Background: T-type Ca 2+ (Cav3.2) channel inhibitors prevent neuronal death during ischemia, but the cytotoxic signals have not been resolved.
Results: Cav3.2 channels generate cytotoxic Ca 2+ elevations in the cytosol and mitochondria of PC12 cells deprived of oxygen and glucose.
Conclusion: Ca 2+ transfer from Cav3.2 channels to mitochondria contributes to ischemic toxicity.
Significance: Cav3.2 channels and the mitochondrial Ca 2+ uniporter (MCU) are potential targets for the treatment of stroke.
T-type Ca 2+ channel inhibitors protect hippocampal CA1 neurons from delayed death after global ischemia in rats, suggesting that Cav3.1, Cav3.2, or Cav3.3 channels generate cytotoxic Ca 2+ elevations during anoxia. To test this hypothesis, we measured the Ca 2+ concentration changes evoked by oxygen and glucose deprivation (OGD) in the cytosol and in the mitochondria of PC12 cells. OGD evoked long-lasting cytosolic Ca 2+ elevations that were reduced by Cav3.2 inhibition (50 μ m Ni 2+) and Cav3.1/Cav3.2 silencing and potentiated by Cav3.2 overexpression. The kinetics of the sustained cytosolic Ca 2+ elevations occurring during OGD directly correlated to the extent of cell death measured 20 h after reoxygenation, which was decreased by Ni 2+ and Cav3.1/Cav3.2 silencing and increased by Cav3.2 overexpression. Ni 2+ and Cav3.1/Cav3.2 silencing delayed the decline of cellular ATP during OGD, consistent with a reduction in the Ca 2+ load actively extruded by plasma membrane Ca 2+ pumps. The cytosolic Ca 2+ elevations were paralleled by mitochondrial Ca 2+ elevations that were also increased by Cav3.2 overexpression and decreased by Ni 2+ but not by Cav3.1/Cav3.2 silencing. Overexpression and silencing of the mitochondrial Ca 2+ uniporter, the major mitochondrial Ca 2+ uptake protein, revealed that the cytotoxicity was correlated to the amplitude of the mitochondrial, rather than the cytosolic, Ca 2+ elevations. Selective activation of T-type Ca 2+ channels evoked both cytosolic and mitochondrial Ca 2+ elevations, but only the mitochondrial responses were reduced by Cav3.1/Cav3.2 silencing. We conclude that the opening of Cav3.2 channels during ischemia contribute to the entry of Ca 2+ ions that are transmitted to mitochondria, resulting in a deleterious mitochondrial Ca 2+ overload.
|Keywords:||stroke, calcium signaling, ion channels, ischemia, mitochondria|