scholarly journals Tracking the sarcoplasmic reticulum membrane voltage in muscle with a FRET biosensor

2018 ◽  
Vol 150 (8) ◽  
pp. 1163-1177 ◽  
Author(s):  
Colline Sanchez ◽  
Christine Berthier ◽  
Bruno Allard ◽  
Jimmy Perrot ◽  
Clément Bouvard ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ryanodine Receptor ◽  
Striated Muscle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Voltage ◽  
Voltage Sensitivity ◽  
Cell Functions

Ion channel activity in the plasma membrane of living cells generates voltage changes that are critical for numerous biological functions. The membrane of the endoplasmic/sarcoplasmic reticulum (ER/SR) is also endowed with ion channels, but whether changes in its voltage occur during cellular activity has remained ambiguous. This issue is critical for cell functions that depend on a Ca2+ flux across the reticulum membrane. This is the case for contraction of striated muscle, which is triggered by opening of ryanodine receptor Ca2+ release channels in the SR membrane in response to depolarization of the transverse invaginations of the plasma membrane (the t-tubules). Here, we use targeted expression of voltage-sensitive fluorescence resonance energy transfer (FRET) probes of the Mermaid family in differentiated muscle fibers to determine whether changes in SR membrane voltage occur during depolarization–contraction coupling. In the absence of an SR targeting sequence, FRET signals from probes present in the t-tubule membrane allow calibration of the voltage sensitivity and amplitude of the response to voltage-clamp pulses. Successful SR targeting of the probes was achieved using an N-terminal domain of triadin, which completely eliminates voltage-clamp–activated FRET signals from the t-tubule membrane of transfected fibers. In fibers expressing SR-targeted Mermaid probes, activation of SR Ca2+ release in the presence of intracellular ethyleneglycol-bis(β-amino-ethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) results in an accompanying FRET signal. We find that this signal results from pH sensitivity of the probe, which detects cytosolic acidification because of the release of protons upon Ca2+ binding to EGTA. When EGTA is substituted with either 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or the contraction blocker N-benzyl-p-toluene sulfonamide, we find no indication of a substantial change in the FRET response caused by a voltage change. These results suggest that the ryanodine receptor–mediated SR Ca2+ efflux is well balanced by concomitant counterion currents across the SR membrane.

scholarly journals High-Throughput Screens to Discover Small-Molecule Modulators of Ryanodine Receptor Calcium Release Channels

2016 ◽  
Vol 22 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Robyn T. Rebbeck ◽  
Maram M. Essawy ◽  
Florentin R. Nitu ◽  
Benjamin D. Grant ◽  
Gregory D. Gillispie ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Calcium Release ◽  
Striated Muscle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Pathological State ◽  
Pharmacological Agents ◽  
Intracellular Ca

Using time-resolved fluorescence resonance energy transfer (FRET), we have developed and validated the first high-throughput screening (HTS) method to discover compounds that modulate an intracellular Ca2+ channel, the ryanodine receptor (RyR), for therapeutic applications. Intracellular Ca2+ regulation is critical for striated muscle function, and RyR is a central player. At resting [Ca2+], an increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes, and neurological disorders. This leaky state of the RyR is an attractive target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin (CaM) or FKBP12.6. Under conditions that mimic a pathological state, we carried out a screen of the 727-compound NIH Clinical Collection, which yielded six compounds that reproducibly changed FRET by >3 SD. Dose–response of FRET and [3H]ryanodine binding readouts reveal that five hits reproducibly alter RyR1 structure and activity. One compound increased FRET and inhibited RyR1, which was only significant at nM [Ca2+], and accentuated without CaM present. These properties characterize a compound that could mitigate RyR1 leak. An excellent Z′ factor and the tight correlation between structural and functional readouts validate this first HTS method to identify RyR modulators.

scholarly journals Measurement of RyR permeability reveals a role of calsequestrin in termination of SR Ca2+ release in skeletal muscle

