Movement of hClC-1 C-termini during common gating and limits on their cytoplasmic location

2011 ◽  
Vol 436 (2) ◽  
pp. 415-428 ◽  
Author(s):  
Linlin Ma ◽  
Grigori Y. Rychkov ◽  
Ekaterina A. Bykova ◽  
Jie Zheng ◽  
Allan H. Bretag
Keyword(s):  
Human Skeletal Muscle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Carboxyl Group ◽  
Conformational Rearrangement ◽  
C Terminus ◽  
Common Gate ◽  
The Common ◽  
Gating Process ◽  
Cytoplasmic Location

Functionally, the dimeric human skeletal muscle chloride channel hClC-1 is characterized by two distinctive gating processes, fast (protopore) gating and slow (common) gating. Of these, common gating is poorly understood, but extensive conformational rearrangement is suspected. To examine this possibility, we used FRET (fluorescence resonance energy transfer) and assessed the effects of manipulating the common-gating process. Closure of the common gate was accompanied by a separation of the C-termini, whereas, with opening, the C-termini approached each other more closely. These movements were considerably smaller than those seen in ClC-0. To estimate the C-terminus depth within the cytoplasm we constructed a pair of split hClC-1 fragments tagged extracellularly and intracellularly respectively. These not only combined appropriately to rescue channel function, but we detected positive FRET between them. This restricts the C-termini of hClC-1 to a position close to its membrane-resident domain. From mutants in which fast or common gating were affected, FRET revealed a close linkage between the two gating processes with the carboxyl group of Glu232 apparently acting as the final effector for both.

scholarly journals Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain

2018 ◽  
Vol 115 (3) ◽  
pp. 546-555 ◽  
Author(s):  
Zeynep Bastug-Özel ◽  
Peter T Wright ◽  
Axel E Kraft ◽  
Davor Pavlovic ◽  
Jacqueline Howie ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adult Rat ◽  
C Terminus ◽  
Complex Forms ◽  
Cardiac Excitation

Abstract Aims Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation–contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium–potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. Methods and results A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by β2-adrenoceptors (β2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific β2-AR/cAMP signals and β2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. Conclusions NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by β2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted β2-AR signalling to cAMP in the vicinity of PLM.

scholarly journals Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

2015 ◽  
Vol 112 (15) ◽  
pp. 4660-4665 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Ewa Prochniewicz ◽  
David D. Thomas
Keyword(s):  
Light Chain ◽  
Contractile Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Efficiency ◽  
Power Stroke ◽  
Time Resolved ◽  
Muscle Disorders ◽  
Essential Light Chain ◽  
C Terminus

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.

Sustained Ca2+ signaling and delayed internalization associated with endothelin receptor heterodimers linked through a PDZ fingerThis article is one of a selection of papers published in the special issue (part 2 of 2) on Forefronts in Endothelin.

2008 ◽  
Vol 86 (8) ◽  
pp. 526-535 ◽  
Author(s):  
Nathan J. Evans ◽  
Jeffery W. Walker
Keyword(s):  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Endothelin Receptor ◽  
Similar Time ◽  
Receptor Dimerization ◽  
C Terminus ◽  
Interaction Interface ◽  
Intracellular Ca

