Background: Depolarization-induced suppression of excitation (DSE) at parallel fiber-Purkinje cell synapse is an endocannabinoid-mediated short-term retrograde plasticity. Intracellular Ca 2+ elevation is critical for the endocannabinoid production and DSE. Nevertheless, how elevated Ca 2+ leads to DSE is unclear.Methodology/Principal Findings: We utilized cytosolic phospholipase A 2 alpha (cPLA 2 a) knock-out mice and whole-cell patch clamp in cerebellar slices to observed the action of cPLA 2 a/arachidonic acid signaling on DSE at parallel fiber-Purkinje cell synapse. Our data showed that DSE was significantly inhibited in cPLA 2 a knock-out mice, which was rescued by arachidonic acid. The degradation enzyme of 2-arachidonoylglycerol (2-AG), monoacylglycerol lipase (MAGL), blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine (AEA), fatty acid amide hydrolase (FAAH), did not affect DSE. These results suggested that 2-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K + , indicating that large conductance Ca 2+ -activated potassium channel (BK) is sufficient to inhibit cPLA 2 a/arachidonic acid-mediated DSE. In addition, we showed that the release of 2-AG was independent of soluble NSF attachment protein receptor (SNARE), protein kinase C and protein kinase A.Conclusions/Significance: Our data first showed that cPLA 2 a/arachidonic acid/2-AG signaling pathway mediates DSE at parallel fiber-Purkinje cell synapse.
Mitochondrial Ca2+ (mtCa2+) uptake via the mtCa2+ uniporter (MCU) complex is a critical factor in determining cell survival or death. We previously reported that the activation of a Ca2+‐ and reactive oxygen species (ROS)‐sensitive protein tyrosine kinase (PTK), proline‐rich tyrosine kinase 2 (Pyk2), under Gq protein–coupled receptor (GqPCR) stimulation increases tyrosine phosphorylation (P‐Y) of MCU and mtCa2+ uptake, followed by the activation of mitochondrial permeability transition pore, and apoptotic cell death in cardiomyocytes. However, further investigation is required to determine 1) the identity of Pyk2‐specific phosphorylation sites within the MCU structure and 2) the functional relevance of P‐Y to MCU channel function as well as mitochondrial functions both in situ and in vivo. In this project, we determined specific Pyk2 phosphorylation site(s) in MCU and tested whether P‐Y at these site(s) modulates MCU channel function using cell lines stably expressing wild‐type (WT)‐ or dephosphomimetic mutants of MCU (MCU‐YFs). Furthermore, we investigated the role of MCU P‐Y in vivo using transgenic (TG) mice with cardiac‐specific overexpression of constitutively active Gαq protein (Gαq TG mice). Through phosphorylation prediction programs, we identified three tyrosine residues as candidate PTK phosphorylation sites, which are conserved across all eukaryotic species. In vitro kinase assays showed that purified active Pyk2 phosphorylated purified full‐length MCU. Next, P‐Y levels at candidate sites were biochemically detected after GqPCR stimulation using cell lines stably expressing Flag‐tagged WT‐ or MCU‐YFs. This in situ assay revealed that only two tyrosine sites increased P‐Y levels in response to GqPCR stimulation. We further assessed mtCa2+ uptake profiles in response to cytosolic Ca2+ elevation in cells stably expressing WT‐ and MCU‐YFs via live cell imaging using mitochondria‐targeted Ca2+‐sensitive biosensors. Using GFP‐tagged MCU‐YFs, we confirmed that all mutant MCUs, like WT‐MCU, were exclusively expressed in mitochondria. Overexpression of one MCU‐YF failed to increase mtCa2+ uptake in response to cytosolic Ca2+ elevation, although the overexpression of WT‐ and the other two MCU‐YFs significantly accelerated mtCa2+ uptake compared to non‐transfected cells. Finally, we demonstrated that P‐Y of MCU occurs in Gαq TG mouse hearts, concomitant with higher Pyk2 activation and apoptotic signaling. In summary, MCU contains Pyk2‐specific phosphorylation site(s) and Pyk2‐dependent P‐Y of MCU increase mtCa2+ uptake via the MCU complex. Moreover, GqPCR‐Pyk2 signaling may induce P‐Y of MCU and cardiomyocyte death in vivo. These findings suggest that inhibition of GqPCR‐Pyk2‐MCU signaling may be a novel therapeutic target to prevent mitochondrial Ca2+ overload, oxidative stress, and cardiomyocyte death during pathophysiological conditions such as heart failure, in which chronic GqPCR stimulation occurs. Support or Funding Information B.S.J was supported by NIH/NIGMS U54GM115677 and P30GM1114750. U.M. was support...
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