Dataset Information

Molecular basis of b-adrenergic regulation of cardiac calcium current

ABSTRACT: Increased cardiac contractility during fight-or-flight response is caused by b-adrenergic augmentation of CaV1.2 channels. It is assumed that this iconic regulation involves phosphorylation of CaV1.2 a1/b-subunits. In transgenic murine hearts expressing fully PKA phosphorylation-deficient mutant a1C/b, this regulation persists, however, suggesting involvement of extra-channel factors. We here show that PKA up-regulates cardiac CaV1.2 channels by phosphorylating the small G-protein Rad, relieving constitutive inhibition of CaV1.2. Peroxidase-catalyzed labeling in mice hearts expressing ascorbate peroxidase conjugated-a1C or b2b with multiplexed quantitative proteomics allowed tracking of thousands of proteins in proximity of CaV1.2.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Larimichthys crocea Xanthomonas oryzae pv. oryzae PXO99A Peniophora incarnata Lysiphlebus fabarum Lentinus crinitus Rhodopseudomonas palustris Coxiella burnetii RSA 493 Stenotrophomonas maltophilia Aspergillus nidulans TN02A3 Nelumbo nucifera Bothrops neuwiedi Actinomycetia Medicago truncatula Pyrococcus abyssi Staphylococcus sp. 1 Tetrahymena thermophila Bacillus subtilis subsp. subtilis str. 168 Oreochromis mossambicus Flavobacterium columnare Burkholderia cenocepacia Rhodococcus jostii RHA1 Aves Desulfovibrio ferrophilus Giardia intestinalis Struthio camelus Schistosoma mansoni Muvirus mu Trypanosoma Streptococcus pneumoniae TIGR4 Sus scrofa Desulfovibrio sp. G11 Lens culinaris Phytophthora capsici Bubalus bubalis Meleagris gallopavo Sciuridae Mus musculus Giardia intestinalis assemblage A Leishmania donovani Paramoeba perurans Cavia porcellus Thermochaetoides thermophila DSM 1495 Streptococcus equi subsp. equi 4047 Candidatus Nitrospira inopinata Pachysolen tannophilus NRRL Y-2460 Pseudomonas putida F1 Leishmania major strain Friedlin Eulimnogammarus cyaneus Capsicum annuum Phytophthora sojae Dirofilaria immitis Leishmania braziliensis MHOM/BR/75/M2904 Boehmeria nivea Ophrys sphegodes Cupiennius salei Phaseolus vulgaris Streptomyces coelicolor Gorilla gorilla Plasmodium knowlesi strain H Streptococcus agalactiae FSL S3-026 Taeniopygia guttata Pseudomonas syringae Scolopendra morsitans Gossypium hirsutum Cervus nippon nippon rhizosphere soil bacteria ensemble Saccharopolyspora erythraea Vitis riparia Latrodectus hesperus Clostridioides difficile 630 Cercopithecidae Entamoeba histolytica HM-1:IMSS-A Saccharomyces cerevisiae Human immunodeficiency virus Strigamia maritima Paramecium tetraurelia Hordeum vulgare Bicyclus anynana Pyricularia oryzae Pectobacterium carotovorum Cyanophora paradoxa Clostridium butyricum E4 str. BoNT E BL5262 Coprococcus eutactus Oryza sativa Japonica Group Candidatus Accumulibacter phosphatis Salmo salar Lingula anatina Helicobacter pylori J99 Lagomorpha Pyrococcus furiosus Lotus japonicus Colinus virginianus Pan troglodytes Theobroma cacao Haemophilus influenzae Sphingomonas sp. Leaf257 Human alphaherpesvirus 1 strain 17 Zymomonas mobilis Anopheles culicifacies A Tuber albidum Pseudomonas