TRPP2 and STIM1 form a microdomain to regulate store-operated Ca2+ entry and blood vessel tone

Abstract BackgroundTRPP2 (Polycystin-2) is a Ca2+ permeable nonselective cationic channel essential for maintaining physiological function in live cells. Stromal interaction molecule 1 (STIM1) is an important Ca2+ sensor in store-operated Ca2+ entry (SOCE). Both TRPP2 and STIM1 are expressed in endoplasmic reticular membrane and participate in Ca2+ signaling, suggesting a physical interaction and functional synergism.MethodsWe performed co-localization, co-immunoprecipitation, and fluorescence resonance energy transfer assay to identify the interactions of TRPP2 and STIM1 in transfected HEK293 cells and native vascular smooth muscle cells (VSMCs). The function of the TRPP2-STIM1 complex in TG or ATP-induced SOCE was explored using specific siRNA. Further, we created TRPP2 CKO mouse to investigate the functional role of TRPP2 in agonist-induced vessel contraction.ResultsTRPP2 and STIM1 form a complex in transfected HEK293 cells and native VSMCs. Genetic manipulations with TRPP2 siRNA, dominant negative TRPP2 or STIM1 siRNA significantly suppressed adenosine triphosphate (ATP) and thapsigargin (TG)-induced intracellular Ca2+ release and SOCE in HEK293 cells. Inositol triphosphate receptor inhibitor 2-aminoethyl diphenylborinate (2APB) abolished ATP-induced Ca2+ release and SOCE in HEK293 cells. In addition, TRPP2 and STIM1 knockdown significantly inhibited ATP- and TG-induced STIM1 puncta formation and SOCE in VSMCs. Importantly, knockdown of TRPP2 and STIM1 or conditional knockout TRPP2 markedly suppressed agonist-induced mouse aorta contraction.Conclusions Our data indicate that TRPP2 and STIM1 are physically associated and form a functional complex to regulate agonist-induced intracellular Ca2+ mobilization, SOCE and blood vessel tone.

Download Full-text

TRPP2 and STIM1 form a microdomain to regulate store-operated Ca2+ entry and blood vessel tone

10.21203/rs.2.18317/v2 ◽

2020 ◽

Author(s):

Jizheng Guo ◽

Ren Zhao ◽

MuYao Zhou ◽

Jie Li ◽

Xiaoqiang Yao ◽

...

Keyword(s):

Blood Vessel ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Dominant Negative ◽

Conditional Knockout ◽

Hek293 Cells ◽

Live Cells ◽

Physical Interaction ◽

Functional Complex ◽

Intracellular Ca

Abstract Background: Polycystin-2 (TRPP2) is a Ca2+ permeable nonselective cationic channel essential for maintaining physiological function in live cells. Stromal interaction molecule 1 (STIM1) is an important Ca2+ sensor in store-operated Ca2+ entry (SOCE). Both TRPP2 and STIM1 are expressed in endoplasmic reticular membrane and participate in Ca2+ signaling, suggesting a physical interaction and functional synergism.Methods: We performed co-localization, co-immunoprecipitation, and fluorescence resonance energy transfer assay to identify the interactions of TRPP2 and STIM1 in transfected HEK293 cells and native vascular smooth muscle cells (VSMCs). The function of the TRPP2-STIM1 complex in thapsigargin (TG) or adenosine triphosphate (ATP)-induced SOCE was explored using specific small interfering RNA (siRNA). Further, we created TRPP2 conditional knockout (CKO) mouse to investigate the functional role of TRPP2 in agonist-induced vessel contraction.Results: TRPP2 and STIM1 form a complex in transfected HEK293 cells and native VSMCs. Genetic manipulations with TRPP2 siRNA, dominant negative TRPP2 or STIM1 siRNA significantly suppressed ATP and TG-induced intracellular Ca2+ release and SOCE in HEK293 cells. Inositol triphosphate receptor inhibitor 2-aminoethyl diphenylborinate (2APB) abolished ATP-induced Ca2+ release and SOCE in HEK293 cells. In addition, TRPP2 and STIM1 knockdown significantly inhibited ATP- and TG-induced STIM1 puncta formation and SOCE in VSMCs. Importantly, knockdown of TRPP2 and STIM1 or conditional knockout TRPP2 markedly suppressed agonist-induced mouse aorta contraction.Conclusions : Our data indicate that TRPP2 and STIM1 are physically associated and form a functional complex to regulate agonist-induced intracellular Ca2+ mobilization, SOCE and blood vessel tone.

Download Full-text

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.

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y08-050 ◽

2008 ◽

Vol 86 (8) ◽

pp. 526-535 ◽

Cited By ~ 29

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.

Download Full-text

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412.v1 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This Quenching Bioluminescent Voltage Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.

Download Full-text

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

Download Full-text

Probing interdomain linkers and protein supertertiary structure in vitro and in live cells with fluorescent protein resonance energy transfer

Journal of Molecular Biology ◽

10.1016/j.jmb.2020.166793 ◽

2021 ◽

pp. 166793

Author(s):

Sujit Basak ◽

Nabanita Sakia ◽

Laura Dougherty ◽

Zhuojun Guo ◽

Fang Wu ◽

...