2011 ◽  
Vol 138 (2) ◽  
pp. 231-247 ◽  
Author(s):  
Monika Sztretye ◽  
Jianxun Yi ◽  
Lourdes Figueroa ◽  
Jingsong Zhou ◽  
Leandro Royer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Voltage Clamp ◽  
Ryanodine Receptor ◽  
Release Flux ◽  
Consequent Reduction ◽  
First Time ◽  
Receptor Channel

The mechanisms that terminate Ca2+ release from the sarcoplasmic reticulum are not fully understood. D4cpv-Casq1 (Sztretye et al. 2011. J. Gen. Physiol. doi:10.1085/jgp.201010591) was used in mouse skeletal muscle cells under voltage clamp to measure free Ca2+ concentration inside the sarcoplasmic reticulum (SR), [Ca2+]SR, simultaneously with that in the cytosol, [Ca2+]c, during the response to long-lasting depolarization of the plasma membrane. The ratio of Ca2+ release flux (derived from [Ca2+]c(t)) over the gradient that drives it (essentially equal to [Ca2+]SR) provided directly, for the first time, a dynamic measure of the permeability to Ca2+ of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, [Ca2+]SR stabilized at ∼35% of its resting level; depletion was therefore incomplete. By 0.4 s of depolarization, the measured permeability decayed to ∼10% of maximum, indicating ryanodine receptor channel closure. Inactivation of the t tubule voltage sensor was immeasurably small by this time and thus not a significant factor in channel closure. In cells of mice null for Casq1, permeability did not decrease in the same way, indicating that calsequestrin (Casq) is essential in the mechanism of channel closure and termination of Ca2+ release. The absence of this mechanism explains why the total amount of calcium releasable by depolarization is not greatly reduced in Casq-null muscle (Royer et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.201010454). When the fast buffer BAPTA was introduced in the cytosol, release flux became more intense, and the SR emptied earlier. The consequent reduction in permeability accelerated as well, reaching comparable decay at earlier times but comparable levels of depletion. This observation indicates that [Ca2+]SR, sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates Ca2+ release.

Dynamic receptor superstructures at the plasma membrane

1997 ◽  
Vol 30 (1) ◽  
pp. 67-106 ◽  
Author(s):  
S. DAMJANOVICH ◽  
R. GÁSPÁR, Jr. ◽  
C. PIERI
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scanning Force Microscopy ◽  
Physical Parameters ◽  
Conformational States ◽  
Lipid Domain ◽  
Receptor Clusters

1. INTRODUCTION 681.1 Receptor patterns in the plasma membrane 681.2 Different types of receptor patterns 712. METHODS TO INVESTIGATE NON-RANDOM RECEPTOR CLUSTERING 732.1 Fluorescence resonance energy transfer 732.2 Flow cytometric energy transfer measurement 782.3 Fluorescence anisotropy and energy transfer 792.4 Photobleaching energy transfer on single cells 812.5 Two-dimensional mapping of receptor superstructures 822.6 Detecting single receptor molecules 852.7 Chemical identification of receptor clusters 862.8 Electron microscopy 872.9 Scanning force microscopy 883. CONFORMATIONAL STATES OF RECEPTORS 903.1 Multi-subunit receptor structures 903.2 Physical parameters influencing conformational states 913.3 Chemical interactions and receptor conformations 924. ON THE ORIGIN OF NATURALLY OCCURRING RECEPTOR CLUSTERS 934.1 Synthesis of receptors and their localization in the plasma membrane 934.2 Lipid domain structure of the plasma membrane 944.3 The validity of the Singer–Nicolson model 945. CONCLUSIONS 966. ACKNOWLEDGEMENTS 967. REFERENCES 97

scholarly journals Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

2008 ◽  
Vol 19 (10) ◽  
pp. 4366-4373 ◽  
Author(s):  
Xinxin Gao ◽  
Jin Zhang
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Lipid Rafts ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spatiotemporal Analysis ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Growth Factor Signaling

