Caveolae and Lipid Rafts: G Protein‐Coupled Receptor Signaling Microdomains in Cardiac Myocytes

Insel, Paul A.; Head, Brian P.; Ostrom, Rennolds S.; Patel, Hemal H.; Swaney, James S.; Tang, Chih‐Min; Roth, David M.

doi:10.1196/annals.1341.015

Cited by 120 publications

(95 citation statements)

References 25 publications

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“…Because it is well-recognized that G s ␣ has a multiplicity of effects in vivo (9,11,12,19,22), we tested whether G s ␣-Cav-3-independent mechanisms existed. Previous work on G protein and caveolin interactions has focused primarily on Cav-1; however, G s ␣ localization to Cav-3 has also been reported (11,12).…”

Section: Discussionmentioning

confidence: 99%

Regulation of caveolar cardiac sodium current by a single G_sα histidine residue

Palygin¹,

Pettus²,

Shibata³

2008

American Journal of Physiology-Heart and Circulatory Physiology

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Palygin OA, Pettus JM, Shibata EF. Regulation of caveolar cardiac sodium current by a single Gs␣ histidine residue. Am J Physiol Heart Circ Physiol 294: H1693-H1699, 2008. First published February 8, 2008 doi:10.1152/ajpheart.01337.2007.-Cardiac sodium channels (voltage-gated Na ϩ channel subunit 1.5) reside in both the plasmalemma and membrane invaginations called caveolae. Opening of the caveolar neck permits resident channels to become functional. In cardiac myocytes, caveolar opening can be stimulated by applying ␤-receptor agonists, which initiates an interaction between the stimulatory G protein subunit-␣ (Gs␣) and caveolin-3. This study shows that, in adult rat ventricular myocytes, a functional Gs␣-caveolin-3 interaction occurs, even in the absence of the caveolin-binding sequence motif of Gs␣. Consistent with previous data, whole cell experiments conducted in the presence of intracellular PKA inhibitor stimulation with ␤-receptor agonists increased the sodium current (INa) by 35.9 Ϯ 8.6% (P Ͻ 0.05), and this increase was mimicked by application of Gs␣ protein. Inclusion of anti-caveolin-3 antibody abolished this effect. These findings suggest that Gs␣ and caveolin-3 are components of a PKA-independent pathway that leads to the enhancement of INa. In this study, alanine scanning mutagenesis of Gs␣ (40THR42), in conjunction with voltage-clamp studies, demonstrated that the histidine residue at position 41 of Gs␣ (H41) is a critical residue for the functional increase of INa. Protein interaction assays suggest that Gs␣FL (full length) binds to caveolin-3, but the enhancement of INa is observed only in the presence of Gs␣ H41. We conclude that Gs␣ H41 is a critical residue in the regulation of the increase in INa in ventricular myocytes.

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Section: Discussionmentioning

confidence: 99%

Regulation of caveolar cardiac sodium current by a single G_sα histidine residue

Palygin¹,

Pettus²,

Shibata³

2008

American Journal of Physiology-Heart and Circulatory Physiology

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“…The first is the subsarcolemmal space associated with the caveolar membrane domains of the cell. The key signaling elements included in that compartment have been found in cholesterol-rich membrane fractions that are associated with caveolin 3, the muscle specific form of caveolin that is involved in creating signaling complexes necessary for producing functional responses (13,21,30). This compartment is assumed to include ϳ1% of the total cytosolic volume and 10% of the plasma membrane surface area (4).…”