pseudoalcaligenes CECT 5344 Bacillus subtilis subsp. subtilis NCIB 3610 = ATCC 6051 = DSM 10 Mustela putorius furo Oncorhynchus tshawytscha Crithidia fasciculata Arachis hypogaea Riptortus pedestris Plasmodium sp. pBT9 Coffea eugenioides Olavius algarvensis Gamma 3 endosymbiont Physcomitrella Lactococcus lactis subsp. cremoris MG1363 Cyclopes didactylus Human alphaherpesvirus 1 strain KOS soil bacterium TS1114R Zea mays Brugia malayi Lactococcus lactis subsp. cremoris UC509.9 Coffea canephora Bothrops jararacussu Phytophthora capsici LT1534 Anopheles culicifacies B Bartonella quintana JK 31 Thalassiosira pseudonana CCMP1335 Ficus carica Human immunodeficiency virus 1 Globicatella sanguinis Crassostrea hongkongensis Tapirus terrestris Olavius algarvensis Delta 1 endosymbiont Severe acute respiratory syndrome coronavirus 2 Cytomegalovirus Rhizobium cf. leguminosarum Haptophyta Thermochaetoides thermophila Rattus norvegicus Homo sapiens subsp. 'Denisova' Betaproteobacteria bacterium RIFCSPLOWO2_02_64_14 [Ruminococcus] gnavus Emiliania huxleyi Pseudomonas putida KT2440 Elaeis guineensis Hendra virus horse/Australia/Hendra/1994 Panicum virgatum Fusarium graminearum GZ3639 Bothrops brazili Corynebacterium glutamicum ATCC 13032 Mycobacterium tuberculosis SP7 Brassica oleracea var. botrytis Crassostrea gigas Bothrops jararaca Halobacterium salinarum Cleome angustifolia Mycolicibacterium smegmatis MC2 155 Escherichia coli O157:NM Arabidopsis thaliana Mycobacterium tuberculosis SP10 Acinetobacter baumannii D1279779 Anopheles stephensi Tuber melanosporum Mel28 Pteropus alecto Capsicum annuum var. annuum Escherichia coli O157:H7 Histomonas meleagridis Anas platyrhynchos Plasmodium falciparum 3D7 Streptococcus pneumoniae R6 Klebsiella pneumoniae Thalassiosira cf. oceanica Apteronotus leptorhynchus Lactobacillus Deinococcus radiodurans R1 Staphylococcus aureus Callinectes sapidus Naja sagittifera Emiliania Faecalibacterium prausnitzii human metagenome Vitis vinifera Oryctolagus cuniculus Brassica napus Brucella melitensis Chlorella sp. SAG 211-18 Burkholderia ubonensis MSMB22 Noccaea caerulescens Pseudotsuga menziesii var. menziesii Enterococcus faecalis Bacillus subtilis subsp. spizizenii ATCC 6633 = JCM 2499 Coffea arabica Bodo saltans Craterostigmus tasmanianus Thermoplasma acidophilum DSM 1728 Archaea Ophrys garganica Acyrthosiphon pisum Capra hircus Mycoplasma gallisepticum S6 Dasypogon diadema Escherichia coli O157:H7 str. TW14359 Canis lupus x Canis lupus familiaris Bothrops moojeni Echinococcus granulosus Rhizophagus irregularis Geobacter sulfurreducens Acinetobacter baumannii ATCC 19606 = CIP 70.34 = JCM 6841 Trypanosoma brucei brucei Pseudomonas aeruginosa PAO1 Mycoplasma pneumoniae M129 Pseudomonas fluorescens SBW25 Penaeus vannamei Ovis aries Olavius algarvensis sulfate-reducing endosymbiont Spirometra erinaceieuropaei Olavius algarvensis Gamma 1 endosymbiont Euzebya tangerina Amaranthus tricolor Dioscorea alata Cryptococcus neoformans var. grubii H99W Ensifer adhaerens Synechocystis sp. PCC 6803 substr. Kazusa Dialister Aspergillus niger NRRL3 Schizosaccharomyces pombe OY26 Botrytis cinerea Roseburia Apostichopus