O− of phenolate ring of RGEPO2 upon K+ binding. The hydrogen bonds are represented by thin red dashed lines, indicating the interaction points between the phenolate ring and surrounding residues/water (in sticks), and are labeled with their respective names. The chromophore’s conformational space in both (i) and (ii) is depicted by gray sticks and double-headed arrows, indicating the range of movement in the absence of bound K⁺. In the right panel (ii), the conformation of K317 in the absence of K⁺ is highlighted in gray sticks. https://doi.org/10.1371/journal.pbio.3002993.g004 In contrast, RGEPO2’s MD simulations identified only one binding pocket under both ionic conditions (20 and 300 mM), in a similar location to the main binding pocket for RGEPO1, but all different amino acids in the binding pocket, which included W148/E149/A150/S151/D350/E352/K317 (Fig 4B). The binding region is located closer to the FP (Fig 4B). The bound ion remained stable for ~50 ns in this binding pocket. This positioning also caused an opening between two beta sheets near the phenolate O− end, destabilizing the FP’s beta-barrel structure and exposing the inner residues more to the solvent as compared to RGEPO1. This shorter timescale is consistent with the slightly lowered affinity of RGEPO2 observed experimentally in mammalian cells. The main K+-binding pockets are located either on the interface between the binding domain and FP in the case of RGEPO1 or in the β-barrel of FP next to the phenolate group of the chromophore. These binding pockets were not previously reported for any other single-FP-based biosensors that utilize other ion-binding domains. However, it was possible to engineer GFP-like FPs to have Zn2+ or Cu2+ binding pockets inside of the β-barrel, directly interacting with the chromophore [39]. To further evaluate the ion selectivity of RGEPOs, we conducted MD simulations under high calcium conditions (300 mM Ca2⁺) for both RGEPO1 and RGEPO2. Although Ca2⁺ was occasionally observed near the chromophore region in both sensors, its binding was transient and substantially less stable than K⁺. In RGEPO1, Ca2⁺ localized to a solvent-exposed region adjacent to the transient K⁺ binding pocket, but did not enter the main K⁺ binding site (S17A–S17C Fig). The phenolate–K317 distance remained only briefly stabilized (~30–40 ns), and the surrounding water shell was not consistently displaced, indicating weak coordination (S17D–S17F Fig). In contrast, RGEPO2 allowed slightly deeper Ca2⁺ entry into the chromophore-adjacent cavity (S17G–S17I Fig), but the binding remained unstable and short-lived. Notably, the H-bond network between Ca2⁺, the chromophore, and K317 was far less persistent than that observed with K⁺ (S17J–S17L Fig). Together, these results demonstrate that although Ca2⁺ can transiently engage with surface-exposed acidic residues near the binding site, its interaction lacks the stability, depth, and configurational consequences required to activate the fluorescence response. This further confirms the selectivity of RGEPOs for K⁺ over biologically relevant divalent cations such as Ca2⁺. To better understand the structural basis underlying this ion selectivity, we next examined the surface charge landscapes of RGEPO1 and RGEPO2. Further analysis of RGEPO1’s surface charge distribution revealed an electronegative region extending from the main binding site to the transient site, indicating a higher likelihood of retaining positively charged ions. The MD shows K+ ions transiently bound to this red region for a brief time (Figs 4C and S18). The more grooved areas, such as the main binding pocket and the transient binding regions, provide increased ion stability due to reduced solvent exposure, resulting in longer ion retention. Comparatively, RGEPO2’s surface lacked a continuous negatively charged region. Instead, it had more negatively charged residues clustered around the main binding pocket, forming a groove that stabilized the cations in that specific area (Fig 4C). Collectively, these results suggest that RGEPO1 has a better performance as a fluorescent sensor than RGEPO2, which is consistent with our experimental results. Even though RGEPO1 and RGEPO2 have completely different main binding pockets, further analysis of the MD results confirmed that both show the same mechanism related to fluorescence sensing. Upon binding of K+ to the main binding pocket of both RGEPO1 and RGEPO2, we observed a chain of hydrogen bonding interactions occurring with the O− position of the phenolate ring of the chromophore. In RGEPO1, these interactions extend from the phenolate ring to K317 and through a water molecule to E149 (Figs 4D and S19). In RGEPO2, the same interactions occur without the solvent mediating the chain (Fig 4D). The H-bonding residence time between phenolate O− and K317 is approximately 80% and 40% of the trajectory for RGEPO1 and RGEPO2, respectively. The analysis of variation of distance between K317 at the sidechain N atom and chromophore phenolate O− atoms upon binding of K+ to the proposed main and transient sites that the distance is lowered (<3 Å) and hydrogen bonding between K317 and O− of chromophore is more stabilized compared to unbound situations (S20 Fig). Additionally, analysis of the water shell around the phenolate O− end of all the systems shows a drop in the number of water molecules upon K+ binding to the binding domain (S21 Fig). To further investigate the binding regions, we examined the structure of the previously reported NMR-resolved K+-binding site in Ec-Kbp of the KRaION1 biosensor (PDB ID: 7PVC). The electrostatic distribution of the binding site clearly differed from that of RGEPO1 and RGEPO2 (S22 Fig), suggesting that the binding regions were altered by the introduced mutations. Furthermore, the electronegative charge distribution introduced by mApple at the interface of the potassium binding domain and the FP compared to that of green FP (PDB ID: 7VCM) made the proposed binding sites more accessible and stable in RGEPO1 and RGEPO2. To determine if the previously reported Kbp binding pocket was still a possibility, we carried out MD simulations by placing the K+ ion at this previously reported Kbp binding pocket. In RGEPO1, the bound ion remained stable in this region throughout the simulation, although another K+ ion diffused freely to the proposed main binding pocket. The proposed transient pocket of RGEPO1 is located close to the previously reported site (~7 Å) (S22D Fig), but importantly neither induces the necessary conformational change in the chromophore and K317 (Fig 4D). Additionally, in RGEPO2, the K+ binding at the previously reported site was unstable, lasting less than 20 ns. Analysis of the distance between the K317(NZ) atom and the O− atom of the chromophore shows no significant difference between the bound and unbound states (S20F Fig). Taken together, this suggests that the observed main binding pockets in RGEPO1 and RGEPO2 are more likely to be involved in the fluorescence mechanism.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002993

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