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.

scholarly journals TCR–pMHC bond conformation controls TCR ligand discrimination

2019 ◽  
Vol 17 (3) ◽  
pp. 203-217 ◽  
Author(s):  
Dibyendu K. Sasmal ◽  
Wei Feng ◽  
Sobhan Roy ◽  
Peter Leung ◽  
Yanran He ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Feedback Loop ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intrinsic Property ◽  
Unanswered Question ◽  
Structural Differences ◽  
Self Peptides

Abstract A major unanswered question is how a TCR discriminates between foreign and self-peptides presented on the APC surface. Here, we used in situ fluorescence resonance energy transfer (FRET) to measure the distances of single TCR–pMHC bonds and the conformations of individual TCR–CD3ζ receptors at the membranes of live primary T cells. We found that a TCR discriminates between closely related peptides by forming single TCR–pMHC bonds with different conformations, and the most potent pMHC forms the shortest bond. The bond conformation is an intrinsic property that is independent of the binding affinity and kinetics, TCR microcluster formation, and CD4 binding. The bond conformation dictates the degree of CD3ζ dissociation from the inner leaflet of the plasma membrane via a positive calcium signaling feedback loop to precisely control the accessibility of CD3ζ ITAMs for phosphorylation. Our data revealed the mechanism by which a TCR deciphers the structural differences among peptides via the TCR–pMHC bond conformation.

scholarly journals Structural Mapping of Divergent Regions in the Type 1 Ryanodine Receptor Using Fluorescence Resonance Energy Transfer

Structure ◽  
2014 ◽  
Vol 22 (9) ◽  
pp. 1322-1332 ◽  
Author(s):  
Mohana Mahalingam ◽  
Tanya Girgenrath ◽  
Bengt Svensson ◽  
David D. Thomas ◽  
Razvan L. Cornea ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Ryanodine Receptor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Mapping ◽  
Fluorescence Resonance

scholarly journals Quality control for unfolded proteins at the plasma membrane

2010 ◽  
Vol 191 (3) ◽  
pp. 553-570 ◽  
Author(s):  
Pirjo M. Apaja ◽  
Haijin Xu ◽  
Gergely L. Lukacs
Keyword(s):  
Quality Control ◽  
Plasma Membrane ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Protein ◽  
Temperature Sensitive ◽  
Potential Contribution ◽  
Unfolded Proteins ◽  
Endosomal Sorting ◽  
V2 Receptor

Cellular protein homeostasis profoundly depends on the disposal of terminally damaged polypeptides. To demonstrate the operation and elucidate the molecular basis of quality control of conformationally impaired plasma membrane (PM) proteins, we constructed CD4 chimeras containing the wild type or a temperature-sensitive bacteriophage λ domain in their cytoplasmic region. Using proteomic, biochemical, and genetic approaches, we showed that thermal unfolding of the λ domain at the PM provoked the recruitment of Hsp40/Hsc70/Hsp90 chaperones and the E2–E3 complex. Mixed-chain polyubiquitination, monitored by bioluminescence resonance energy transfer and immunoblotting, is responsible for the nonnative chimera–accelerated internalization, impaired recycling, and endosomal sorting complex required for transport–dependent lysosomal degradation. A similar paradigm prevails for mutant dopamine D4.4 and vasopressin V2 receptor removal from the PM. These results outline a peripheral proteostatic mechanism in higher eukaryotes and its potential contribution to the pathogenesis of a subset of conformational diseases.

scholarly journals Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Stijn van Dorp ◽  
Ruoyi Qiu ◽  
Ucheor B Choi ◽  
Minnie M Wu ◽  
Michelle Yen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Structural Basis ◽  
Resting Cells ◽  
Dual Roles ◽  
Er Calcium

The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it towards the plasma membrane to activate Orai and SOCE after store depletion.

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