Enigma homolog 1 scaffolds protein kinase D1 to regulate the activity of the cardiac L-type voltage-gated calcium channel

Autor: Masahiko Hoshijima, Sébastien Wälchli, Miki Iwata, Yasuhiro Ikeda, Andrés D. Maturana, Stephan Ryser, Shun'ichi Kuroda, Werner Schlegel, Johannes Van Lint, Katsuyuki Tanizawa
Rok vydání: 2008
Předmět:
Patch-Clamp Techniques
Calcium Channels
L-Type
Physiology
Genetic Vectors
chemistry.chemical_element
Calcium
Biology
urologic and male genital diseases
Article
Adenoviridae
Membrane Potentials
Phenylephrine
Two-Hybrid System Techniques
Physiology (medical)
Animals
Humans
Immunoprecipitation
Myocytes
Cardiac
Calcium Signaling
Patch clamp
RNA
Small Interfering
Protein kinase A
Cells
Cultured
Protein Kinase C
Protein kinase C
Adaptor Proteins
Signal Transducing
Calcium signaling
Voltage-dependent calcium channel
Calcium channel
LIM Domain Proteins
Molecular biology
female genital diseases and pregnancy complications
Protein Structure
Tertiary
Rats
Cell biology
Animals
Newborn
chemistry
Mutation
RNA Interference
Rabbits
Protein kinase D1
Cardiology and Cardiovascular Medicine
Adrenergic alpha-Agonists
Protein Kinases
HeLa Cells
Protein Binding
Zdroj: Cardiovascular Research. 78:458-465
ISSN: 1755-3245
0008-6363
Popis: Aims In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signals. From a yeast two-hybrid assay, we have identified Enigma Homolog 1 (ENH1) as a new binding partner of PKD1. Since in neurons, ENH1, associated with protein kinase Cepsilon, was shown to modulate the activity of N-type calcium channels, and the pore-forming subunit of the cardiac L-type voltage-gated calcium channel, alpha1C, possesses a potential phosphorylation site for PKD1, we studied here a possible role of ENH1 and PKD1 in the regulation of the cardiac L-type voltage-gated calcium channel. Methods and results PKD1-interacting proteins were searched by yeast two-hybrid screening. In vivo protein interactions in cardiomyocytes isolated from heart ventricles of newborn rats were tested by co-immunoprecipitation. Small interfering RNA and a dominant negative mutant of PKD1 were delivered into cardiomyocytes by use of an adenovirus. Calcium currents were measured by the patch-clamp technique. Both ENH1 and PKD1 interact with alpha1C in cardiomyocytes. This interaction is increased upon stimulation. Silencing of ENH1 prevented the binding of PKD1 to alpha1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the alpha-adrenergic-induced increase of L-type calcium currents. Conclusion We found a new binding partner, ENH1, and a new target, alpha1C, for PKD1 in neonatal rat cardiomyocytes. We propose a model where ENH1 scaffolds PKD1 to alpha1C in order to form a signalling complex that regulates the activity of cardiac L-type voltage-gated Ca(2+) channels.
Databáze: OpenAIRE
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