Signaling Microdomains Research Articles

A novel signalling role for NAADP in arterial smooth muscle

In arterial smooth muscle, Ca2+ is a pivotal modulator of vascular tone. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizer, releasing Ca2+ from lysosomal‐type acidic stores. NAADP can act as an agonist by binding to two pore channels, with the structural analogue Ned‐19 acting as an antagonist. We investigated the actions of NAADP in arterial smooth muscle using isolated rat mesenteric arteries. In pressurized arteries, confocal imaging of the intrinsic fluorescence of Ned‐19 demonstrated an overlap with a subpopulation of acidic organelles labeled with Lysotracker Green. In arteries mounted on the wire myograph, tone generated with the thromboxane mimetic U46619 was rapidly reversed when exposed to the cell‐permeant acetoxymethylester of NAADP (NAADP‐AM). This relaxation occurred in both endothelium‐intact and ‐denuded preparations, and was sensitive to an inhibitor of large‐conductance K+ channels (BKCa, iberiotoxin). Finally, we used immunohistochemistry in pressurized arteries to characterize the distribution of BKCa channels and lysosomal associated membrane protein (LAMP‐2). In conclusion, these data support a signalling microdomain coupling NAADP‐mediated Ca2+ release from acidic stores to opening of BKCa channels, suggesting a novel potential mechanism for modulation of vascular tone. Supported by the British Heart Foundation and Wellcome Trust, UK.

Multiscale computational models of microvascular reactivity from ion channels to intercellular signaling

Detailed compartmental, continuous, and multicellular computational models of smooth muscle (SM) and endothelial cell (EC) electrophysiology were developed and validated from patch clamp and calcium (Ca2+) imaging data. These models examine localized Ca2+ events from individual ion channels, role of signaling microdomains, effect of ion channel distribution, and homo‐ and hetero‐cellular signaling in regulation of vascular tone. Activation of TRPV4 channels in EC generates sparklets that activate colocalized IKCa channels and EDHF. EDHF is transmitted robustly by hyperpolarizing current through gap junctions, but less so by extracellular potassium. During SM stimulation, diffusion of IP3, and to smaller degree Ca2+, from SM to EC projections induces localized Ca2+ events and EDHF, which closes the myoendothelial feedback loop. Spatial heterogeneities in Ca2+ handling components generate oscillatory Ca2+ waves. In vasomotion, Ca2+ oscillations in SM can be synchronized by electrical or IP3 coupling through SM gap junctions. Conducted relaxation is mediated mainly by electrotonic spread of local hyperpolarization along the endothelium, but EC Ca2+ spread from the stimulus site and facilitation mechanism along the conduction can amplify the response. In conclusion, vasoreactivity involves complex signaling, which can be analyzed with the aid of computational models. Supported by NIH SC1HL95101.

Mitochondria in Hypoxic Pulmonary Vasoconstriction

Mitochondria in Hypoxic Pulmonary Vasoconstriction

Calcium signalling microdomains and the t-tubular system in atrial mycoytes: potential roles in cardiac disease and arrhythmias.

The atria contribute 25% to ventricular stroke volume and are the site of the commonest cardiac arrhythmia, atrial fibrillation (AF). The initiation of contraction in the atria is similar to that in the ventricle involving a systolic rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). There are, however, substantial inter-species differences in the way systolic Ca(2+) is regulated in atrial cells. These differences are a consequence of a well-developed and functionally relevant transverse (t)-tubule network in the atria of large mammals, including humans, and its virtual absence in smaller laboratory species such as the rat. Where T-tubules are absent, the systolic Ca(2+) transient results from a 'fire-diffuse-fire' sequential recruitment of Ca(2+) release sites from the cell edge to the centre and hence marked spatiotemporal heterogeneity of systolic Ca(2+). Conversely, the well-developed T-tubule network in large mammals ensures a near synchronous rise of [Ca(2+)](i). In addition to synchronizing the systolic rise of [Ca(2+)](i), the presence of T-tubules in the atria of large mammals, by virtue of localization of the L-type Ca(2+) channels and Na(+)-Ca(2+) exchanger antiporters on the T-tubules, may serve to, respectively, accelerate changes in the amplitude of the systolic Ca(2+) transient during inotropic manoeuvres and lower diastolic [Ca(2+)](i). On the other hand, the presence of T-tubules and hence wider cellular distribution of the Na(+)-Ca(2+) exchanger may predispose the atria of large mammals to Ca(2+)-dependent delayed afterdepolarizations (DADs); this may be a determining factor in why the atria of large mammals spontaneously develop and maintain AF.

