Mitochondrial Ca²⁺ Processing by a Unit of Mitochondrial Ca²⁺ Uniporter and Na⁺/Ca²⁺ Exchanger Supports the Neuronal Ca²⁺ Influx via Activated Glutamate Receptors

Abstract

The current study demonstrates that in hippocampal neurons mitochondrial Ca²⁺ processing supports Ca²⁺ influx via ionotropic glutamate (Glu) receptors. We define mitochondrial Ca²⁺ processing as Ca²⁺ uptake via mitochondrial Ca²⁺ uniporter (MCU) combined with subsequent Ca²⁺ release via mitochondrial Na⁺/Ca²⁺ exchanger (NCX). Our tool is to measure the Ca²⁺ influx rate in primary hippocampal co-cultures, i.e. neurons and astrocytes, by fluorescent digital microscopy, using a Fura-2-quenching method where we add small amounts of Mn²⁺ in the superfusion medium. Thus, Ca²⁺ influx is measured with Mn²⁺ in the bath. Ru360 as inhibitor of mitochondrial Ca²⁺ uptake through MCU strongly reduces the rate of Ca²⁺ influx in Glu-stimulated primary hippocampal neurons. Similarly, the Ca²⁺ influx rate in Glu-stimulated neurons declines after suppression of potential-dependent MCU, when we depolarize mitochondria with rotenone. With inhibition of Ca²⁺ release from mitochondria via NCX using CGP-37157 the Ca²⁺ influx via N-methyl-d-aspartate (NMDA)- and kainate-sensitive receptors is slowed down. Working jointly as mitochondrial Ca²⁺ processing unit, MCU and NCX, apparently sustain the Ca²⁺ throughput of activated Glu-sensitive receptors. Our results revise the role frequently attributed to mitochondria in neuronal Ca²⁺ homeostasis, where mitochondria function mainly as Ca²⁺ buffer, and prevent excessively high cytosolic Ca²⁺ concentration increase during neuronal activity. The mechanism to control Ca²⁺ influx in neurons, as discovered in this study, highlights mitochondrial Ca²⁺ processing as a promising pharmacological target. We discuss this pathway in relation to the endoplasmic reticulum-related mechanisms of Ca²⁺ processing.