Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α

Jyoti Gulia, Manuel F Navedo, Peichun Gui, Jun Tzu Chao, Jose L. Mercado, Luis Fernando Santana, Michael J. Davis

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The activity of persistent Ca2+ sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca2+ entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca2+ sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca2+ sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca2+ sparklets? With the use of total internal reflection fluorescence microscopy, Ca2+ 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 Ca2+ sparklets. Persistent Ca2+ 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 Ca2+ sparklet activity. We could not detect a significant change in persistent Ca2+ 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 Ca2+ sparklets. However, our results indicate that persistent Ca2+ sparklet activity is promoted by the action of c-Src on residue Y2122 of the Cav1.2c COOH terminus.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume305
Issue number5
DOIs
StatePublished - Sep 1 2013

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L-Type Calcium Channels
Fluorescence Microscopy
Protein Isoforms
Cell Count
Phosphorylation

Keywords

  • Calcium entry
  • L-type calcium channel
  • Patch clamp
  • PKC
  • TIRF microscopy

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α. / Gulia, Jyoti; Navedo, Manuel F; Gui, Peichun; Chao, Jun Tzu; Mercado, Jose L.; Santana, Luis Fernando; Davis, Michael J.

In: American Journal of Physiology - Cell Physiology, Vol. 305, No. 5, 01.09.2013.

Research output: Contribution to journalArticle

Gulia, Jyoti ; Navedo, Manuel F ; Gui, Peichun ; Chao, Jun Tzu ; Mercado, Jose L. ; Santana, Luis Fernando ; Davis, Michael J. / Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α. In: American Journal of Physiology - Cell Physiology. 2013 ; Vol. 305, No. 5.
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AU - Santana, Luis Fernando

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AB - The activity of persistent Ca2+ sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca2+ entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca2+ sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca2+ sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca2+ sparklets? With the use of total internal reflection fluorescence microscopy, Ca2+ 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 Ca2+ sparklets. Persistent Ca2+ 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 Ca2+ sparklet activity. We could not detect a significant change in persistent Ca2+ 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 Ca2+ sparklets. However, our results indicate that persistent Ca2+ sparklet activity is promoted by the action of c-Src on residue Y2122 of the Cav1.2c COOH terminus.

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