Source 1primary paper2025MED
Toolkit/CfRhPDE1
CfRhPDE1
Also known as: C. flexa RhPDE1
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
C. flexa RhPDE1 (CfRhPDE1) revealed the highest cGMP affinity and the most pronounced light regulation
Usefulness & Problems
Why this is useful
CfRhPDE1 is a photomodulated rhodopsin-phosphodiesterase from Choanoeca flexa used to control cGMP with light. In the reported mammalian-cell system it serves as the cGMP-degrading component.; light-controlled reduction of cGMP; bidirectional optogenetic cGMP control when paired with a rhodopsin-guanylyl-cyclase; subsecond regulation of cGMP signaling in mammalian cells
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CfRhPDE1 is a photomodulated rhodopsin-phosphodiesterase from Choanoeca flexa used to control cGMP with light. In the reported mammalian-cell system it serves as the cGMP-degrading component.
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light-controlled reduction of cGMP
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bidirectional optogenetic cGMP control when paired with a rhodopsin-guanylyl-cyclase
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subsecond regulation of cGMP signaling in mammalian cells
Problem solved
It addresses the limited optogenetic utility of earlier RhPDEs with low light-dependent modulation by providing stronger light regulation and high cGMP affinity.; provides a photomodulated phosphodiesterase with stronger light regulation than earlier low-modulation RhPDEs; helps close a gap in optogenetic control of cyclic nucleotides by enabling light-driven cGMP degradation
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It addresses the limited optogenetic utility of earlier RhPDEs with low light-dependent modulation by providing stronger light regulation and high cGMP affinity.
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provides a photomodulated phosphodiesterase with stronger light regulation than earlier low-modulation RhPDEs
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helps close a gap in optogenetic control of cyclic nucleotides by enabling light-driven cGMP degradation
Problem links
helps close a gap in optogenetic control of cyclic nucleotides by enabling light-driven cGMP degradation
LiteratureIt addresses the limited optogenetic utility of earlier RhPDEs with low light-dependent modulation by providing stronger light regulation and high cGMP affinity.
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It addresses the limited optogenetic utility of earlier RhPDEs with low light-dependent modulation by providing stronger light regulation and high cGMP affinity.
provides a photomodulated phosphodiesterase with stronger light regulation than earlier low-modulation RhPDEs
LiteratureIt addresses the limited optogenetic utility of earlier RhPDEs with low light-dependent modulation by providing stronger light regulation and high cGMP affinity.
Source:
It addresses the limited optogenetic utility of earlier RhPDEs with low light-dependent modulation by providing stronger light regulation and high cGMP affinity.
Published Workflows
Objective: Identify a Choanoeca flexa rhodopsin-phosphodiesterase suitable for optogenetic cGMP control and combine it with a rhodopsin-guanylyl-cyclase and channel readout to achieve bidirectional dual-color cGMP modulation in mammalian cells.
Why it works: The workflow first enriches for a RhPDE variant with strong light regulation and high cGMP affinity, then tests interoperability with a light-activated guanylyl cyclase and channel readout to achieve bidirectional control, while spectroscopy and fast recordings probe the mechanistic basis of activation.
light-dependent phosphodiesterase control of cGMPM-state-linked activation of the phosphodiesterase domain via rhodopsin-PDE couplingvariant screeningcoexpression in mammalian cellsspectroscopic characterizationfast functional recordings
Stages
- 1.screening of Choanoeca flexa RhPDE variants(broad_screen)
This stage identifies the most optogenetically suitable RhPDE variant from the Choanoeca flexa family.
Selection: photomodulation, cGMP affinity, and degree of light regulation
- 2.multicomponent cellular functional demonstration(confirmatory_validation)
This stage confirms that the selected RhPDE can function with a rhodopsin-guanylyl-cyclase and channel readout in mammalian cells to achieve the intended bidirectional cGMP control.
Selection: ability to support bidirectional dual-color modulation of cGMP levels and ion channel conductance in mammalian cells
- 3.mechanistic characterization(functional_characterization)
This stage probes how light activation is transmitted from the rhodopsin domain to the phosphodiesterase domain.
