Simultaneous Monitoring of Ionophore- and Inhibitor-mediated Plasma and Mitochondrial Membrane Potential Changes in Cultured Neurons

David G Nicholls

doi:10.1074/jbc.m510916200

Abstract

Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV.

Highlights

Tion that it will generate a stable, moderately elevated, cytoplasmic free Ca2ϩ concentration, [Ca2ϩ]c with maintained cell viability
Ionomycin intercalates into the inner mitochondrial membrane where it provides an additional pathway for Ca2ϩ efflux from the matrix in parallel with the native Ca2ϩ/Naϩ antiporter, setting up a protondissipating, i.e. uncoupling, Ca2ϩ cycle that is controlled by the activity of the mitochondrial Ca2ϩ uniporter and by [Ca2ϩ]c [1]
An important ambiguity surrounds the widespread use of cationic, membrane permeant, fluorescent probes as mitochondrial membrane potential indicators [3,4,5] because their uptake and equilibrium accumulation within the mitochondrial matrix of the cell is responsive to the plasma membrane potential, ⌬␺p, and the mitochondrial membrane potential, ⌬␺m

Summary

Introduction

Tion that it will generate a stable, moderately elevated, cytoplasmic free Ca2ϩ concentration, [Ca2ϩ]c with maintained cell viability. The bioenergetic consequences of this are usually ignored, they could have profound effects on cellular function Protonophores such as FCCP that are widely employed to depolarize mitochondria in intact cells can affect plasma membrane potentials at higher concentrations [2]. Low probe loadings that avoid matrix quenching must be employed to follow slow changes in potential as well as to estimate pre-existing values of ⌬␺m in cell populations (reviewed in Ref. 12). Under these latter conditions there is serious ambiguity as to whether the observed change in fluorescence is due to a difference in ⌬␺p, ⌬␺m, or both. The curve-fitting spreadsheet may be used for single-probe studies of ⌬␺m, where ⌬␺p is invariant or its changes can be estimated

Methods

Results

Discussion

Conclusion