japonicus Candida albicans Cervus elaphus elaphus Gynandropsis gynandra Chlamydomonas reinhardtii Parasynechococcus marenigrum WH 8102 Bothrops leucurus Trypanosoma brucei brucei TREU927 Mycolicibacterium smegmatis Bradyrhizobium japonicum Dentex dentex Allexis Olavius algarvensis Delta 4 endosymbiont Bombyx mori Solanum lycopersicum Palaemonetes sp. common grass shrimp associated circular virus Exiguobacterium sp. SH31 Synechocystis sp. PCC 6803 Brassica oleracea var. gemmifera Eukaryota Apis mellifera ligustica Trichechus manatus Chromatiales bacterium OalgGamma1 Deltaproteobacteria bacterium RIFCSPLOWO2_12_FULL_50_11 Pseudomonas stutzeri Channa striata Bothrops cotiara Malassezia sympodialis ATCC 42132 Ictalurid herpesvirus 1 Picea abies unclassified Marseilleviridae Medicago sativa environmental samples Euprymna scolopes Schizosaccharomyces pombe 927 Gammarus fossarum Calicophoron daubneyi Equus caballus Marchantia polymorpha subsp. ruderalis Chlamydia trachomatis D/UW-3/CX Ruminiclostridium papyrosolvens DSM 2782 Photobacterium profundum SS9 Mycobacterium tuberculosis SP1 Oryzias latipes Tequatrovirus T4 Tuber magnatum Daphnia magna environmental samples Vibrio natriegens NBRC 15636 = ATCC 14048 = DSM 759 Macaca mulatta Aspergillus flavus NRRL3357 Eremothecium gossypii Bacteria Medicago truncatula var. truncatula Vibrio anguillarum Entamoeba histolytica Bovine leukemia virus Clostridioides difficile R20291 Ictidomys tridecemlineatus Schizophyllum commune H4-8 Pseudomonas putida BIRD-1 Aedes aegypti aegypti Glycine max Hippopotamus amphibius Moraxella catarrhalis Paracoccus denitrificans PD1222 Orycteropus afer (Vitis vinifera x Vitis berlandieri) x Vitis berlandieri Strigomonas culicis Schizosaccharomyces pombe Hydra vulgaris Rattus rattus Chlorocebus sabaeus Candidatus Thioglobus autotrophicus Daphnia pulex Acinetobacter baumannii Mycobacterium tuberculosis H37Rv Schizosaccharomyces pombe DM3650 Triticum aestivum Ceratopteris richardii environmental samples Tradescantia spathacea Sus scrofa domesticus Mycobacterium tuberculosis SP22 Clavibacter michiganensis subsp. nebraskensis NCPPB 2581 Vicia faba Nicotiana tabacum Listeria monocytogenes Mesocricetus auratus Bursaphelenchus xylophilus Acanthoscurria geniculata Ctenopharyngodon idella Trichoderma sp. 1 YW-2011 Acipenser transmontanus Neurospora crassa Bacillus subtilis Olavius algarvensis Bartonella quintana Human alphaherpesvirus 1 Granulicella sp. Cyclotella cryptica Oreochromis niloticus niloticus Angiostrongylus cantonensis Dendroaspis polylepis polylepis Herpes simplex virus unknown type Acidithiobacillus ferrooxidans ATCC 53993 Scutigera coleoptrata Sorghum bicolor Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 Paucilactobacillus suebicus Potentilla saundersiana Avena strigosa unidentified Ostreococcus tauri Escherichia coli Cecropia peltata Onchocerca volvulus albimanus section Populus tremula x Populus alba Coronavirus PREDICT CoV-2 Giardia lamblia ATCC 50803 Oryza sativa Indica Group Cryptochiton stelleri Lactobacillus johnsonii N6.2 Streptomyces coelicolor A3(2) Bothrops insularis Schizosaccharomyces pombe 972h- Human herpesvirus 