Protein phosphatase1 regulates normal automaticity of heart pacemaker node cells

蛋白磷酸酶1调节心脏起搏器节点细胞的正常自律性

• 批准号: 8931599
• 负责人: Edward Lakatta
• 金额: $ 38.01万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8931599
• 关键词: Action Potentials; Adenylate Cyclase; Binding; Biochemical; Biological Assay; Buffers; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcineurin; Calmodulin; Cations; Cell physiology; Cells; Co-Immunoprecipitations; Code; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Detection; Dose; Equilibrium; Far-Western Blotting; Fluo 4; Generations; Heart; Homeostasis; Image; Immunoprecipitation; Kinetics; Lead; Left ventricular structure; Length; Mass Spectrum Analysis; Measures; Mediating; Membrane; Messenger RNA; Methods; Modeling; Muscle Cells; Okadaic Acid; Oryctolagus cuniculus; Pacemakers; Phosphoric Monoester Hydrolases; Phosphorylation; Potassium Channel; Promega; Protein Phosphatase 2A Regulatory Subunit PR53; Protein Phosphatase Inhibitor; Protein phosphatase; Proteins; Pump; Reaction; Regulation; Relative (related person); Right atrial structure; Role; Ryanodine Receptors; Saponin; Saponins; Sarcoplasmic Reticulum; Scaffolding Protein; Signal Transduction; Sinoatrial Node; Sodium-Calcium Exchanger; Surface; Techniques; Time; Transcript; Ventricular; Western Blotting; Work; biophysical properties; calyculin A; feeding; improved; inhibitor/antagonist; interest; models and simulation; novel strategies; phospholamban; phosphoric diester hydrolase; protein phosphatase 2C; research study; voltage clamp

In freshly isolated rabbit SANC, Protein Phosphatase Inhibition (PPI) by the PP1/2A inhibitor, Calyculin A, (100-500 nM) reduced PP activity by 90%, and increased basal PLB phosphorylation at Thr17 and Ser16 by about 2.5-fold. PPI increased: the rate of spontaneous Ca2+ release of the LCR ensemble (measured via confocal fluo-4 imaging) by nearly four-fold in saponin-permeabilized SANC; the L-type Ca2+ current (ICaL) amplitude by 30% in voltage-clamped, single SANC; and the LCR size in spontaneously firing single, intact SANC. PPI also decreased the LCR period, and this reduction predicted a concurrent 25% reduction in the spontaneous AP cycle length. We found that PP are important regulators of PDE functioning: addition of PP inhibitors to the reaction buffer increased total PDE activity approximately 50 % in both SANC and VM lysates. We found that PP1 is present in SANC via Western Blot technique. A numerical model simulation of the effect of PPI on SANC firing rate, incorporating experimental observed changes in ICaL and PLB phosphorylation effects on SR Ca2+ pumping, closely predicted the experimental results. We measured expression of different types of PPs and PP1 inhibitors mRNAs in SANC, LV and RA cells. Conclusion: Basal PP activity modulates spontaneous SANC AP firing rate, in part at least, by modulating ICaL, PLB phosphorylation, and SR-generated LCRs. We identified transcripts coding PP1, PP2A, PP2B and PP1 inhibitors in VM, RA and SANC. We found that the level of all of these transcripts except PP2B was significantly lower in SANC compared to VM. In order to see partitioning of different PPs in the total picture of cell ability to dephosphorylate proteins, we have modified Promega ProFluor Ser/Thr PPase assay, and now we are able to measure PP activity in cell lysates in the same conditions for PP1, PP2A, PP2B and PP2C. We found that PP2A is a dominant PP in SANC. In order to differentiate between PP1 and PP2A, we studied dose-dependent effects of Calyculin A and Okadaic acid at low nanomolar concentrations and found that PP2A is more sensitive to these inhibitors than PP1. We found that PP1 is present in SANC, but its relative activity is low. We tried to discern the PP1 partitioning by using I-1, the most specific PP1 inhibitor. Unfortunately, this approach did not help, mainly due to the high level of PP2A activity in cells, contamination in the activated I-1 and low level of PP1 (relatively to the total variability). Knowing that in cells PP are often located in different microenvironments (for example they can be bound to scaffolding proteins), now we are developing a new approach to investigate role of PPs in cell functioning. Currently we are developing methods of immunoprecipitation of the most important complexes in SANC and VM in order to detect via co-immunoprecipitation (with further Western blot and mass spectrometry analysis) which PPs are bound to them and are involved in their functioning. Initially, we took SERCA and mAKAP as our targets. This method is especially important because even low abundant phosphatase can have an important and specific role in cell regulation. Currently we continue our work to develop and improve co-immunoprecipitation techniques. The most recent experiments demonstrated that activity of PPs depends tremendously on cations present in phosphatase reaction. We have developed conditions in which PP2A activity is not so high but at the same time PP1 and PP2B activities appear much higher than previously. Using Calyculin A and Okadaic acid at low nanomolar concentrations under the new conditions helped us demonstrate a much clearer presence of PP1 and PP2B activities in SANC. These data also prove the importance of intracellular microenvironment cation balance in the activities of PPs.

在新分离的兔SANC中，PP1/2A抑制剂Calyculin A （100-500 nM）抑制蛋白磷酸酶（PPI）可使PP活性降低90%，并使PLB Thr17和Ser16的基础磷酸化增加约2.5倍。PPI增加：在皂素渗透的SANC中，LCR集合的自发Ca2+释放率（通过共聚焦荧光成像测量）增加了近四倍；l型Ca2+电流（ICaL）幅值在电压箝位的单一SANC中提高30%；以及自燃单一完整SANC的LCR大小。PPI也缩短了LCR周期，这种减少预示着自发性AP周期长度同时减少了25%。