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Updated: Aug 13, 2026
Pull-down of Calmodulin-binding Proteins
Published on: January 23, 2012
Mode-specific inhibition of sodium-calcium exchange during protein phosphatase blockade
M Condrescu1, B M Hantash, Y Fang
1Department of Pharmacology, University of Medicine and Dentistry of New Jersey, The New Jersey Medical School, Newark, New Jersey 07103, USA.
Abstract:
The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na(+)/Ca(2+) exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated (45)Ca(2+) uptake or Ba(2+) influx by 50-75%. Half-maximal inhibition of (45)Ca(2+) uptake was observed at 15 nM calyculin A. The nonselective protein kinase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhibitor, nearly completely blocked exchange-mediated Ba(2+) influx. Chinese hamster ovary cells expressing a mutant exchanger in which 420 out of 520 amino acid residues were deleted from the central hydrophilic domain of the exchanger remained sensitive to the inhibitory effects of calyculin A and okadaic acid. Surprisingly, Na(o)(+)-dependent Ca(2+) efflux appeared to be only modestly inhibited, if at all, by calyculin A or okadaic acid. We conclude that protein hyperphosphorylation during protein phosphatase blockade selectively inhibits the Ca(2+) influx mode of Na(+)/Ca(2+) exchange, probably by an indirect mechanism that does not involve phosphorylation of the exchanger itself.
Insights
Protein phosphatase inhibitors calyculin A and okadaic acid selectively block calcium influx via Na+/Ca2+ exchange. This inhibition likely occurs indirectly, not through direct phosphorylation of the exchanger itself.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Physiology
Background:
- Na+/Ca2+ exchanger (NCX) plays a critical role in cellular calcium homeostasis.
- Protein phosphorylation is a key regulator of ion transport.
- Protein phosphatases counteract kinase activity, influencing protein function.
Purpose of the Study:
- To investigate the impact of protein phosphatase inhibitors on Na+/Ca2+ exchange activity.
- To determine if inhibition of Na+/Ca2+ exchange by these agents involves direct phosphorylation of the exchanger.
- To elucidate the specific mode of Na+/Ca2+ exchange affected by protein phosphatase blockade.
Main Methods:
- Utilized Chinese hamster ovary (CHO) cells transfected with the bovine cardiac Na+/Ca2+ exchanger.
- Assessed Na+/Ca2+ exchange activity by measuring 45Ca2+ uptake and Ba2+ influx.
- Employed protein phosphatase inhibitors (calyculin A, okadaic acid) and protein kinase inhibitors (K252a, staurosporine).
- Investigated effects on both wild-type and mutant Na+/Ca2+ exchangers with a deleted central hydrophilic domain.
Main Results:
- Calyculin A significantly inhibited Na+/Ca2+ exchange-mediated 45Ca2+ uptake and Ba2+ influx in a dose-dependent manner.
- Okadaic acid demonstrated potent inhibition of exchange-mediated Ba2+ influx.
- Inhibition persisted even in cells expressing a mutant exchanger lacking a large portion of its central domain.
- Na+ gradient-driven Ca2+ efflux was only modestly affected by the inhibitors.
Conclusions:
- Protein hyperphosphorylation induced by phosphatase blockade selectively inhibits the Ca2+ influx mode of Na+/Ca2+ exchange.
- The inhibitory mechanism is likely indirect and does not involve direct phosphorylation of the Na+/Ca2+ exchanger itself.
- These findings highlight a regulatory pathway influencing cardiac Na+/Ca2+ exchanger function.
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