Selection: spectroscopic and fast functional evidence for photocycle-state coupling to PDE-domain activation
Steps
- 1.screen Choanoeca flexa RhPDE variants for photomodulated PDE activity
Identify photomodulated RhPDE candidates from Choanoeca flexa.
A suitable RhPDE had to be found before building a multicomponent bidirectional cGMP control system.
- 2.select CfRhPDE1 as the lead variant based on cGMP affinity and light regulationlead screened variant
Choose the best-performing RhPDE for downstream optogenetic system assembly.
Lead selection follows screening so that only the strongest candidate is taken into cellular validation.
- 3.coexpress CfRhPDE1 with the Catenaria anguillulae rhodopsin-guanylyl-cyclase and an olfactory cyclic nucleotide-gated ion channel in ND7/23 cellscoexpressed system components
Assemble the multicomponent optogenetic system in mammalian cells.
Cellular coexpression is required after lead selection to test whether the chosen RhPDE interoperates with the cyclase and channel readout.
- 4.measure bidirectional dual-color modulation of cGMP levels and ion channel conductancesystem under test
Confirm that the assembled system achieves the intended bidirectional optical control output.
Functional measurement follows coexpression because the system must first be assembled in cells before its output can be tested.
- 5.perform spectroscopic characterization and fast functional recordings to infer photocycle-state couplingmechanistically characterized RhPDE
Probe how the photocycle state triggers functional changes in the phosphodiesterase domain.
Mechanistic analysis follows functional demonstration to explain the basis of the observed optogenetic behavior.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A reusable architecture pattern for arranging parts into an engineered system.
Mechanisms
cgmp hydrolysislight-gated phosphodiesterase activityphotocycle-state-dependent allosteric coupling between rhodopsin and phosphodiesterase domainsTarget processes
recombinationselectionsignalingInput: Light
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: spectral hardware requirementoperating role: sensor
The demonstrated bidirectional system coexpressed CfRhPDE1 with a rhodopsin-guanylyl-cyclase from Catenaria anguillulae and an olfactory cyclic nucleotide-gated ion channel in ND7/23 cells.; optogenetic use in the demonstrated system required coexpression with a rhodopsin-guanylyl-cyclase and a cyclic nucleotide-gated ion channel; light-dependent behavior depends on the rhodopsin-phosphodiesterase photocycle
By itself it does not provide bidirectional cGMP control; the abstract only demonstrates that in combination with a cyclase and channel readout.; requires coexpression with other components for bidirectional dual-color cGMP control
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
Coexpression of CfRhPDE1 with a Catenaria anguillulae rhodopsin-guanylyl-cyclase and an olfactory cyclic nucleotide-gated ion channel in ND7/23 cells enabled bidirectional dual-color modulation of cGMP levels and ion channel conductance.
By coexpressing CfRhPDE1 with the rhodopsin-guanylyl-cyclase from the fungus Catenaria anguillulae and a cyclic nucleotide-gated ion channel from olfactory neurons in ND7/23 cells, we demonstrate bidirectional dual-color modulation of cGMP levels and ion channel conductance.
RhPDEs can regulate cGMP signaling in mammalian cells on a subsecond timescale, helping close a gap in optogenetics for bidirectional cyclic nucleotide control.
This demonstrates that RhPDEs can regulate cGMP signaling in mammalian cells on a subsecond timescale, closing a present gap in optogenetics and assisting researchers in setting up multicomponent optogenetic systems for bidirectional control of cyclic nucleotides.
Spectroscopic characterization and fast functional recordings suggest that the M-state of the photocycle initiates functional changes in the phosphodiesterase domain through rapid rhodopsin-PDE coupling.
Together with spectroscopic characterization, our fast functional recordings suggest that the M-state of the photocycle initiates functional changes in the phosphodiesterase domain via rapid rhodopsin-PDE coupling.
CfRhPDE1 shows efficient expression and a 3.5 s active-state lifetime, providing high photosensitivity to host cells.
With efficient expression and 3.5 s lifetime of the active state, this protein provides high photosensitivity to the host cells.
active state lifetime 3.5 s
Screening of Choanoeca flexa RhPDE variants identified four photomodulated phosphodiesterases, with CfRhPDE1 showing the highest cGMP affinity and the strongest light regulation among them.
