Source 1primary paper2025MED
Toolkit/RGEPOs
RGEPOs
Also known as: red genetically encoded potassium indicators
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2.
Usefulness & Problems
Why this is useful
RGEPOs are red genetically encoded fluorescent indicators for monitoring potassium ion dynamics. The abstract states they were used for real-time visualization of intracellular and extracellular K+ signals, including subsecond dynamics.; real-time visualization of potassium ion dynamics; monitoring intracellular and extracellular K+ dynamics; subsecond potassium imaging in vivo and ex vivo
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RGEPOs are red genetically encoded fluorescent indicators for monitoring potassium ion dynamics. The abstract states they were used for real-time visualization of intracellular and extracellular K+ signals, including subsecond dynamics.
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real-time visualization of potassium ion dynamics
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monitoring intracellular and extracellular K+ dynamics
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subsecond potassium imaging in vivo and ex vivo
Problem solved
They address the limited availability of tools for tracking intracellular and extracellular potassium in intact biological systems. This expands the ability to study physiological K+ dynamics in vivo.; limited availability of detection tools for tracking intracellular and extracellular K+
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They address the limited availability of tools for tracking intracellular and extracellular potassium in intact biological systems. This expands the ability to study physiological K+ dynamics in vivo.
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limited availability of detection tools for tracking intracellular and extracellular K+
Problem links
limited availability of detection tools for tracking intracellular and extracellular K+
LiteratureThey address the limited availability of tools for tracking intracellular and extracellular potassium in intact biological systems. This expands the ability to study physiological K+ dynamics in vivo.
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They address the limited availability of tools for tracking intracellular and extracellular potassium in intact biological systems. This expands the ability to study physiological K+ dynamics in vivo.
Published Workflows
Sensitive red fluorescent indicators for real-time visualization of potassium ion dynamics in vivo.
2025Objective: Develop red genetically encoded potassium indicators suitable for real-time visualization of intracellular and extracellular K+ dynamics in intact biological systems and in vivo.
Why it works: The workflow combines microbial directed evolution with subsequent mammalian-cell optimization, then applies the resulting indicators in relevant neural and in vivo contexts. The abstract also states that molecular dynamics simulations were used to interpret potassium-binding mechanisms.
potassium binding-linked fluorescence responsedistinct potassium-binding pockets and structural featuresdirected evolution in Escherichia colioptimization in mammalian cellsmolecular dynamics simulation
Stages
- 1.Directed evolution in Escherichia coli(selection)
The abstract states that the indicators were developed through directed evolution in Escherichia coli as an initial engineering stage.
Selection: Development of novel red genetically encoded potassium indicators through directed evolution.
- 2.Optimization in mammalian cells(secondary_characterization)
The abstract explicitly states that optimization in mammalian cells followed directed evolution in E. coli.
Selection: Subsequent optimization of indicator performance in mammalian cells.
- 3.Cell-based characterization in HEK293FT cells(functional_characterization)
The abstract reports affinities and localization-specific responses in HEK293FT cells before broader biological deployment.
Selection: Measure K+-specific fluorescence response and affinity of RGEPO1 and RGEPO2 in HEK293FT cells.
- 4.Application in neural preparations and awake mouse(confirmatory_validation)
The abstract describes deployment of the indicators in progressively more intact neural systems to demonstrate real-time potassium imaging capability.
Selection: Demonstrate real-time monitoring of subsecond K+ dynamics in cultured neurons, astrocytes, acute brain slices, and awake mouse brain.
- 5.Molecular dynamics analysis of potassium-binding mechanisms(secondary_characterization)
The abstract states that molecular dynamics simulations provided insights into potassium-binding mechanisms and distinct binding pockets.
Selection: Use molecular dynamics simulations to analyze potassium-binding mechanisms and structural features of RGEPO1 and RGEPO2.
Steps
- 1.Perform directed evolution in Escherichia coliengineered indicators
Generate improved red genetically encoded potassium indicator variants.
The abstract presents directed evolution in E. coli as the first engineering step before mammalian-cell optimization.
- 2.Optimize indicator variants in mammalian cellsengineered indicators
Improve performance of the indicators in mammalian cellular context.
This step follows E. coli directed evolution because the abstract explicitly states subsequent optimization in mammalian cells.
- 3.Measure localization-specific K+-responsive fluorescence and affinity in HEK293FT cellsindicators under characterization
Quantify K+-specific fluorescence response and affinity in a mammalian cell line.
