scholarly journals Dynamic rearrangement of the intrinsic ligand regulates KCNH potassium channels

2018 ◽  
Vol 150 (4) ◽  
pp. 625-635 ◽  
Author(s):  
Gucan Dai ◽  
Zachary M. James ◽  
William N. Zagotta
Keyword(s):  
Potassium Channels ◽  
Danio Rerio ◽  
Cyclic Nucleotides ◽  
Metal Ion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Binding Cavity ◽  
Noncanonical Amino Acid

KCNH voltage-gated potassium channels (EAG, ERG, and ELK) play significant roles in neuronal and cardiac excitability. They contain cyclic nucleotide-binding homology domains (CNBHDs) but are not directly regulated by cyclic nucleotides. Instead, the CNBHD ligand-binding cavity is occupied by an intrinsic ligand, which resides at the intersubunit interface between the N-terminal eag domain and the C-terminal CNBHD. We show that, in Danio rerio ELK channels, this intrinsic ligand is critical for voltage-dependent potentiation (VDP), a process in which channel opening is stabilized by prior depolarization. We demonstrate that an exogenous peptide corresponding to the intrinsic ligand can bind to and regulate zebrafish ELK channels. This exogenous intrinsic ligand inhibits the channels before VDP and potentiates the channels after VDP. Furthermore, using transition metal ion fluorescence resonance energy transfer and a fluorescent noncanonical amino acid L-Anap, we show that there is a rearrangement of the intrinsic ligand relative to the CNBHD during VDP. We propose that the intrinsic ligand switches from antagonist to agonist as a result of a rearrangement of the eag domain–CNBHD interaction during VDP.

scholarly journals Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET

2016 ◽  
Vol 147 (2) ◽  
pp. 201-216 ◽  
Author(s):  
William N. Zagotta ◽  
Moshe T. Gordon ◽  
Eric N. Senning ◽  
Mika A. Munari ◽  
Sharona E. Gordon
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Transition Metal ◽  
Fluorescence Resonance Energy Transfer ◽  
Metal Ion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Noncanonical Amino Acid

Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co2+ bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.

scholarly journals Molecular mechanism of voltage-dependent potentiation of KCNH potassium channels

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Gucan Dai ◽  
William N Zagotta
Keyword(s):  
Potassium Channels ◽  
Metal Ion ◽  
Voltage Dependence ◽  
Direct Interaction ◽  
Voltage Sensitivity ◽  
State Dependent ◽  
Voltage Dependent ◽  
Noncanonical Amino Acid ◽  
The Brain ◽  
Hyperpolarizing Shift

EAG-like (ELK) voltage-gated potassium channels are abundantly expressed in the brain. These channels exhibit a behavior called voltage-dependent potentiation (VDP), which appears to be a specialization to dampen the hyperexitability of neurons. VDP manifests as a potentiation of current amplitude, hyperpolarizing shift in voltage sensitivity, and slowing of deactivation in response to a depolarizing prepulse. Here we show that VDP of D. rerio ELK channels involves the structural interaction between the intracellular N-terminal eag domain and C-terminal CNBHD. Combining transition metal ion FRET, patch-clamp fluorometry, and incorporation of a fluorescent noncanonical amino acid, we show that there is a rearrangement in the eag domain-CNBHD interaction with the kinetics, voltage-dependence, and ATP-dependence of VDP. We propose that the activation of ELK channels involves a slow open-state dependent rearrangement of the direct interaction between the eag domain and CNBHD, which stabilizes the opening of the channel.

scholarly journals Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels

2018 ◽  
Vol 115 (47) ◽  
pp. 12051-12056 ◽  
Author(s):  
Akil A. Puckerin ◽  
Donald D. Chang ◽  
Zunaira Shuja ◽  
Papiya Choudhury ◽  
Joachim Scholz ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Channel Blockers ◽  
Wild Type ◽  
Voltage Dependent ◽  
Somatosensory Neurons ◽  
Potential Applications ◽  
Voltage Dependent Calcium Channels ◽  
Potential Therapeutics

