Protein phosphatase1 regulates normal automaticity of heart pacemaker node cells

在新鲜分离的兔SANC中，蛋白磷酸酶(PPI)被PP1/2A抑制剂Calyculin A(100-500 nM)抑制，PP活性降低90%，Thr17和Ser16的基础PLB磷酸化增加约2.5倍。PPI增加：在皂苷通透性SANC中，LCR整体的自发钙释放速率(通过共聚焦Fluo-4成像测量)增加了近4倍；在电压钳制的单个SACC中，L型钙电流(ICAL)的幅度增加了30%；在自发放电的单个SANC中，LCR的大小增加了近4倍。PPI也缩短了LCR周期，这种缩短预示着自发AP周期长度同时减少25%。PPI对SANC放电速率影响的数值模型模拟结合了实验观察到的对肌浆网钙泵的磷酸化和PLB效应的变化，与实验结果吻合较好。 我们检测了不同类型的PPS和PP1抑制剂在SANC、LV和RA细胞中的表达。 结论：因此，基础PP活动至少部分地通过调节CIC、PLB磷酸化和SR产生的LCRs来调节自发的SANC AP的放电频率。我们在VM、RA和SANC中发现了编码PP1、PP2A、PP2B和PP1抑制物的转录本。我们发现，除PP2B外，SANC中所有这些转录本的水平都显著低于VM。 为了在细胞脱磷能力的总体图像中看到不同PPS的分配，我们改进了Promega ProFluor Ser/Thr PPase实验，现在我们能够在相同条件下测量细胞裂解产物中PP1、PP2A、PP2B和PP2C的活性。我们发现PP2A是SANC中占主导地位的PP。为了区分PP1和PP2A，我们研究了低纳摩尔浓度下Calyculin A和冈田酸的剂量依赖效应，发现PP2A对这些抑制剂比PP1更敏感。我们发现，如果SANC细胞有PP1，其相对活性就低。我们试图通过使用最特异的PP1抑制剂I-1来找出PP1的分配。不幸的是，这种方法无济于事，主要是因为细胞中PP2A活性高，激活的I-1受到污染，PP1水平低(相对于总变异性)。 了解到在细胞中PP通常位于不同的微环境中(例如，它们可以与支架蛋白结合)，现在我们正在开发一种新的方法来研究PPS在细胞功能中的作用。目前，我们正在开发SANC和Vm中最重要的复合体的免疫沉淀方法，以便通过免疫共沉淀(进一步的Western Blot和MS分析)检测哪些PPS与它们结合并参与它们的功能。一开始，我们把SERCA和MAKAP作为我们的目标。这种方法特别重要，因为即使是低丰度的磷酸酶也可以在细胞中发挥重要和特定的作用。 此外，我们还研究了PPS在SANC和VM中PDE活性调节中的重要性。

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. 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: Thus, 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 level of all of these transcripts except PP2B is significantly smaller in SANC compare 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-dependence effects of Calyculin A and Okadaic acid at low nanomolar concentrations and found that PP2A is more sensitive to these inhibitors then PP1. We found that if SANC cells have PP1, its relative activity is low. We tried to find out 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, contaminations 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 microenvironment (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. For the beginning 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. In addition we also investigate importance of PPs in PDE activity regulation in SANC and VM.