Measuring mitochondrial Ca2+ efflux in isolated mitochondria and permeabilized cells

Abstract

Mitochondrial Ca²⁺ efflux is essential for mitochondrial and cell Ca²⁺ homeostasis. Mitochondrial inner membrane Ca²⁺/H⁺ and Na⁺/Li⁺/Ca²⁺ (NCLX) exchangers are known today to be plastic transporters, with important roles in physiological responses and pathological states. Until now, however, no consensus protocols were available to measure mitochondrial Ca²⁺ efflux, and we find that some published protocols may induce mitochondrial permeability transition, underestimating the effects of these exchangers. In this work we describe a method to measure Na⁺-sensitive and insensitive mitochondrial Ca²⁺ efflux activity in isolated mitochondria and permeabilized cells using the Ca²⁺ Green indicator and a fluorimeter. A checklist is provided to avoid artefacts as well as pinpoint adaptations necessary in specific experimental models.

Cite:

Serna JDC, Ramos VM, Cabral-Costa JV, Vilas-Boas EA, Amaral AG, Ohya G, Caldeira da Silva CC, Kowaltowski AJ (2022) Measuring mitochondrial Ca²⁺ efflux in isolated mitochondria and permeabilized cells. Bioenerg Commun 2022.7. https://doi.org/10.26124/bec:2022-0007