Proceedings of The Physiological Society

King's College London (2011) Proc Physiol Soc 22, PC05

Poster Communications

Effect of Hydrogen Peroxide on the Delayed Rectifier Potassium Current in Dissociated Hippocampal CA1 Neurons

S. Hasan1, W. Al-Shuaib1, Z. Redzic1

1. Department of Physiology, Faculty of Medicine, Kuwait University, Kuwait, Kuwait.


  • Concentration response curve for H2O2 inhibition of IKDR. CA1 cells were treated with increasing H2O2 concentrations (10 - 1300

  • The effect of DFO on the H2O2 - induced decrease in IKDR. A: Addition of DFO simultaneously with H2O2 showed no decrease in IKDR currents. B: Addition of DFO alone for up to 5 minutes showed no change in current. Addition of H2O2 for 6 minutes showed no change in IKDR. C. H2O2 addition for 6 minutes caused a decrease in IKDR that was not reversed by DFO. Currents were elicited during eleven 500 msec clamp potentials between - 40 mV (bottom trace) and + 60 mV (top trace) in steps of 10 mV. Holding potential was - 80 mV.

Introduction: Hydrogen peroxide (H2O2) is a reactive oxygen species that is generated as part of normal cellular metabolism. When in excess, H2O2 is known to cause damage to various biomolecules. Application of H2O2 has been frequently used in previous studies to induce oxidative stress conditions. The present study examined the effect of H2O2 on the delayed rectifier potassium current (IKDR), a voltage - dependent current of paramount importance for neuronal excitability. Methods: Young adult Sprague Dawley rats were housed in the Animal Resources Centre at the Faculty of Medicine, Kuwait University; animals were humanely treated in accordance with the rules of animal care in this institution. Rats were sacrificed by terminal anaesthesia with ether and hippocampal CA1 neurons were isolated as explained earlier [1]. Whole cell voltage - clamp experiments were performed on freshly dissociated neurons before and after treatment with H2O2 using the method explained earlier [2]; in some cases different antioxidants and reagents were used in order to reveal the mechanism behind the H2O2 induced changes in IKDR. Data were acquired using the p-CLAMP10 software (Axon Instruments, USA) and analyzed using the GraphPad PRISM 5 software (GraphPad Sowtware Inc., USA). The data were compared using a two-tailed unpaired Student’s t-test and a difference between groups (n=5-6 per group) was considered statistically significant if p<0.05. Results: The external application of H2O2 inhibited IKDR in hippocampal CA1 neurons in a concentration dependent manner (Figure 1). H2O2 reduced IKDR’s amplitude and voltage dependence. Desferoxamine (DFO), an iron - chelator that prevents hydroxyl radical generation, prevented H2O2 - induced reduction in IKDR (Figure 2). Application of the cysteine-SH oxidizing agent, 5,5 -dithio-bis-nitrobenzoic acid (DTNB) mimicked the effect of H2O2, whereas SH - reducing agents dithiothreitol (DTT) and glutathione (GSH) reversed and prevented the inhibition in IKDR respectively. Addition of reducing agents DTT and GSH alone did not affect IKDR. Membrane impermeable oxidative and reducing agents had effects only when added intracellularly. Conclusions: H2O2 reduced IKDR, and this reduction was prevented by DFO thus identifying hydroxyl radicals as the intermediate oxidants responsible for the decrease in current amplitude. The reversal of H2O2 - induced reduction of IKDR by SH - reducing agents identified SH groups as oxidative targets. Thus, the oxidative modulation of IKDR by H2O2 was probably via hydroxyl radicals which targeted free SH groups of cysteine residues found in the intracellular aspect of the delayed rectifier potassium channel protein.

Where applicable, experiments conform with Society ethical requirements