Mitochondrial Ca ²⁺ Uptake from Plasma Membrane Cav3.2 Protein Channels Contributes to Ischemic Toxicity in PC12 Cells*

Abstract

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.

Abstract

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.