An electron transfer-dependent membrane potential in chromaffin vesicle ghosts

Abstract

Adrenal medullary chromaffin-vesicle membranes contain a transmembrane electron carrier that may provide reducing equivalents for intravesicular dopamine beta-hydroxylase in vivo. This electron transfer system can generate a membrane potential (inside positive) across resealed chromaffin-vesicle membranes (ghosts) by passing electrons from an internal electron donor to an external electron acceptor. Both ascorbic acid and isoascorbic acid are suitable electron donors. As an electron acceptor, ferricyanide elicits a transient increase in membrane potential at physiological temperatures. A stable membrane potential can be produced by coupling the chromaffin-vesicle electron-transfer system to cytochrome oxidase by using cytochrome c. The membrane potential is generated by transferring electrons from the internal electron donor to cytochrome c. Cytochrome c is then reoxidized by cytochrome oxidase. In this coupled system, the rate of electron transfer can be measured as the rate of oxygen consumption. The chromaffin-vesicle electron-transfer system reduces cytochrome c relatively slowly, but the rate is greatly accelerated by low concentrations of ferrocyanide. Accordingly, stable electron transfer dependent membrane potentials require cytochrome c, oxygen, and ferrocyanide. They are abolished by the cytochrome oxidase inhibitor cyanide. This membrane potential drives reserpine-sensitive norepinephrine transport, confirming the location of the electron-transfer system in the chromaffin-vesicle membrane. This also demonstrates the potential usefulness of the electron transfer driven membrane potential for studying energy-linked processes in this membrane.

Lobeline Displaces [3H]Dihydrotetrabenazine Binding and Releases [3H]Dopamine from Rat Striatal Synaptic Vesicles: Comparison with d‐Amphetamine

“…Furthermore, it is well established that VMAT activity in vesic!es requires both a proton gradient and a membrane potential for monoamine transport (Johnson andScarpa, 1976, 1979;Kanner et al, 1980;Harnadek et a!., 1985;Russell et a!., 1985;Brownstein and Hoffman, 1994). d-Amphetamine has been reported to disrupt the vesicular proton gradient required for VMAT2 activity, releasing DA from synaptic vesicles of P. corneus giant DA cells, increasing the cytosolic concentration of DA, and promoting its reverse transport (Sulzer and Rayport, 1990;Sulzer et a!., 1995).…”

Rate of transmembrane electron transfer in chromaffin-vesicle ghosts

“…We have observed that electron transfer from internal ascorbate to external cytochrome c will generate a membrane potential only if ferrocyanide or ferricyanide is present (Harnadek et al, 1985). We suggested that these anions stimulate the rate of electron transfer.…”

“…Electron transfer from ascorbate trapped inside chromaffin-vesicle ghosts to external cytochrome c can be observed by monitoring the membrane potential (inside positive) created by charge movement (Njus et al, 1983;Harnadek et al, 1985). A more direct method, however, is to measure the reduction of cytochrome c spectrophotometrically.…”

“…To show that the chromaffin-vesicle membrane has a transmembrane electron carrier, we (Njus et al, 1983;Harnadek et al, 1985) measured the membrane potential created when electrons are passed from an internal electron donor (ascorbic acid) to an external electron acceptor (ferricyanide or cytochrome c). A more direct and quantitative assay for •This work was supported by National Institutes of Health Grant GM-30500.…”

The ascorbate‐driven reduction of extracellular ascorbate free radical by the erythrocyte is an electrogenic process

“…Other transmembrane redox processes were indeed shown to be electrogenic. In cells and vesicles from different cell types and species, the reduction of extracellular substrates like ferricyanide was found to affect the membrane potential, resulting in depolarization [20–24].…”