G protein-coupled receptors (GPCRs), including endothelin receptor A (ETA) and B (ETB), may form dimers or higher-order oligomers that profoundly influence signaling. Here we examined a PDZ finger motif within the C-terminus of ETA and its role in heterodimerization with ETB, and in homodimerization with itself, when expressed in HEK293 cells. Receptor dimerization was monitored by (i) fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) (FRET donor) and tetracysteine/FlAsH (FRET acceptor) fused to the C-termini of ET receptors, and (ii) coimmunoprecipitation of ET receptors after mild detergent solubilization. Mutations in a PDZ finger motif at threonine403/serine404 eliminated FRET and reduced coimmunoprecipitation of heterodimers and homodimers. Functional consequences were evaluated by measuring mobilization of intracellular Ca2+ and internalization of receptors in response to a 10 nmol/L ET-1 challenge. PDZ mutations converted a sustained Ca2+ signal mediated by ETA:ETB heterodimers into a transient response, similar to that observed for homodimers or monomers. Heterodimers containing PDZ mutations were seen to internalize in a similar time domain (approximately 5 min) to the transient Ca2+ elevation and with similar kinetics to internalization of ETA homodimers or monomers. Without the PDZ mutations, heterodimers did not internalize over 15 min, suggesting the intriguing possibility that sustained Ca2+ signaling was a consequence (at least in part) of delayed internalization. The results are consistent with structural models of ETA-receptor dimerization that place threonine403/serine404 of the PDZ finger motif at the interaction interface between heterodimers and homodimers. Sustained Ca2+ signaling and delayed endocytosis of ETA:ETB heterodimers argues strongly for a unique dimer interface that impacts transmembrane signaling and internalization.

Thrombin Modulates PAR1-PAR4 Heterodimers

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2203-2203 ◽  
Author(s):  
Maria de la Fuente ◽  
Amal Arachiche ◽  
Marvin T. Nieman
Keyword(s):  
Conflicts Of Interest ◽  
Transmembrane Helix ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Bimolecular Fluorescence Complementation ◽  
Physical Interaction ◽  
Signaling Specificity ◽  
C Terminus ◽  
The Stability

Abstract Abstract 2203 Thrombin is a potent platelet agonist. Thrombin activates platelets and other cells of the cardiovascular system by cleaving its receptors, protease activated receptor 1 (PAR1), PAR4 or both. PARs are G-protein coupled receptors that activate cellular signaling through Gq and G12/13. There is structural evidence that GPCRs, as a class, function as dimers and that dimerization can alter signaling specificity. Our previous studies have determined that PAR4 forms homodimers and have mapped the homodimer interface to transmembrane helix 4 (TM4). We have also shown that coexpression of PAR1 with PAR4 lowers the threshold for PAR4 activation by thrombin ∼10-fold. The purpose of the current study is to examine the physical interaction between PAR1 and PAR4 and how these interactions influence PAR1's ability to enhance PAR4 activation. The PAR1-PAR4 heterodimers were examined by bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC). Similar to our previous studies with PAR4 homodimers, PAR1 homodimers were constitutive and did not require receptor activation. In contrast, PAR1-PAR4 heterodimers were not detected under basal conditions. However, when the cells were stimulated with 10 nM thrombin, we were able to detect a strong interaction between PAR1 and PAR4. We next examined if PAR1-PAR4 heterodimers would be induced by stimulating PAR1 or PAR4 individually with their agonist peptides TFLLRN (100 μM) or AYPGKF (500 μM), respectively. The agonist peptides were unable to induce heterodimers when added to the cells individually or simultaneously. These data demonstrate that PAR1 and PAR4 require allosteric changes induced by receptor cleavage by thrombin to mediate heterodimer formation. To examine this further, we removed 37 amino acids from the C-terminus of PAR1, which disrupts the eighth helix. The truncated PAR1 was able to form constitutive heterodimers with PAR4 and these heterodimers were unaffected by thrombin. These data suggest that PAR1 is the allosteric modulator of the PAR1-PAR4 heterodimers. Finally, the stability of the constitutive PAR1 and PAR4 homodimers was unchanged in response to thrombin or the agonist peptides. Taken together, these data suggest that PAR1 and PAR4 have a dynamic interaction depending on the context of their expression. Since PAR1 is an attractive antiplatelet target, the molecular interactions of this receptor on the cells surface must be taken into account when developing and characterizing these antagonists. Disclosures: No relevant conflicts of interest to declare.

