Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes

• Published in: Journal of molecular and cellular cardiology 2014-03, Vol.68 (100), p.47-55
• Main Authors: Bryant, Simon, Kimura, Tomomi E, Kong, Cherrie H.T, Watson, Judy J, Chase, Anabelle, Suleiman, M. Saadeh, James, Andrew F, Orchard, Clive H
• Format: Article
• Language: English
• Subjects: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester - pharmacology; Adrenergic beta-2 Receptor Agonists - pharmacology; Animals; Calcium Channels, L-Type - metabolism; Calcium Signaling; Cardiovascular; Caveolin 3 - metabolism; Cyclic AMP-Dependent Protein Kinases - antagonists & inhibitors; Cyclic AMP-Dependent Protein Kinases - metabolism; Ethanolamines - pharmacology; Heart Ventricles - cytology; Isoquinolines - pharmacology; Male; Myocytes, Cardiac - enzymology; Original; Patch-Clamp Techniques; Phosphorylation; Protein Binding; Protein Kinase Inhibitors - pharmacology; Protein Processing, Post-Translational; Rats; Rats, Wistar; Sarcolemma - enzymology; Sulfonamides - pharmacology; t-tubules; β2-Adrenoceptors
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2013.12.026

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.