Through screening various RhPDE variants from C. flexa, we identified four photomodulated PDEs of which C. flexa RhPDE1 (CfRhPDE1) revealed the highest cGMP affinity and the most pronounced light regulation with Km values of 1.9 and 4.4 μM in light and darkness.
Km 1.9 μMKm 4.4 μM
Approval Evidence
1 source5 linked approval claimsfirst-pass slug cfrhpde1
C. flexa RhPDE1 (CfRhPDE1) revealed the highest cGMP affinity and the most pronounced light regulation
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application resultsupports
Coexpression of CfRhPDE1 with a Catenaria anguillulae rhodopsin-guanylyl-cyclase and an olfactory cyclic nucleotide-gated ion channel in ND7/23 cells enabled bidirectional dual-color modulation of cGMP levels and ion channel conductance.
By coexpressing CfRhPDE1 with the rhodopsin-guanylyl-cyclase from the fungus Catenaria anguillulae and a cyclic nucleotide-gated ion channel from olfactory neurons in ND7/23 cells, we demonstrate bidirectional dual-color modulation of cGMP levels and ion channel conductance.
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application scopesupports
RhPDEs can regulate cGMP signaling in mammalian cells on a subsecond timescale, helping close a gap in optogenetics for bidirectional cyclic nucleotide control.
This demonstrates that RhPDEs can regulate cGMP signaling in mammalian cells on a subsecond timescale, closing a present gap in optogenetics and assisting researchers in setting up multicomponent optogenetic systems for bidirectional control of cyclic nucleotides.
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mechanistic inferencesupports
Spectroscopic characterization and fast functional recordings suggest that the M-state of the photocycle initiates functional changes in the phosphodiesterase domain through rapid rhodopsin-PDE coupling.
Together with spectroscopic characterization, our fast functional recordings suggest that the M-state of the photocycle initiates functional changes in the phosphodiesterase domain via rapid rhodopsin-PDE coupling.
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performance propertysupports
CfRhPDE1 shows efficient expression and a 3.5 s active-state lifetime, providing high photosensitivity to host cells.
With efficient expression and 3.5 s lifetime of the active state, this protein provides high photosensitivity to the host cells.
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screening resultsupports
Screening of Choanoeca flexa RhPDE variants identified four photomodulated phosphodiesterases, with CfRhPDE1 showing the highest cGMP affinity and the strongest light regulation among them.
Through screening various RhPDE variants from C. flexa, we identified four photomodulated PDEs of which C. flexa RhPDE1 (CfRhPDE1) revealed the highest cGMP affinity and the most pronounced light regulation with Km values of 1.9 and 4.4 μM in light and darkness.
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Comparisons
Source-stated alternatives
The abstract contrasts CfRhPDE1 with earlier RhPDEs first discovered in Salpingoeca rosetta, whose low light-dependent modulation limited optogenetic application.
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The abstract contrasts CfRhPDE1 with earlier RhPDEs first discovered in Salpingoeca rosetta, whose low light-dependent modulation limited optogenetic application.
Source-backed strengths
highest cGMP affinity among screened C. flexa RhPDE variants; most pronounced light regulation among screened variants; efficient expression; 3.5 s active-state lifetime associated with high photosensitivity
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highest cGMP affinity among screened C. flexa RhPDE variants
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most pronounced light regulation among screened variants
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efficient expression
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3.5 s active-state lifetime associated with high photosensitivity
Compared with optogenetic
The abstract contrasts CfRhPDE1 with earlier RhPDEs first discovered in Salpingoeca rosetta, whose low light-dependent modulation limited optogenetic application.
Shared frame: source-stated alternative in extracted literature
Strengths here: highest cGMP affinity among screened C. flexa RhPDE variants; most pronounced light regulation among screened variants; efficient expression.
Relative tradeoffs: requires coexpression with other components for bidirectional dual-color cGMP control.
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The abstract contrasts CfRhPDE1 with earlier RhPDEs first discovered in Salpingoeca rosetta, whose low light-dependent modulation limited optogenetic application.
Ranked Citations
- 1.