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.

National Institute of Health (NIH)
National Institute on Aging (NIA)
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