Mitochondrial Inner Membrane Electrophysiology Assessed by Rhodamine-123 Transport and Fluorescence

Abstract

Rhodamine-123 is widely used to make dynamic measurements of mitochondrial membrane potential both in vitro and in situ. Yet data interpretation is difficult due to a lack of quantitative understanding of how membrane potential and measured fluorescence are related. To develop such understanding, a model for dye transport across the mitochondrial inner membrane and partition into the membrane was developed. The model accounts for experimentally measured dye selfquenching and was integrated into a model of mitochondrial electrophysiology to estimate transients in mitochondrial membrane potential from kinetic fluorescence measurements. Our analysis indicates that (i) R123 fluorescence peaks at concentrations near 50 μM due to selfquenching; (ii) measured fluorescence intensity and membrane potential are related by a nonlinear calibration curve sensitive to certain experimental details, including total concentration of dye and mitochondria in suspensions; and (iii) the time courses of membrane potential and electron transport fluxes following a perturbation (i.e. addition of ADP) significantly differ from observed transients in fluorescence intensity. These findings are consistent with the model predictions that mitochondria display a characteristic time of response to changes in substrate concentration of less than 0.1 s, corresponding to the time scale over which the rate of ATP synthesis changes to meet changes in ADP concentration.

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes

“…On the other hand, if we used the lower surface area for the R123 studies, the time course required to obtain data sufficient for interpretation would be too long to be consistent with high cell viability. The dye concentrations selected for further study were within the linear dye concentration vs. fluorescence intensity range, i.e., below the aggregation threshold concentration for both dyes (10 nM for R123 and 20 nM for TMRE; data not shown) (21,26).…”

“…6). The dye flux across plasma (J 1 ) or inner mitochondrial membrane (J 2 ) is represented by a modified one-dimensional Goldman-Hodgkin-Katz equation (26). Because the fractional loss of dye from the extracellular medium in Fig.…”

“…On the other hand, pulmonary endothelial ⌬ p is implicated in regulating channel-mediated calcium entry as a key signaling response to mechanical stimuli, vasoactive substances, oxidative stress, ischemia, and hypoxia (14,17,31,49,59,67,70). Thus the ability to quantify ⌬ m and ⌬ p is important for characterization of mechanisms underlying pulmonary endothelial responses to injury and adaptation and for evaluation of the utility of therapeutics directed at restoration of normal metabolic, signaling, and bioenergetic function.While various techniques have been reported for evaluating ⌬ m in different cell types, a typical approach has been to use cationic fluorescent dyes that accumulate in the mitochondrial matrix driven by the voltage gradient across the inner mitochondrial membrane (22,26,56,58,64). Such studies generally have involved measurements of fluorescence intensity within cells or isolated mitochondria.…”

“…Because such measurements are easily confounded by the propensity of the dyes to undergo self-aggregation, quenching, and photobleaching and/or to exert phototoxic effects, the outcomes have been predominantly confined to qualitative changes in ⌬ m (22,26,28,56,58,64). In addition, the vast majority of studies have involved mitochondrial isolation, cell permeabilization, or other conditions that have little relevance for intact cells; yet, when intact cells have been studied, the contributions of processes other than ⌬ m [e.g., the multidrug transporter Pglycoprotein (Pgp) and/or ⌬ p ] to dye disposition have often been overlooked (6,21,26,46,68). Patch clamping, perhaps considered the gold standard for measuring ⌬ m , also involves cell disruption (1,14,29,31,62).…”

“…While various techniques have been reported for evaluating ⌬ m in different cell types, a typical approach has been to use cationic fluorescent dyes that accumulate in the mitochondrial matrix driven by the voltage gradient across the inner mitochondrial membrane (22,26,56,58,64). Such studies generally have involved measurements of fluorescence intensity within cells or isolated mitochondria.…”