The abstract reports HEK293FT-cell characterization after mammalian-cell optimization and before deployment in neural and in vivo systems.
- 4.Deploy RGEPOs in cultured neurons, astrocytes, acute brain slices, and awake mice for real-time K+ imagingimaging indicators
Validate real-time monitoring of subsecond potassium dynamics in relevant biological systems.
This application step follows cell-line characterization to test the indicators in more physiologically relevant and intact systems.
- 5.Use molecular dynamics simulations to analyze potassium-binding mechanismssimulated indicators
Interpret structural features and potassium-binding pockets of the indicators.
The abstract presents molecular dynamics simulations as a later analysis that provided mechanistic insight into the developed indicators.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A reusable architecture pattern for arranging parts into an engineered system.
Techniques
No technique tags yet.
Target processes
No target processes tagged yet.
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: sensor
These tools require genetic expression in the biological system of interest and fluorescence imaging. The abstract also indicates that localization/targeting differs between family members for extracellular membrane versus cytoplasmic measurements.; genetically encoded expression is required; distinct targeting/localization is needed for extracellular versus cytoplasmic measurements
Independent follow-up evidence is still limited. Validation breadth across biological contexts is still narrow. Independent reuse still looks limited, so the evidence base may be fragile. No canonical validation observations are stored yet, so context-specific performance remains under-specified.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
RGEPOs were used for real-time monitoring of subsecond potassium dynamics in cultured neurons, astrocytes, acute brain slices, and awake mice in intracellular and extracellular environments.
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.
RGEPO1 and RGEPO2 were developed through directed evolution in Escherichia coli followed by optimization in mammalian cells.
through a combination of directed evolution in Escherichia coli and subsequent optimization in mammalian cells
Using RGEPOs, the authors visualized intracellular and extracellular potassium transients during seizures in the brains of awake mice.
Using RGEPOs, we were able, for the first time, to visualize intracellular and extracellular potassium transients during seizures in the brains of awake mice.
RGEPO1 is targeted to the extracellular membrane and shows a positive K+-specific fluorescence response with 2.4 mM affinity in HEK293FT cells.
RGEPO1, targeted to the extracellular membrane... exhibited positive K+-specific fluorescence response with affinities of 2.4 ... mM in HEK293FT cells
affinity 2.4 mM
RGEPO2 is localized in the cytoplasm and shows a positive K+-specific fluorescence response with 43.3 mM affinity in HEK293FT cells.
RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of ... 43.3 mM in HEK293FT cells
affinity 43.3 mM
Molecular dynamics simulations suggested that RGEPO1 and RGEPO2 have distinct potassium-binding pockets and structural features.
molecular dynamics simulations provided new insights into the potassium-binding mechanisms of RGEPO1 and RGEPO2, revealing distinct K+-binding pockets and structural features
The study developed two novel red genetically encoded potassium indicators, RGEPO1 and RGEPO2.
In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2
Approval Evidence
1 source3 linked approval claimsfirst-pass slug rgepos
In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2.
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application scopesupports
RGEPOs were used for real-time monitoring of subsecond potassium dynamics in cultured neurons, astrocytes, acute brain slices, and awake mice in intracellular and extracellular environments.
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.
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first visualizationsupports
Using RGEPOs, the authors visualized intracellular and extracellular potassium transients during seizures in the brains of awake mice.
Using RGEPOs, we were able, for the first time, to visualize intracellular and extracellular potassium transients during seizures in the brains of awake mice.
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tool developmentsupports
The study developed two novel red genetically encoded potassium indicators, RGEPO1 and RGEPO2.
In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2
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Comparisons
Source-stated alternatives
The upstream source-discovery summary identifies prior genetically encoded potassium indicators such as KIRIN, GINKO, KRaION, GEPII, and HaloKbp1 as nearby alternatives or lineage comparators.
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The upstream source-discovery summary identifies prior genetically encoded potassium indicators such as KIRIN, GINKO, KRaION, GEPII, and HaloKbp1 as nearby alternatives or lineage comparators.
Source-backed strengths
red genetically encoded potassium indicators; used for real-time monitoring of subsecond K+ dynamics across multiple preparations; enabled visualization of potassium transients during seizures in awake mouse brain
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red genetically encoded potassium indicators
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used for real-time monitoring of subsecond K+ dynamics across multiple preparations
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enabled visualization of potassium transients during seizures in awake mouse brain
Ranked Citations
- 1.