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            -Adrenergic Receptor Redistribution in Heart Failure Changes cAMP Compartmentation

Nikolaev, Viacheslav O.; Moshkov, Alexey; Lyon, Alexander R.; Miragoli, Michele; Novák, Pavel; Paur, Helen; Lohse, Martin J.; Korchev, Yuri E.; Harding, Siân E.; Gorelik, Julia

doi:10.1126/science.1185988

Cited by 512 publications

(585 citation statements)

References 29 publications

(3 reference statements)

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“…This suggests that in the targeted sensor, the end-point ratio in the bound state might be altered, for example, due to reduced conformational change in the sensor. An increase in local cAMP levels in the SERCA2a microdomain was not detectable after b 2 -AR stimulation, which is in line with previous studies showing no effect of b 2 -AR-cAMP on PLN phosphorylation 27 and its stringent compartmentation at the T-tubular membranes 28,29 . However, the relatively small b 2 -AR-cAMP signals are potentially difficult to resolve, especially using the less sensitive Epac1-PLN sensor, which is certainly a limitation of our study.…”

Section: Discussionsupporting

confidence: 91%

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

et al. 2015

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3 0 ,5 0 -cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced b-adrenergic receptor-cAMP signalling to SERCA.

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Section: Discussionsupporting

confidence: 91%

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

et al. 2015

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“…We then applied our method to the GABA B receptor, a prototypical family-C GPCR, consisting of heteromers between GABA B1 and GABA B2 subunits (10)(11)(12). Initially, we transfected CHO cells with N-terminally SNAPtagged GABA B1 and GABA B2 subunits (31). As expected, the SNAP-tagged GABA B1 subunit alone did not reach the cell surface, whereas the SNAP-tagged GABA B2 subunit alone was detectable on the cell surface, where it formed a mixture of mono-/di-/oligomers with high lateral mobility (Fig.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization

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Wagner

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the β 1 -adrenergic receptor (β 1 AR), the β 2 -adrenergic receptor (β 2 AR), and the γ-aminobutyric acid (GABA B ) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for β 1 ARs (monomers/dimers) and highest for GABA B receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas β 1 -/ β 2 ARs were apparently freely diffusing on the cell surface, GABA B receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA B receptor complexes. These data provide a spatiotemporal characterization of β 1 -/β 2 ARs and GABA B receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.live cell imaging | protein-protein interactions

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“…As the nanopipette-surface distance decreases, the solution resistance in the probe-surface gap increases which, in turn, reduces the ion current. This decrease in ion current is used as a non-contact signal to sense the nanopipette-surface distance and ultimately for topographical imaging, [14][15][16] proving particularly effective for soft samples. 10,12 SICM is typically operated in aqueous solutions with relatively high ionic strength.…”

Section: Introductionmentioning

confidence: 99%

Surface Charge Mapping with a Nanopipette

et al. 2014

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Nanopipettes are emerging as simple but powerful tools for probing chemistry at the nanoscale.In this contribution the use of nanopipettes for simultaneous surface charge mapping and topographical imaging is demonstrated, using a scanning ion conductance microscopy (SICM) format. When a nanopipette is positioned close to a surface in electrolyte solution the direct ion current (DC), driven by an applied bias between a quasi-reference counter electrode (QRCE) in the nanopipette and a second QRCE in the bulk solution is sensitive to surface charge. The charge sensitivity arises because the diffuse double layers at the nanopipette and the surface interact, creating a perm-selective region which becomes increasingly significant at low ionic strengths (10 mM 1:1 aqueous electrolyte herein). This leads to a polarity-dependent ion current and surface-induced rectification as the bias is varied. Using distance-modulated SICM, which induces an alternating ion current component (AC) by periodically modulating the distance between the nanopipette and the surface, the effect of surface charge on the DC and AC is explored and rationalized. The impact of surface charge on the AC phase (with respect to the driving sinusoidal signal) is highlighted in particular; this quantity shows a shift that is highly sensitive to interfacial charge and provides the basis for visualizing charge simultaneously with topography. The studies herein highlight the use of nanopipettes for functional imaging with applications from cell biology to materials characterization where understanding surface charge is of key importance.3

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β ₂ -Adrenergic Receptor Redistribution in Heart Failure Changes cAMP Compartmentation

Cited by 512 publications

References 29 publications

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization

Surface Charge Mapping with a Nanopipette

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β 2 -Adrenergic Receptor Redistribution in Heart Failure Changes cAMP Compartmentation

Cited by 512 publications

References 29 publications

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization

Surface Charge Mapping with a Nanopipette

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β ₂ -Adrenergic Receptor Redistribution in Heart Failure Changes cAMP Compartmentation