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Regulation of ATP-induced calcium release in COS-7 cells by calcineurin.

ABSTRACT: Experiments were conducted to examine the role of calcineurin in regulating Ca(2+) fluxes in mammalian cells. In COS-7 cells, increasing concentrations (1-10 microM) of ATP triggered intracellular Ca(2+) release in a dose-dependent manner. Treatment of the cells with calcineurin inhibitors such as cyclosporin A (CsA), deltamethrin and FK506 resulted in an enhancement of ATP-induced intracellular Ca(2+) release. Measurement of calcineurin-specific phosphatase activity in vitro demonstrated a high level of endogenous calcineurin activities in COS-7 cells, which was effectively inhibited by the addition of deltamethrin or CsA. The expression of constitutively active calcineurin (CnADeltaCaMAI) inhibited the ATP-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), in both the presence and the absence of extracellular Ca(2+). These results suggest that the constitutively active calcineurin prevented Ca(2+) release from the intracellular stores. In the calcineurin-transfected cells, treatment with CsA restored the calcineurin-mediated inhibition of intracellular Ca(2+) release. Protein kinase C-mediated phosphorylation of Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)R] was partly inhibited by the extracts prepared from the vector-transfected cells and completely inhibited by those from cells co-transfected with CnADeltaCaMAI and calcineurin B. On the addition of 10 microM CsA, the inhibited phosphorylation of Ins(1,4,5)P(3)R was restored in both the vector-transfected cells and the calcineurin-transfected cells. These results show direct evidence that Ca(2+) release through Ins(1, 4,5)P(3)R in COS-7 cells is regulated by calcineurin-mediated dephosphorylation.

SUBMITTER: Bandyopadhyay A

PROVIDER: S-EPMC1221051 | biostudies-other | 2000 May

REPOSITORIES: biostudies-other

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Project description:In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights into the calcium signaling mechanisms involved in early developmental processes.

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Regulation of ATP-induced calcium release in COS-7 cells by calcineurin.

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