Section: Methodsmentioning

confidence: 99%

Cytoplasmic cAMP concentrations in intact cardiac myocytes

Iancu

Gopalakrishnan

Warrier

et al. 2008

American Journal of Physiology-Cell Physiology

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.-In cardiac myocytes there is evidence that activation of some receptors can regulate protein kinase A (PKA)-dependent responses by stimulating cAMP production that is limited to discrete intracellular domains. We previously developed a computational model of compartmentalized cAMP signaling to investigate the feasibility of this idea. The model was able to reproduce experimental results demonstrating that both ␤ 1-adrenergic and M2 muscarinic receptor-mediated cAMP changes occur in microdomains associated with PKA signaling. However, the model also suggested that the cAMP concentration throughout most of the cell could be significantly higher than that found in PKA-signaling domains. In the present study we tested this counterintuitive hypothesis using a freely diffusible fluorescence resonance energy transferbased biosensor constructed from the type 2 exchange protein activated by cAMP (Epac2-camps). It was determined that in adult ventricular myocytes the basal cAMP concentration detected by the probe is ϳ1.2 M, which is high enough to maximally activate PKA. Furthermore, the probe detected responses produced by both ␤1 and M2 receptor activation. Modeling suggests that responses detected by Epac2-camps mainly reflect what is happening in a bulk cytosolic compartment with little contribution from microdomains where PKA signaling occurs. These results support the conclusion that even though ␤1 and M2 receptor activation can produce global changes in cAMP, compartmentation plays an important role by maintaining microdomains where cAMP levels are significantly below that found throughout most of the cell. This allows receptor stimulation to regulate cAMP activity over concentration ranges appropriate for modulating both higher (e.g., PKA) and lower affinity (e.g., Epac) effectors.␤-adrenergic receptor signaling; muscarinic receptor signaling; live cell imaging; fluorescence resonance energy transfer; biosensors MANY DIFFERENT NEUROTRANSMITTERS and hormones control a wide range of cellular processes by regulating the production of a common second messenger, cAMP (32). This signaling pathway plays a particularly important role in sympathetic regulation of cardiac function, where ␤ 1 -adrenergic receptor (␤ 1 AR) activation generates significant changes in the electrical, mechanical, and metabolic properties of the heart by stimulating the production of cAMP and activating protein kinase A (PKA) (4). Although other G protein-coupled receptors are also able to affect cAMP production in the heart, they do not all produce the same responses. This has led to the hypothesis that cAMP production is compartmentalized and that different receptors can regulate cAMP production in specific microdomains (31).The recent development of several different biosensors capable of monitoring cAMP activity in intact living cells has provided a means of obtaining more direct proof that compartmentation occurs (20,33,37). Some of these sensors are targeted to specific subcellular locations. This includes the fluorescence resonance energ...

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“…However, the plasma membrane compartment of these cells is not homogeneous but rather divided into subcompartments, including caveolae and non-caveolar lipid rafts, which contain distinct sets of receptors and signaling proteins (Insel et al, 2005). To further characterize the membrane subcompartment where PDE4B is active, we determined whether PDE4B ablation affects the signaling of different G-protein-coupled receptors (GPCRs).…”

Section: Pde4b Controls B 1 Ar But Not B 2 Ar or Epr Responses In Carmentioning

confidence: 99%

PDE4B mediates local feedback regulation of β1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes

Mika

Richter

Westenbroek

et al. 2014

Journal of Cell Science

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Multiple cAMP phosphodiesterase (PDE) isoforms play divergent roles in cardiac homeostasis but the molecular basis for their nonredundant function remains poorly understood. Here, we report a novel role for the PDE4B isoform in b-adrenergic (bAR) signaling in the heart. Genetic ablation of PDE4B disrupted bAR-induced cAMP transients, as measured by FRETsensors, at the sarcolemma but not in the bulk cytosol of cardiomyocytes. This effect was further restricted to a subsarcolemmal compartment because PDE4B regulates b 1 AR-, but not b 2 AR-or PGE2-induced responses. The spatially restricted function of PDE4B was confirmed by its selective effects on PKA-mediated phosphorylation patterns. PDE4B limited the PKA-mediated phosphorylation of key players in excitationcontraction coupling that reside in the sarcolemmal compartment, including L-type Ca 2+ channels and ryanodine receptors, but not phosphorylation of distal cytosolic proteins. b 1 AR-but not b 2 ARligation induced PKA-dependent activation of PDE4B and interruption of this negative feedback with PKA inhibitors increased sarcolemmal cAMP. Thus, PDE4B mediates a crucial PKAdependent feedback that controls b 1 AR-dependent cAMP signals in a restricted subsarcolemmal domain. Disruption of this feedback augments local cAMP/PKA signals, leading to an increased intracellular Ca 2+ level and contraction rate.

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Caveolae and Lipid Rafts: G Protein‐Coupled Receptor Signaling Microdomains in Cardiac Myocytes

Cited by 120 publications

References 25 publications

Regulation of caveolar cardiac sodium current by a single G_sα histidine residue

Regulation of caveolar cardiac sodium current by a single G_sα histidine residue

Cytoplasmic cAMP concentrations in intact cardiac myocytes

PDE4B mediates local feedback regulation of β1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes

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Caveolae and Lipid Rafts: G Protein‐Coupled Receptor Signaling Microdomains in Cardiac Myocytes

Cited by 120 publications

References 25 publications

Regulation of caveolar cardiac sodium current by a single Gsα histidine residue

Regulation of caveolar cardiac sodium current by a single Gsα histidine residue

Cytoplasmic cAMP concentrations in intact cardiac myocytes

PDE4B mediates local feedback regulation of β1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes

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Regulation of caveolar cardiac sodium current by a single G_sα histidine residue

Regulation of caveolar cardiac sodium current by a single G_sα histidine residue