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Related Experiment Videos

A noninvasive fluorimetric procedure for measurement of membrane potential. Quantification of the NADPH

A Jankowski1, S Grinstein

  • 1Cell Biology Programme, Research Institute, The Hospital for Sick Children, Toronto, Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada.

The Journal of Biological Chemistry
|September 3, 1999
PubMed
Summary

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Activated neutrophils exhibit significant plasma membrane depolarization due to NADPH oxidase activity. This electrogenic effect, measured via manganese uptake, reaches +58 mV, potentially activating voltage-gated channels.

Area of Science:

  • Cellular electrophysiology
  • Immunology
  • Biochemistry

Background:

  • NADPH oxidase activity in neutrophils causes plasma membrane depolarization.
  • Previous methods using fluorescent dyes were unreliable due to organelle interference.
  • The magnitude and consequences of this depolarization were previously unknown.

Purpose of the Study:

  • To accurately measure the plasma membrane potential of activated neutrophils.
  • To determine the extent of depolarization caused by NADPH oxidase.
  • To investigate the functional consequences of this depolarization.

Main Methods:

  • Developed a novel method using voltage-driven Mn(2+) uptake across the plasma membrane.
  • Measured Mn(2+) influx via Indo-1 fluorescence quenching in thapsigargin-treated cells.

Related Experiment Videos

  • Calibrated membrane potential using the Na(+) ionophore SQI-Pr and inhibited NADPH oxidase with diphenylene iodonium.

    • Main Results:

    • Confirmed intracellular generation of oxidase products using dihydrorhodamine.
    • Demonstrated that Mn(2+) influx varies with membrane potential and NADPH oxidase activation.
    • Activated neutrophils showed a sustained depolarization to +58 ± 6 mV, over 100 mV from resting potential.
    • Depolarization was significantly reduced upon NADPH oxidase inhibition.

    Conclusions:

    • NADPH oxidase generates a substantial plasma membrane depolarization in neutrophils.
    • This depolarization is sufficient to activate voltage-gated channels, including H+ conductance.
    • The novel Mn(2+) uptake method provides a reliable way to measure surface membrane potential.