ArticleThe Journal of general physiology2005
Generation of functional fluorescent BK channels by random insertion of GFP variants.
Article in The Journal of general physiology, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 28 papers.
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Who cites it
28 citing papers in PubMed.
- Control of Tetrazine Bioorthogonal Reactivity by Rotaxanation.Angewandte Chemie (International ed. in English) · 2025Article
- GRIN2B disease-associated mutations disrupt the function of BK channels and NMDA receptor signaling nanodomains.The Journal of general physiology · 2025Article
- Structural mapping of patient-associated KCNMA1 gene variants.Biophysical journal · 2024Article
- Visualizing the invisible: novel approaches to visualizing bacterial proteins and host-pathogen interactions.Frontiers in bioengineering and biotechnology · 2024Review
- Flow cytometry based-FRET: basics, novel developments and future perspectives.Cellular and molecular life sciences : CMLS · 2022Review
- NMDA receptor-BK channel coupling regulates synaptic plasticity in the barrel cortex.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Comparative gain-of-function effects of theJournal of neurophysiology · 2020Article
- Targeted insertional mutagenesis libraries for deep domain insertion profiling.Nucleic acids research · 2020Article
- The contribution of voltage clamp fluorometry to the understanding of channel and transporter mechanisms.The Journal of general physiology · 2019Review
- EGFP insertional mutagenesis reveals multiple FXR2P fibrillar states with differing ribosome association in neurons.Biology open · 2019Article
- Effects of Single Nucleotide Polymorphisms in HumanFrontiers in molecular neuroscience · 2019Article
- Article
- Regulatory γ1 subunits defy symmetry in functional modulation of BK channels.Proceedings of the National Academy of Sciences of the United States of America · 2018Article
- Physiological Roles and Therapeutic Potential of CaFrontiers in molecular neuroscience · 2018Review
- Impact of fluorescent protein fusions on the bacterial flagellar motor.Scientific reports · 2017Article
- Understanding the conformational motions of RCK gating rings.The Journal of general physiology · 2017Review
- A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging.Annual review of physiology · 2017Review
- Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring.Proceedings of the National Academy of Sciences of the United States of America · 2016Article
- Engineering Permissive Insertion Sites in the Bacteriophage Phi29 DNA-Linked Terminal Protein.PloS one · 2016Article
- Article
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Authors and funding
3 authors.
Funding
Abstract
The yellow and cyan variants of green fluorescent protein (GFP) constitute an excellent pair for fluorescence resonance energy transfer (FRET) and can be used to study conformational rearrangements of proteins. Our aim was to develop a library of fluorescent large conductance voltage- and Ca2+-gated channels (BK or slo channels) for future use in FRET studies. We report the results of a random insertion of YFP and CFP into multiple sites of the alpha subunit of the hslo channel using a Tn5 transposon-based technique. 55 unique fluorescent fusion proteins were obtained and tested for cell surface expression and channel function. 19 constructs are expressed at the plasma membrane and show voltage and Ca2+-dependent currents. In 16 of them the voltage and Ca2+ dependence is very similar to the wild-type channel. Two insertions in the Ca2+ bowl and one in the RCK2 domain showed a strong shift in the G-V curve. The remaining 36 constructs were retained intracellularly; a solubility assay suggests that these proteins are not forming intracellular aggregates. The "success rate" of 19 out of 55 hslo insertion constructs compares very favorably with other studies of random GFP fusions.
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