5 strain AD169 Piper nigrum Phytophthora infestans Streptomyces avidinii Citrobacter rodentium Enterobacter cloacae Bordetella pertussis Tohama I Enterococcus mundtii CRL35 Plasmodium falciparum uncultured marine microorganism Trypanosoma cruzi Bradyrhizobium diazoefficiens USDA 110 Campylobacter fetus subsp. fetus 82-40 Synechococcus elongatus Hepatitis E virus type 1 Naja naja Trichoderma reesei QM9414 Aspergillus niger Olavius algarvensis spirochete endosymbiont Sedum plumbizincicola Trypanosoma brucei Lacticaseibacillus rhamnosus GG Dendroaspis angusticeps Paenibacillus sp. Saccharum hybrid cultivar Sinorhizobium meliloti BL225C Leishmania major Candida albicans SC5314 Oryctolagus cuniculus algirus Blumeria hordei DH14 Spirochaeta sp. ELBA Streptococcus dysgalactiae subsp. equisimilis SK1249 Vitis Acinetobacter baumannii ACICU Human herpesvirus 2 strain 186 Oryctolagus cuniculus cuniculus [Candida] glabrata Lactococcus phage Toxoplasma gondii type III Physcomitrium patens Burkholderia cenocepacia K56-2Valvano Clostridium autoethanogenum Hebomoia glaucippe Nephila clavipes Alteromonadales Streptococcus pneumoniae SP14-BS69 Xenopus laevis Mycobacterium tuberculosis SP13 anaerobic digester metagenome Naja mandalayensis Tarenaya hassleriana Brachyspiraceae Paracoccidioides brasiliensis Pb18 Streptococcus pneumoniae D39 hybrid Macaca fascicularis Microcystis aeruginosa Phytophthora pisi Oncorhynchus mykiss Acanthamoeba castellanii Burkholderia sp. 2332 Ursus arctos arctos Leishmania infantum JPCM5 Chlorocebus aethiops Equus asinus Morchella importuna Helianthus annuus Aerococcus urinae NBRC 15544 = CCUG 36881 Brucella abortus Mycoplasma pneumoniae M129-B7 Mycoplasmopsis agalactiae Tuber indicum Austrolebias Chlamydia trachomatis 434/Bu Fasciola hepatica Apis mellifera Bos taurus Viruses Drosophila melanogaster Catenibacterium Pyricularia oryzae B157 Teleogryllus oceanicus Acanthoscurria gomesiana Camelus dromedarius Populus yunnanensis Pseudomonas aeruginosa Ustilago maydis Cricetulus griseus Mytilus edulis Sphingobium chlorophenolicum Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044 Passiflora edulis Heloderma suspectum suspectum Cryptococcus neoformans var. grubii H99 Caenorhabditis elegans Daktulosphaira vitifoliae Mengo virus Oryza sativa Beta vulgaris subsp. vulgaris mixed culture metagenome Drosophila Rhizobium sp. Leaf68 Populus trichocarpa Strawberry leafy fruit phytoplasma Vibrio cholerae O1 biovar El Tor str. N16961 Cupriavidus metallidurans Trichoderma reesei RUT C-30 Scophthalmus maximus Brassica oleracea var. italica Octopodiformes Listeria monocytogenes EGD-e Ophrys x arachnitiformis subsp. archipelagi Tuber aestivum Mycobacterium tuberculosis Brucella Gallus gallus Salmo sp. Homo sapiens Trichomonas gallinae Aspergillus flavus Malus domestica Helicobacter pylori 26695 Mylodon darwinii Toxoplasma gondii Butyrivibrio fibrisolvens Mycobacterium marinum ATCC BAA-535 Lithobius forficatus Mycobacterium avium subsp. paratuberculosis K-10 Canis lupus familiaris Dioscorea oppositifolia Staphylococcus haemolyticus Danio rerio Ascaris suum Acinetobacter baumannii AB307-0294 Quercus ilex

