title: Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes.
abstract: L-type Ca channels (LTCC), which play a key role in cardiac excitation-contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation.
authors.affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: Bryant
authors.affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Tomomi E
authors.initials: TE
authors.lastname: Kimura
authors.affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Cherrie H T
authors.initials: CH
authors.lastname: Kong
authors.affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Judy J
authors.initials: JJ
authors.lastname: Watson
authors.affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Anabelle
authors.initials: A
authors.lastname: Chase
authors.affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: M Saadeh
authors.initials: MS
authors.lastname: Suleiman
authors.affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK. Electronic address: a.james@bristol.ac.uk.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: James
authors.affiliation: School of Physiology and Pharmacology, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK. Electronic address: clive.orchard@bristol.ac.uk.
authors.forename: Clive H
authors.initials: CH
authors.lastname: Orchard
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology; D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling; D051244:Caveolin 3 / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004983:Ethanolamines / Q000494:pharmacology; D006352:Heart Ventricles / Q000166:cytology*; D007546:Isoquinolines / Q000494:pharmacology; D008297:Male; D032383:Myocytes, Cardiac / Q000201:enzymology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D012508:Sarcolemma / Q000201:enzymology; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D020746:Calcium Channels, L-Type; C495549:Cav3 protein, rat; D051244:Caveolin 3; D004983:Ethanolamines; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; C002904:zinterol; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
keywords: Ca; Phosphorylation; t-tubules; β(2)-Adrenoceptors