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

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-? 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-? 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-?/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 Y²¹³?F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y²¹²²F but not Y²¹³?F 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-?, regardless of whether WT or Y²¹²²F Cav1.2c was used, or after PP2 application, suggesting that PKC-? 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.

SUBMITTER: Gulia J

PROVIDER: S-EPMC3761150 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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

Gulia Jyoti J Navedo Manuel F MF Gui Peichun P Chao Jun-Tzu JT Mercado Jose L JL Santana Luis F LF Davis Michael J MJ

American journal of physiology. Cell physiology 20130626 5

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⁺ ...[more]

PMID: 23804206

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Project description:Objective: L-type calcium channels (LTCC) homeostatically regulate calcium on a beat by beat basis, but also provide Ca that over long time scales may contribute to transcriptional regulation. We previously showed that sustained LTCC blockade (CCB) elicits LTCC remodeling in ventricular cardiac myocytes (CM). Here we hypothesize that sustained CCB has broad effects on the expression of genes involved in calcium handling. Methods and Results: Therefore, we subjected adult mice to sustained CCB for 24 hours and performed gene expression profiling. In comparison to vehicle-only control animals, 231 genes were up-regulated, and 111 genes were down-regulated by sustained LTCC blockade (p <0.01). Gene ontology analysis suggested that the CaMKIIdelta signaling pathway was up-regulated in these cells. Unexpectedly, phosphorylation of phospholamban (PLN) at threonine17 (Thr17), an index of CaMKIIdelta activity, was not changed by sustained CCB; however, the degree of phosphorylation of the neighboring PLN-Ser16 substrate site for PKA was significantly reduced by sustained CCB compared to control. Gene expression profiling suggested no change in PKA, but it showed that protein phosphatase 2A (PP2A) mRNA increased, and immunoblots demonstrated that PP2Ac-alpha protein was significantly increased by sustained CCB. Consistent with elevated PP2Ac-alpha protein expression LTCC exhibited decreased phosphorylation of the C-terminal Ser1928 PKA substrate site. Conclusions: We conclude that sustained CCB elicits a spectrum of transcriptional events, including compensatory up-regulation of LTCC and PP2Ac-alpha. Although this study is restricted to mouse, these results suggest the new hypothesis that clinically-relevant sustained LTCC blockade in humans results in changes in gene regulation in the heart. Keywords: L-type calcium channel, calcium channel blockade, verapamil Female and male ICR mice (12-14 weeks age) weighing between 25 and 30 grams were anesthetized with a ketamine/xylazine mixture ( i.p.) allowing the subcutaneous implantation of miniosmotic pumps (Alzet, model 2001). The pumps were filled with either verapamil or vehicle (0.02% ascorbic acid). Control animals carried mini-pumps with vehicle and control animals were investigated in parallel with each set of experimental animals. Mini pumps delivered verapamil at 3.6 mg/kg/day for 24 (RNA)-48 (protein) hours. After treatment animals were anesthetized and weighed. Hearts were excised, rinsed, blotted dry, weighed, and then frozen on dry ice and the stored at -80oC until studied. Animals were anesthetized and euthanized according to animal protocols approved by the University of Kentucky Institutional Animal Care and Use Committee. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 1996). Left ventricular free wall from female mice was rapidly excised and either snap frozen at -80oC or used immediately for RNA isolation. Three VER treated mice and 3 vehicle treated mice were used to generate RNA for microarray. Total RNA was isolated using the RNAqueous -4PCR kit (Ambion) and quantitated spectrophotometrically at 260nm. Contaminating genomic DNA was eliminated by DNase treatment (Ambion). RNA quality was assessed using the Agilent 2100 Bioanalyzer. Microarray data was obtained using the Affymetrix 430 V2 GeneChip (representing 45,101 probe sets), in accordance with the manufacturer’s specifications.

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

Publications

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

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