Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α.
Am J Physiol Cell Physiol
; 305(5): C568-77, 2013 Sep.
Article
em En
| MEDLINE
| ID: mdl-23804206
ABSTRACT
The activity of persistent Ca²âº sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca²âº entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca²âº sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current 1) Does c-Src activation enhance persistent Ca²âº sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca²âº sparklets? With the use of total internal reflection fluorescence microscopy, Ca²âº sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca²âº sparklets. Persistent Ca²âº sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Cav1.2c constructs mutated at one of two COOH-terminal residues (Y²¹²²F and Y²¹³9F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y²¹²²F but not Y²¹³9F Cav1.2c abrogated the potentiating effect of c-Src on Ca²âº sparklet activity. We could not detect a significant change in persistent Ca²âº sparklet activity or density in cells coexpressing Cav1.2c + PKC-α, regardless of whether WT or Y²¹²²F Cav1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca²âº sparklets. However, our results indicate that persistent Ca²âº sparklet activity is promoted by the action of c-Src on residue Y²¹²² of the Cav1.2c COOH terminus.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cálcio
/
Quinases da Família src
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Canais de Cálcio Tipo L
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Proteína Quinase C-alfa
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Neurônios
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Am J Physiol Cell Physiol
Assunto da revista:
FISIOLOGIA
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Estados Unidos