Keyword(s):

Energy Transfer ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells

Download Full-text

Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana

International Journal of Molecular Sciences ◽

10.3390/ijms22041596 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1596

Author(s):

Elsa Ronzier ◽

Claire Corratgé-Faillie ◽

Frédéric Sanchez ◽

Christian Brière ◽

Tou Cheu Xiong

Keyword(s):

Arabidopsis Thaliana ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Physical Interaction ◽

Fluorescence Lifetime Imaging ◽

Dependent Manner ◽

In Planta ◽

Knock Out ◽

Calcium Dependent ◽

Dependent Protein

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

Download Full-text

Fluorescent Protein-Based Autophagy Biosensors

Materials ◽

10.3390/ma14113019 ◽

2021 ◽

Vol 14 (11) ◽

pp. 3019

Author(s):

Heejung Kim ◽

Jihye Seong

Keyword(s):

Energy Transfer ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Treatment Strategies ◽

Resonance Energy ◽

Live Cells ◽

Spectral Profile ◽

Spatiotemporal Resolution ◽

Future Directions ◽

Complex Process

Autophagy is an essential cellular process of self-degradation for dysfunctional or unnecessary cytosolic constituents and organelles. Dysregulation of autophagy is thus involved in various diseases such as neurodegenerative diseases. To investigate the complex process of autophagy, various biochemical, chemical assays, and imaging methods have been developed. Here we introduce various methods to study autophagy, in particular focusing on the review of designs, principles, and limitations of the fluorescent protein (FP)-based autophagy biosensors. Different physicochemical properties of FPs, such as pH-sensitivity, stability, brightness, spectral profile, and fluorescence resonance energy transfer (FRET), are considered to design autophagy biosensors. These FP-based biosensors allow for sensitive detection and real-time monitoring of autophagy progression in live cells with high spatiotemporal resolution. We also discuss future directions utilizing an optobiochemical strategy to investigate the in-depth mechanisms of autophagy. These cutting-edge technologies will further help us to develop the treatment strategies of autophagy-related diseases.

Download Full-text

AIE-Driven Fluorescent Polysaccharide Polymersome as Enzyme-responsive FRET Nanoprobe to Study the Real-time Delivery Aspects in Live-Cells

Polymer Chemistry ◽

10.1039/d0py01085e ◽

2021 ◽

Author(s):

Nilesh Umakant Deshpande ◽

Mishika Virmani ◽

Manickam Jayakannan

Keyword(s):

Energy Transfer ◽

Confocal Microscopy ◽

Real Time ◽

Fluorescence Resonance Energy Transfer ◽

Imaging Techniques ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Intracellular Enzyme ◽

Bio Imaging

We report aggregation induced emission (AIE) driven polysaccharide polymersome as fluorescence resonance energy transfer (FRET) nanoprobes to study their intracellular enzyme-responsive delivery by real-time live-cell confocal microscopy bio-imaging techniques. AIE...

Download Full-text

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811024115 ◽

2018 ◽

Vol 115 (47) ◽

pp. 12051-12056 ◽

Cited By ~ 4

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.

Download Full-text

Minireview: GPCR and G Proteins: Drug Efficacy and Activation in Live Cells

Molecular Endocrinology ◽

10.1210/me.2008-0204 ◽

2009 ◽

Vol 23 (5) ◽

pp. 590-599 ◽

Cited By ~ 51

Author(s):

Jean-Pierre Vilardaga ◽

Moritz Bünemann ◽

Timothy N. Feinstein ◽

Nevin Lambert ◽

Viacheslav O. Nikolaev ◽

...

Keyword(s):

Energy Transfer ◽

G Protein ◽

G Proteins ◽

Intracellular Signaling ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Receptor Activation ◽

Live Cells ◽

Mechanical Stimuli ◽

G Protein Coupled

Abstract Many biochemical pathways are driven by G protein-coupled receptors, cell surface proteins that convert the binding of extracellular chemical, sensory, and mechanical stimuli into cellular signals. Their interaction with various ligands triggers receptor activation that typically couples to and activates heterotrimeric G proteins, which in turn control the propagation of secondary messenger molecules (e.g. cAMP) involved in critically important physiological processes (e.g. heart beat). Successful transfer of information from ligand binding events to intracellular signaling cascades involves a dynamic interplay between ligands, receptors, and G proteins. The development of Förster resonance energy transfer and bioluminescence resonance energy transfer-based methods has now permitted the kinetic analysis of initial steps involved in G protein-coupled receptor-mediated signaling in live cells and in systems as diverse as neurotransmitter and hormone signaling. The direct measurement of ligand efficacy at the level of the receptor by Förster resonance energy transfer is also now possible and allows intrinsic efficacies of clinical drugs to be linked with the effect of receptor polymorphisms.

Download Full-text