Quantitative interactions between the A‐type K+ current and inositol trisphosphate receptors regulate intraneuronal Ca2+ waves and synaptic plasticity

The A-type potassium current has been implicated in the regulation of several physiological processes. Here, we explore a role for the A-type potassium current in regulating the release of calcium through inositol trisphosphate receptors (InsP3R) that reside on the endoplasmic reticulum (ER) of hippocampal pyramidal neurons. To do this, we constructed morphologically realistic, conductance-based models equipped with kinetic schemes that govern several calcium signalling modules and pathways, and constrained the distributions and properties of constitutive components by experimental measurements from these neurons. Employing these models, we establish a bell-shaped dependence of calcium release through InsP3Rs on the density of A-type potassium channels, during the propagation of an intraneuronal calcium wave initiated through established protocols. Exploring the sensitivities of calcium wave initiation and propagation to several underlying parameters, we found that ER calcium release critically depends on dendritic diameter and that wave initiation occurred at branch points as a consequence of a high surface area to volume ratio of oblique dendrites. Furthermore, analogous to the role of A-type potassium channels in regulating spike latency, we found that an increase in the density of A-type potassium channels led to increases in the latency and the temporal spread of a propagating calcium wave. Next, we incorporated kinetic models for the metabotropic glutamate receptor (mGluR) signalling components and a calcium-controlled plasticity rule into our model and demonstrate that the presence of mGluRs induced a leftward shift in a Bienenstock-Cooper-Munro-like synaptic plasticity profile. Finally, we show that the A-type potassium current could regulate the relative contribution of ER calcium to synaptic plasticity induced either through 900 pulses of various stimulus frequencies or through theta burst stimulation. Our results establish a novel form of interaction between active dendrites and the ER membrane, uncovering a powerful mechanism that could regulate biophysical/biochemical signal integration and steer the spatiotemporal spread of signalling microdomains through changes in dendritic excitability.

The Other (Muscarinic) Acetylcholine Receptors in Sympathetic Ganglia: Actions and Mechanisms

Acetylcholine released from preganglionic sympathetic fibers can activate two types of acetylcholine receptors in sympathetic neurons, nicotinic and muscarinic. The former are ligand-gated ion channels responsible for direct synaptic transmission; the latter are G protein-coupled receptors that mediate various indirect modulatory effects. Most mammalian sympathetic neurons express three muscarinic receptor subtypes, M1, M2, and M4; some also express M3 receptors. Activation of M1 receptors stimulates the G protein Gq and causes a slow postsynaptic depolarization and an increase in the excitability, ultimately leading to an asynchronous action potential discharge, which can “break through” the nicotinic ganglion block. This is largely mediated by closure of voltage-gated K+ channels (the M channels) composed of Kv7.2 and Kv7.3 subunits and results from hydrolysis and depletion of membrane phosphatidylinositol-4,5-bisphosphate. Activation of M2 receptors hyperpolarizes and inhibits the postsynaptic neuron by opening G protein-gated inwardlyrectifying Kir K+ channels via the G protein Gi. M4 receptors inhibit N-type (CaV(2)) calcium channels via the G protein Go. In the postganglionic neuron somata, this enhances the excitability by reducing calcium-dependent potassium currents. Conversely, in postganglionic processes and axon terminals, CaV(2)-mediated inhibition reduces norepinephrine release and inhibits postganglionic transmission. Different muscarinic receptors may be anatomically segregated with their cognate G proteins and (in some cases) ion channels in signalling microdomains.

Small-conductance Ca2+-activated K+ channels modulate action potential-induced Ca2+ transients in hippocampal neurons.

In hippocampal pyramidal neurons, voltage-gated Ca(2+) channels open in response to action potentials. This results in elevations in the intracellular concentration of Ca(2+) that are maximal in the proximal apical dendrites and decrease rapidly with distance from the soma. The control of these action potential-evoked Ca(2+) elevations is critical for the regulation of hippocampal neuronal activity. As part of Ca(2+) signaling microdomains, small-conductance Ca(2+)-activated K(+) (SK) channels have been shown to modulate the amplitude and duration of intracellular Ca(2+) signals by feedback regulation of synaptically activated Ca(2+) sources in small distal dendrites and dendritic spines, thus affecting synaptic plasticity in the hippocampus. In this study, we investigated the effect of the activation of SK channels on Ca(2+) transients specifically induced by action potentials in the proximal processes of hippocampal pyramidal neurons. Our results, obtained by using selective SK channel blockers and enhancers, show that SK channels act in a feedback loop, in which their activation by Ca(2+) entering mainly through L-type voltage-gated Ca(2+) channels leads to a reduction in the subsequent dendritic influx of Ca(2+). This underscores a new role of SK channels in the proximal apical dendrite of hippocampal pyramidal neurons.

Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

A-kinase anchoring proteins (AKAPs) bind the regulatory subunits of protein kinase A (PKA) and localize the holoenzyme to discrete signaling microdomains in multiple subcellular compartments. Despite emerging evidence for a nuclear pool of PKA that rapidly responds to activation of the PKA signaling cascade, only a few AKAPs have been identified that localize to the nucleus. Here we show a PKA-binding domain in the amino terminus of Chd8, and demonstrate subcellular colocalization of Chd8 with RII. RII overlay and immunoprecipitation assays demonstrate binding between Chd8-S and RIIα. Binding is abrogated upon dephosphorylation of RIIα. By immunofluorescence, we identified nuclear and perinuclear pools of Chd8 in HeLa cells and rat neonatal cardiomyocytes. We also show high levels of Chd8 mRNA in RNA extracted from post-natal rat hearts. These data add Chd8 to the short list of known nuclear AKAPs, and implicate a function for Chd8 in post-natal rat cardiac development.

Scaffolding Proteins of the Post-synaptic Density Contribute to Synaptic Plasticity by Regulating Receptor Localization and Distribution: Relevance for Neuropsychiatric Diseases

Synaptic plasticity represents the long lasting activity-related strengthening or weakening of synaptic transmission, whose well-characterized types are the long term potentiation and depression. Despite this classical definition, however, the molecular mechanisms by which synaptic plasticity may occur appear to be extremely complex and various. The post-synaptic density (PSD) of glutamatergic synapses is a major site for synaptic plasticity processes and alterations of PSD members have been recently implicated in neuropsychiatric diseases where an impairment of synaptic plasticity has also been reported. Among PSD members, scaffolding proteins have been demonstrated to bridge surface receptors with their intracellular effectors and to regulate receptors distribution and localization both at surface membranes and within the PSD. This review will focus on the molecular physiology and pathophysiology of synaptic plasticity processes, which are tuned by scaffolding PSD proteins and their close related partners, through the modulation of receptor localization and distribution at post-synaptic sites. We suggest that, by regulating both the compartmentalization of receptors along surface membrane and their degradation as well as by modulating receptor trafficking into the PSD, postsynaptic scaffolding proteins may contribute to form distinct signaling micro-domains, whose efficacy in transmitting synaptic signals depends on the dynamic stability of the scaffold, which in turn is provided by relative amounts and post-translational modifications of scaffolding members. The putative relevance for neuropsychiatric diseases and possible pathophysiological mechanisms are discussed in the last part of this work.

Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure to be published in May, 2013

Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure to be published in May, 2013

Announcement: Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure: to be published in May, 2013

Announcement: Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure: to be published in May, 2013

Abstract 111: High Activity Gating of Caveolae-Targeted L-Type Ca 2+ Channels Can Initiate Pathological Hypertrophy

Introduction: The source of Ca 2+ that activates pathological cardiac hypertrophy is not clearly defined. We hypothesize that high activity gating of L-type Ca 2+ channels (LTCCs) localized in caveolae signaling microdomains stabilized by caveolin-3 (Cav-3) provides the Ca 2+ influx that locally activates calcineurin (Cn)-mediated nuclear factor of activated T-cells (NFAT) to induce hypertrophic gene expression. We generated novel reagents that specifically activate or inhibit the gating of LTCCs in caveolae for analysis of the hypertrophic program, as well as any potential effect on ICa and contraction. Methods and Results: We targeted the known LTCC inhibitory protein Rem or an LTCC subunit (β2a) that promotes high activity gating specifically to caveolae by fusing them to a caveolin-binding domain peptide (termed Cav-Rem and Cav-β2a). We infected adult feline left ventricular myocytes (AFLVMs) with adenoviruses containing Cav-Rem or Cav-β2a for membrane localization and functional studies. NFAT nuclear translocation was determined by co-infecting AFLVMs with ad-NFAT-GFP and either ad-Cav-Rem or ad-Cav-β2a and pacing cells to induce Ca2+ influx mediated nuclear NFAT-GFP translocation. Membrane fractionation experiments showed that Cav-3 membrane domains contain 26.2 +/- 12.7% of membrane targeted LTCCs and blocking these with Cav-Rem eliminated a small fraction of the LTCC current (<15%) and almost all Ca2+ influx induced NFAT nuclear translocation (>90%), but did not reduce myocyte contractility. Conversely, selective enhancement of LTCC activity within caveolae with Cav-β2a caused a significant increase in NFAT nuclear translocation (>70%) but had no significant effect on contractility. Conclusions: We provide proof of concept that specific Cav-targeted reagents can be used to enhance or inhibit LTCC activity within caveolae microdomains to amplify or block the hypertrophic response. Our results suggest that high activity gating of LTCCs, which is known to be present in the hypertrophied failing human heart, can activate signaling pathways linked to cardiac hypertrophy. Selectively inhibiting these caveolae localized LTCCs could be a novel mechanism to block pathological hypertrophy without reducing cardiac contractility.

