Mitochondrial pH Monitored by a New Engineered Green Fluorescent Protein Mutant

Abstract

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 pK a (pK a ) 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 Ca 2؉ 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 Ca 2؉ accumulation by this organelle. Finally, we demonstrate that Ca 2؉ mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.

Intracellular pH homeostasis and serotonin-induced pH changes inCalliphorasalivary glands: the contribution of V-ATPase and carbonic anhydrase

“…Two observations indicate, however, that BCECF does not accumulate in the mitochondrial matrix in our preparations, but rather binds to the mitochondrial outer surface: first, incubation of ␤-escinpermeabilized preparations with membrane-impermeable BCECF stains mitochondria in a similar way as BCECF-AM loading of intact preparations and, second, a resting pH i of ~7.5 (see below) argues against the possibility that we have recorded intramitochondrial pH. In the mitochondrial matrix, pH amounts to >8.0 (Abad et al, 2004). These observations and our conclusion that the punctate fluorescence in permeabilzed salivary glands monitors pH changes in the bath reliably support a previous suggestion, derived from a slightly different experimental approach in other cell types (Weinlich et al, 1998), that BCECF reports cytoplasmic pH despite its non-homogeneous intracellular distribution.…”

Peroxisomes as Novel Players in Cell Calcium Homeostasis

“…According to these data, if the Ca 2ϩ influx may take part along with a hardly monitorable H ϩ efflux, the subsequent peroxisomal acidification of the lumen suggests that Ca 2ϩ re-extrusion occurs, directly or indirectly, in exchange with H ϩ . Such a prolonged compensatory response of the organelle in part recalls what happens in mitochondria where transitory alkalinization of the mitochondrial matrix occurs when Ca 2ϩ -mobilizing agents are added (29).…”

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

“…First, it was found that absorbance and°uorescence of GFP mutants strongly depend on pH value both in vitro as well as in intracellular compartments of live cells. 39,40 Probable reason could be that pH can shift equilibrium between two existing GFP ground states, which has di®erent spectroscopic characteristics. 39 For instance, the response of°uorescence of GFP mutant GFP-F64L/S65T to pH changes is rapid and reversible in the range between 6.5 and down to 5.0.…”