Mitochondrial pH Monitored by a New Engineered Green Fluorescent Protein Mutant

• Published in: The Journal of biological chemistry Vol. 279; no. 12; pp. 11521 - 11529
• Main Authors: Abad, María F. Cano, Di Benedetto, Giulietta, Magalhães, Paulo J., Filippin, Luisa, Pozzan, Tullio
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.03.2004; American Society for Biochemistry and Molecular Biology
• Subjects: Chromatography, Affinity; Green Fluorescent Proteins; HeLa Cells; Humans; Hydrogen-Ion Concentration; Luminescent Proteins - genetics; Mitochondria - chemistry; Mutation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M306766200

We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pKa (pKa′) around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca2+ ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca2+ accumulation by this organelle. Finally, we demonstrate that Ca2+ mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.