Announcement: Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure

Announcement: Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure

Announcement: Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure

Announcement: Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure

Myoendothelial Contacts, Gap Junctions, and Microdomains: Anatomical Links to Function?

In several species and in many vascular beds, ultrastructural studies describe close contact sites between the endothelium and smooth muscle of <∼20nm. Such sites are thought to facilitate the local action of signaling molecules and/or the passage of current, as metabolic and electrical coupling conduits between the arterial endothelium and smooth muscle. These sites have the potential for bidirectional communication between the endothelium and smooth muscle, as a key pathway for coordinating vascular function. The aim of this brief review is to summarize the literature on the ultrastructural anatomy and distribution of key components of MECC sites in arteries. In addition to their traditional role of facilitating electrical coupling between the two cell layers, data on the role of MECC sites in arteries, as signaling microdomains involving a spatial localization of channels, receptors and calcium stores are highlighted. Diversity in the density and specific characteristics of MECC sites as signaling microdomains suggests considerable potential for functional diversity within and between arteries in health and disease.

Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity

Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery. TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SK(Ca) (apamin) or IK(Ca) (TRAM-34) block, Pyr3 abolished the residual respective IK(Ca)- and SK(Ca)-dependent EDH-mediated vasodilation. The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.

Announcement: Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure

Announcement: Spotlight on T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure

Dynamics of receptor trafficking in tumorigenicity

The transport and sorting of cell surface receptors into membrane-bound intracellular compartments is crucial for cellular homeostasis. Defects in receptor trafficking are associated with several diseases, including cancer. Recent advances in our understanding of mechanisms that control receptor trafficking have highlighted the involvement of membrane trafficking in cell signaling, as well as in biological processes, including cell migration and invasion. In this review, we summarize current knowledge of how cargos, focusing on receptor tyrosine kinases (RTKs) and integrins, are dynamically transported through the endosomal pathway for recycling, and how this promotes spatially restricted signaling microdomains associated with distinct biological responses. We discuss mechanisms through which dysregulation of membrane trafficking contributes to tumorigenesis and potential therapeutic approaches.

A Caveolae-Targeted L-Type Ca 2+ Channel Antagonist Inhibits Hypertrophic Signaling Without Reducing Cardiac Contractility

The source of Ca(2+) to activate pathological cardiac hypertrophy is not clearly defined. Ca(2+) influx through the L-type Ca(2+) channels (LTCCs) determines "contractile" Ca(2+), which is not thought to be the source of "hypertrophic" Ca(2+). However, some LTCCs are housed in caveolin-3 (Cav-3)-enriched signaling microdomains and are not directly involved in contraction. The function of these LTCCs is unknown. To test the idea that LTCCs in Cav-3-containing signaling domains are a source of Ca(2+) to activate the calcineurin-nuclear factor of activated T-cell signaling cascade that promotes pathological hypertrophy. We developed reagents that targeted Ca(2+) channel-blocking Rem proteins to Cav-3-containing membranes, which house a small fraction of cardiac LTCCs. Blocking LTCCs within this Cav-3 membrane domain eliminated a small fraction of the LTCC current and almost all of the Ca(2+) influx-induced NFAT nuclear translocation, but it did not reduce myocyte contractility. We provide proof of concept that Ca(2+) influx through LTCCs within caveolae signaling domains can activate "hypertrophic" signaling, and this Ca(2+) influx can be selectively blocked without reducing cardiac contractility.

Organization of cAMP signalling microdomains for optimal regulation by Ca2+ entry

Cross-talk between cAMP and Ca2+ signalling pathways plays a critical role in cellular homoeostasis. Several AC (adenylate cyclase) isoforms, catalysing the production of cAMP from ATP, display sensitivity to submicromolar changes in intracellular Ca2+ and, as a consequence, are key sites for Ca2+ and cAMP interplay. Interestingly, these Ca2+-regulated ACs are not equally responsive to equivalent Ca2+ rises within the cell, but display a remarkable selectivity for regulation by SOCE (store-operated Ca2+ entry). Over the years, considerable efforts at investigating this phenomenon have provided indirect evidence of an intimate association between Ca2+-sensitive AC isoforms and sites of SOCE. Now, recent identification of the molecular components of SOCE [namely STIM1 (stromal interaction molecule 1) and Orai1], coupled with significant advances in the generation of high-resolution targeted biosensors for Ca2+ and cAMP, have provided the first detailed insight into the organization of the cellular microdomains associated with Ca2+-regulated ACs. In the present review, I summarize the findings that have helped to provide our most definitive understanding of the selective regulation of cAMP signalling by SOCE.