pH Changes in the Mitochondrial Matrix and Cytosol under Glutamate Deregulation of Ca2+ Homeostasis in Cultured Rat Hippocampal Neurons

Abstract

The effect of high concentrations of glutamate (Glu) on primary cultures of neurons from the rat brain led to a strong depolarization of mitochondria, which developed synchronously with a secondary increase in the intracellular free Ca²⁺ concentration (delayed calcium deregulation, DCD). Simultaneously with measurements of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), pH was measured in the mitochondrial matrix (pH_m) and cytosol (pH_c) of neurons when exposed to a toxic dose of Glu (100 µM). For this purpose, pH-sensitive green fluorescent protein mtYFP in mitochondria and pH-sensitive red fluorescent protein mKate in cytosol were expressed in primary cultures from the hippocampus of newborn rats. The resulting neuronal culture was loaded with the Ca²⁺ indicator Fura-FF; [Ca²⁺]_i, pH_m and pH_c were simultaneously measured in those neurons that expressed both mtYFP and mKate. It was found that during the first phase of the [Ca²⁺]_i response to Glu, when partial depolarization of mitochondria was observed, there was an increase in the pH gradient between the mitochondrial matrix and the cytosol (ΔpH), which compensated for the decrease in the electrical component of the mitochondrial potential (∆Ψ_m), thereby maintaining the constancy of the electrochemical potential of mitochondria. The development of DCD led to an abrupt decrease in ∆Ψ_m and ΔpH in the soma of neurons; however, a complete collapse of ΔpH was not observed. This may mean that DCD was not caused by a nonspecific megapore in the inner mitochondrial membrane (mPTP), as is commonly believed. Alternatively, part of the mitochondria in the soma of neurons could retain the barrier properties of the inner membrane and did not form mPTP even with the development of DCD and reaching a high [Ca²⁺]_i plateau.