Features of MOG required for recognition by patients with MOG antibody-associated disorders

Brain ◽  
2021 ◽  
Author(s):  
Caterina Macrini ◽  
Ramona Gerhards ◽  
Stephan Winklmeier ◽  
Lena Bergmann ◽  
Simone Mader ◽  
...  
Keyword(s):  
Complement Activation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Features ◽  
Full Length ◽  
Target Antigen ◽  
Terminal Part ◽  
Hydrophobic Domain ◽  
C Terminus ◽  
Better Than

Abstract Antibodies (Abs) to myelin oligodendrocyte glycoprotein (MOG) define a distinct disease entity. Here we aimed to understand essential structural features of MOG required for recognition by autoantibodies from patients. We produced the N-terminal part of MOG in a conformationally correct form; this domain was insufficient to identify patients with MOG-Abs by ELISA even after site-directed binding. This was neither due to a lack of lipid embedding nor to a missing putative epitope at the C-terminus, which we confirmed to be an intracellular domain. When MOG was displayed on transfected cells, patients with MOG-Abs recognized full-length MOG much better than its N-terminal part with the first hydrophobic domain (p < 0.0001). Even antibodies affinity-purified with the extracellular part of MOG recognized full-length MOG better than the extracellular part of MOG after transfection. The second hydrophobic domain of MOG enhanced the recognition of the extracellular part of MOG by antibodies from patients as seen with truncated variants of MOG. We confirmed the pivotal role of the second hydrophobic domain by fusing the intracellular part of MOG from the evolutionary distant opossum to the human extracellular part; the chimeric construct restored the antibody-binding completely. Further, we found that in contrast to 8-18C5, MOG-Abs from patients bound preferentially as F(ab’)2 rather than Fab. It was previously found that bivalent binding of human IgG1, the prominent isotype of MOG-Abs, requires that its target antigen is displayed at a distance of 13-16 nm. We found that, upon transfection, molecules of MOG did not interact so closely to induce a Förster resonance energy transfer (FRET) signal, indicating that they are more than 6 nm apart. We propose that the intracellular part of MOG holds the monomers apart at a suitable distance for bivalent binding; this could explain why a cell-based assay is needed to identify MOG-Abs. Our finding that MOG-Abs from most patients require bivalent binding has implications for understanding the pathogenesis of MOG-antibody-associated-disorders. Since bivalently bound antibodies have been reported to only poorly bind C1q, we speculate that the pathogenicity of MOG-Abs is mostly mediated by other mechanisms than complement activation. Therefore, therapeutic inhibition of complement activation should be less efficient in MOG-Ab associated disorders than in patients with Abs to aquaporin-4.

scholarly journals AKAP79/150 recruits the transcription factor NFAT to regulate signaling to the nucleus by neuronal L-type Ca2+ channels

2019 ◽  
Vol 30 (14) ◽  
pp. 1743-1756 ◽  
Author(s):  
Jonathan G. Murphy ◽  
Kevin C. Crosby ◽  
Philip J. Dittmer ◽  
William A. Sather ◽  
Mark L. Dell’Acqua
Keyword(s):  
Hippocampal Neurons ◽  
Leucine Zipper ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Activated T Cells ◽  
Signaling Complex ◽  
Electrophysiological Recordings ◽  
C Terminus ◽  
Anchoring Protein

In neurons, regulation of activity-dependent transcription by the nuclear factor of activated T-cells (NFAT) depends upon Ca2+ influx through voltage-gated L-type calcium channels (LTCC) and NFAT translocation to the nucleus following its dephosphorylation by the Ca2+-dependent phosphatase calcineurin (CaN). CaN is recruited to the channel by A-kinase anchoring protein (AKAP) 79/150, which binds to the LTCC C-terminus via a modified leucine-zipper (LZ) interaction. Here we sought to gain new insights into how LTCCs and signaling to NFAT are regulated by this LZ interaction. RNA interference–mediated knockdown of endogenous AKAP150 and replacement with human AKAP79 lacking its C-terminal LZ domain resulted in loss of depolarization-stimulated NFAT signaling in rat hippocampal neurons. However, the LZ mutation had little impact on the AKAP–LTCC interaction or LTCC function, as measured by Förster resonance energy transfer, Ca2+ imaging, and electrophysiological recordings. AKAP79 and NFAT coimmunoprecipitated when coexpressed in heterologous cells, and the LZ mutation disrupted this association. Critically, measurements of NFAT mobility in neurons employing fluorescence recovery after photobleaching and fluorescence correlation spectroscopy provided further evidence for an AKAP79 LZ interaction with NFAT. These findings suggest that the AKAP79/150 LZ motif functions to recruit NFAT to the LTCC signaling complex to promote its activation by AKAP-anchored calcineurin.