SUBMITTER: Marian Kalocsay

LAB HEAD: Marian Kalocsay

PROVIDER: PXD014500 | Pride | 2019-11-19

REPOSITORIES: Pride

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Publications

Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Liu Guoxia G Papa Arianne A Katchman Alexander N AN Zakharov Sergey I SI Roybal Daniel D Hennessey Jessica A JA Kushner Jared J Yang Lin L Chen Bi-Xing BX Kushnir Alexander A Dangas Katerina K Gygi Steven P SP Pitt Geoffrey S GS Colecraft Henry M HM Ben-Johny Manu M Kalocsay Marian M Marx Steven O SO

Nature 20200122 7792

Increased cardiac contractility during the fight-or-flight response is caused by β-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and β subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of β-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mec ...[more]

PMID: 31969708

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Project description:KEY POINTS:Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca2+ ] in a biphasic manner. Submicromolar [Ca2+ ] activates the channel via constitutively-bound calmodulin, whereas higher [Ca2+ ] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site-directed mutagenesis, we identified serine-465 as the site conferring PKA-dependent effects on SK2 channel function. PKA phosphorylation attenuates ISK rectification by reducing the Ca2+ /voltage-dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+ ]i . This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. ABSTRACT:Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post-translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease-related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti-pan-serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 ?mol L-1 ). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L-1 ), which was eliminated by inhibition of PKA (1 ?mol L-1 ). Beta-adrenergic stimulation (isoproterenol, 100 nmol L-1 ) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild-type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine-465 as the site that elicits PKA-dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+ /voltage-dependent inhibition of the channels at high [Ca2+ ] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA-dependent phosphorylation at serine-465.

Project description:We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.

Project description:Activation of the protease-activated receptor 2 (PAR2) or the transient receptor potential vanilloid type 1 (TRPV1) channels expressed in cardiac sensory afferents containing calcitonin gene-related peptide (CGRP) and/or substance P (SP) has been proposed to play a protective role in myocardial ischemia-reperfusion (I/R) injury. However, the interaction between PAR2 and TRPV1 is largely unknown. Using gene-targeted TRPV1-null mutant (TRPV1(-/-)) or wild-type (WT) mice, we test the hypothesis that TRPV1 contributes to PAR2-mediated cardiac protection via increasing the release of CGRP and SP. Immunofluorescence labeling showed that TRPV1 coexpressed with PAR2, PKC-epsilon, or PKAc in cardiomyocytes, cardiac blood vessels, and perivascular nerves in WT but not TRPV1(-/-) hearts. WT or TRPV1(-/-) hearts were Langendorff perfused with the selective PAR2 agonist, SLIGRL, in the presence or absence of various antagonists, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). The recovery rate of coronary flow, the maximum rate of left ventricular pressure development, left ventricular end-diastolic pressure, and left ventricular developed pressure were evaluated after I/R. SLIGRL improved the recovery of hemodynamic parameters, decreased lactate dehydrogenase release, and reduced the infarct size in both WT and TRPV1(-/-) hearts (P < 0.05). The protection of SLIGRL was significantly surpassed for WT compared with TRPV1(-/-) hearts (P < 0.05). CGRP(8-37), a selective CGRP receptor antagonist, RP67580, a selective neurokinin-1 receptor antagonist, PKC-epsilon V1-2, a selective PKC-epsilon inhibitor, or H-89, a selective PKA inhibitor, abolished SLIGRL protection by inhibiting the recovery of the rate of coronary flow, maximum rate of left ventricular pressure development, and left ventricular developed pressure, and increasing left ventricular end-diastolic pressure in WT but not TRPV1(-/-) hearts. Radioimmunoassay showed that SLIGRL increased the release of CGRP and SP in WT but not TRPV1(-/-) hearts (P < 0.05), which were prevented by PKC-epsilon V1-2 and H-89. Thus our data show that PAR2 activation improves cardiac recovery after I/R injury in WT and TRPV1(-/-) hearts, with a greater effect in the former, suggesting that PAR2-mediated protection is TRPV1 dependent and independent, and that dysfunctional TRPV1 impairs PAR2 action. PAR2 activation of the PKC-epsilon or PKA pathway stimulates or sensitizes TRPV1 in WT hearts, leading to the release of CGRP and SP that contribute, at least in part, to PAR2-induced cardiac protection against I/R injury.

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Dataset Information

Molecular basis of b-adrenergic regulation of cardiac calcium current

Dataset's files

Publications

Mechanism of adrenergic Ca<sub>V</sub>1.2 stimulation revealed by proximity proteomics.

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