Genetically encoded inhibitors for voltage-dependent Ca2+ (CaV) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca2+ (CaV1/CaV2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary CaVβ-subunits. To investigate latent CaVβ-independent components of inhibition, we coexpressed each RGK individually with CaV1 (CaV1.2/CaV1.3) or CaV2 (CaV2.1/CaV2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β2a or a mutant version (β2a,TM) that does not bind RGKs. All four RGKs strongly inhibited CaV1/CaV2 channels reconstituted with WT β2a. By contrast, when channels were reconstituted with β2a,TM, Rem inhibited only CaV1.2, Rad selectively inhibited CaV1.2 and CaV2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT CaVβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted CaV1.2 while Rad[R208A/L235A] inhibited CaV1.2/CaV2.2 but not CaV1.3/CaV2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous CaV1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for CaV1.2 and CaV1.2/CaV2.2 channels. Thus, we have exploited latent β-binding–independent Rem and Rad inhibition of specific CaV1/CaV2 channels to develop selective GECCIs with properties unmatched by current small-molecule CaV channel blockers.

scholarly journals Visualizing conformational dynamics of proteins in solution and at the cell membrane

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sharona E Gordon ◽  
Mika Munari ◽  
William N Zagotta
Keyword(s):  
Membrane Proteins ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Computational Method ◽  
Intact Cells ◽  
Distance Distributions ◽  
Amber Codon Suppression ◽  
Noncanonical Amino Acid

Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

Coumarin-based Fluorescent Probes for Bioimaging: Recent Applications and Developments

2021 ◽  
Vol 25 ◽  
Author(s):  
Jinhua Xie ◽  
Le Wang ◽  
Xiqi Su ◽  
João Rodrigues
Keyword(s):  
Fluorescent Probes ◽  
Photoinduced Electron Transfer ◽  
Metal Ion ◽  
Intramolecular Charge Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Biological Sensing ◽  
Ion Imaging ◽  
Future Perspectives

: Coumarin-based derivatives have easy modification and tunable properties and have been synthesized and applied in many areas including pro-drugs, biomedical materials, and chemical and biological sensing. In this review, recent advances of coumarin-based fluorescent probes for biosensing are reported for metal ion imaging. Metal detection in living cells will be highlighted with representative examples together with fluorescence response mechanisms such as photoinduced electron transfer (PET), intramolecular charge transfer (ICT), and fluorescence resonance energy transfer (FRET). Some future perspectives are also briefly mentioned.

scholarly journals Fluorescence Resonance Energy Transfer Analysis of Recombination Signal Sequence Configuration in the RAG1/2 Synaptic Complex

2007 ◽  
Vol 27 (13) ◽  
pp. 4745-4758 ◽  
Author(s):  
Mihai Ciubotaru ◽  
Aleksei N. Kriatchko ◽  
Patrick C. Swanson ◽  
Frank V. Bright ◽  
David G. Schatz
Keyword(s):  
Energy Transfer ◽  
Metal Ion ◽  
Signal Sequence ◽  
Resonance Energy Transfer ◽  
High Mobility ◽  
Resonance Energy ◽  
High Mobility Group Protein ◽  
Synaptic Complex ◽  
Recombination Signal ◽  
Group Protein

ABSTRACT A critical step in V(D)J recombination is the synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins to generate the paired complex (PC). Using a facilitated ligation assay and substrates that vary the helical phasing of the RSSs, we provide evidence that one particular geometric configuration of the RSSs is favored in the PC. To investigate this configuration further, we used fluorescent resonance energy transfer (FRET) to detect the synapsis of fluorescently labeled RSS oligonucleotides. FRET requires an appropriate 12/23 RSS pair, a divalent metal ion, and high-mobility-group protein HMGB1 or HMGB2. Energy transfer between the RSSs was detected with all 12/23 RSS end positions of the fluorescent probes but was not detected when probes were placed on the two ends of the same RSS. Energy transfer was confirmed to originate from the PC by using an in-gel FRET assay. The results argue against a unique planar configuration of the RSSs in the PC and are most easily accommodated by models in which synapsed 12- and 23-RSSs are bent and cross one another, with implications for the organization of the RAG proteins and the DNA substrates at the time of cleavage.

scholarly journals Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast

2004 ◽  
Vol 380 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Pierre DOUETTE ◽  
Rachel NAVET ◽  
Fabrice BOUILLENNE ◽  
Alain BRANS ◽  
Claudine SLUSE-GOFFART ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Metal Ion ◽  
Binding Capacity ◽  
Uncoupling Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Homology Model ◽  
Structure Characterization ◽  
Uncoupling Protein 1 ◽  
Functional Protein

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-β-d-maltoside-solubilized UCP1–His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of α-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an α-helical degree of approx. 68%, which is at least 25% higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) α-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.

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