scholarly journals α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

2016 ◽  
Vol 27 (16) ◽  
pp. 2542-2553 ◽  
Author(s):  
Peiyao Li ◽  
Yong Miao ◽  
Adish Dani ◽  
Monika Vig
Keyword(s):  
Calcium Release ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Embryonic Fibroblasts ◽  
Unique Role ◽  
C Terminus ◽  
Primary Mouse ◽  
N Terminus ◽  
Variable Stoichiometry

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP–deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP–depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1.

scholarly journals HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag

2009 ◽  
Vol 84 (3) ◽  
pp. 1585-1596 ◽  
Author(s):  
Joëlle V. Fritz ◽  
Denis Dujardin ◽  
Julien Godet ◽  
Pascal Didier ◽  
Jan De Mey ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Physiology ◽  
Fluorescence Lifetime Imaging ◽  
M Cell ◽  
C Terminus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55Gag. Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G2/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55Gag-TC. This TC motif is tethered to the C terminus of Pr55Gag and does not interfere with Pr55Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55Gag-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55Gag-TC mutants confirm that the 41LXXLF domain of Gag-p6 is essential for Pr55Gag-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag-Vpr complexes are still formed when Pr55Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag-Vpr recognition and complex formation occur early during Pr55Gag assembly.

Real-time analysis of amyloid fibril formation of α-synuclein using a fibrillation-state-specific fluorescent probe of JC-1

2009 ◽  
Vol 418 (2) ◽  
pp. 311-323 ◽  
Author(s):  
Jung-Ho Lee ◽  
In-Hwan Lee ◽  
Young-Jun Choe ◽  
Sungsoo Kang ◽  
Hui Young Kim ◽  
...  
Keyword(s):  
Real Time ◽  
Amyloid Fibrils ◽  
Specific Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Real Time Analysis ◽  
High Throughput Drug Screening ◽  
Amyloid Fibril Formation ◽  
The Common

α-Synuclein is a pathological component of PD (Parkinson's disease) by participating in Lewy body formation. JC-1 (5,5′,6,6′-tetrachloro-1,1,3,3′-tetraethylbenzimidazolyl carbocyanine iodide) has been shown to interact with α-synuclein at the acidic C-terminal region with a Kd of 2.6 μM. JC-1 can discriminated between the fibrillation states of α-synuclein (monomeric, oligomeric intermediate and fibrillar forms) by emitting the enhanced binding fluorescence of different colours at 590, 560 and 538 nm respectively with the common excitation at 490 nm. The fibrillation-state-specific interaction of JC-1 allowed us to perform real-time analyses of the α-synuclein fibrillation in the presence of iron as a fibrillation inducer, rifampicin as a fibrillation inhibitor, baicalein as a defibrillation agent and dequalinium as a protofibril inducer. In addition, various α-synuclein fibrils with different morphologies prepared with specific ligands such as metal ions, glutathione, eosin and lipids were monitored with their characteristic JC-1-binding fluorescence spectra. FRET (fluorescence resonance energy transfer) between thioflavin-T and JC-1 was also employed to specifically identify the amyloid fibrils of α-synuclein. Taken together, we have introduced JC-1 as a powerful and versatile probe to explore the molecular mechanism of the fibrillation process of α-synuclein in vitro. It could be also useful in high-throughput drug screening. The specific α-synuclein interaction of JC-1 would therefore contribute to our complete understanding of the molecular aetiology of PD and eventual development of diagnostic/therapeutic strategies for various α-synucleinopathies.

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