Inhibition of Qi site of mitochondrial complex III with antimycin A decreases persistent and transient sodium currents via reactive oxygen species and protein kinase C in rat hippocampal CA1 cells

Experimental Neurology 194 (2005) 484 – 494 www.elsevier.com/locate/yexnr

Inhibition of Qi site of mitochondrial complex III with antimycin A decreases persistent and transient sodium currents via reactive oxygen species and protein kinase C in rat hippocampal CA1 cells Bin Lai, Li Zhang, Lian-Yan Dong, Yan-Hua Zhu, Feng-Yan Sun, Ping Zheng* State Key Laboratory of Medical Neurobiology, Fudan University Shanghai Medical College, 138 Yixueyuan Road, Shanghai 200032, People’s Republic of China Received 30 November 2004; revised 14 March 2005; accepted 18 March 2005 Available online 22 April 2005

Abstract Hypoxia-induced inhibition of Qi site of mitochondrial complex III under hypoxia has received attention, but its downstream pathways remain unclear. In this paper, we used Qi site inhibitor antimycin A to mimic the inhibition of the Qi site of mitochondrial complex III and studied the effects of the inhibition of this site on persistent sodium currents, transient sodium currents, and neuronal excitability in rat hippocampal CA1 cells with whole cell patch-clamp methods. The results showed that antimycin A decreased the amplitude of both persistent and transient sodium currents; antioxidant 2-mercaptopropionylglycine or 1,10 phenanthroline abolished the effect of antimycin A; the complex III Qo site inhibitor stigmatellin, the protein kinase C inhibitor chelerythrine, but not the protein kinase A inhibitor H89, canceled the effect of antimycin A; antimycin A decreased the amplitude of both persistent and transient sodium currents only at more depolarized membrane potentials and the decrease percentage of both persistent and transient sodium currents after antimycin A at potentials above 50 mV increased with the change in potentials toward more depolarized direction; exogenous application of H2O2 inhibited the amplitude of both persistent and transient sodium currents; the amount of current required to trigger spikes was increased and the number of spikes produced by varying levels of currents was decreased by antimycin A. These results suggest that the inhibition of Qi site of mitochondrial complex III decreases both persistent and transient sodium currents via reactive oxygen species and protein kinase C in rat hippocampal CA1 cells. D 2005 Elsevier Inc. All rights reserved. Keywords: Hippocampus; Whole cell patch-clamp; Mitochondrial; Complex III; Qi site; Antimycin A; Sodium currents; Excitability; Reactive oxygen species; Protein kinase C

Introduction The brain is very sensitive to hypoxia. Alterations in oxygen tension elicit a variety of functional responses in neurons, including altered mitochondrial respiratory chain, altered ion channel, altered intracellular environment, altered gene expression, and altered release of neurotransmitters, etc. (Boutilier, 2001; Duranteau et al., 1998; Lipton, 1999; Nicholls and Budd, 2000). Among them, the alteration in mitochondrial respiratory chain is one focus of

* Corresponding author. Fax: +86 21 64174579. E-mail address: [email protected] (P. Zheng). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.03.005

study. The inhibition of the complex I, complex II, complex III, and complex IV of the mitochondrial respiratory chain during hypoxia has been observed (Agani et al., 2000; Almeida et al., 2002; Bolanos et al., 1998; Brooks et al., 2000; Brown and Borutaite, 1999; Chavez et al., 1995; Dagani et al., 1989; Delgado-Esteban et al., 2002; Horakova et al., 1998; Vaux et al., 2001; Srinivas et al., 2001; Wagner et al., 1990), especially the inhibition of the complex III has received considerable attention because evidence points to a role for this complex as the O2 sensor during hypoxia (Chandel et al., 2000). The complex III has two centers: the Qi center, located in the inner membrane and facing the mitochondrial matrix, and the Qo center, oriented toward the intermembrane space

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(Chen et al., 2003). The inhibition of the complex III under hypoxia has been known to occur at both the Qi and Q0 site (Chandel et al., 2000). Among them, the Qi site inhibition is considered to be a direct response to hypoxia (Chen et al., 2003). However, the downstream pathways of the inhibition of the Qi still require to be studied. One downstream pathway of the inhibition of the Qi site of the complex III has been proposed to be that hypoxia inhibits the Qi site of the complex III, producing redox changes in the electron carriers and increasing the generation of reactive oxygen species (ROS), which lead to activation of hypoxia-inducible factor-1a and changes in the expression of hypoxia-responsive genes (Chandel et al., 2000c). However, this pathway mainly involves alterations in gene expression, whereas it is still unknown whether the inhibition of the Qi site of the complex III has an impact on the function of sodium channels and neuronal excitability. We hypothesize that in addition to the initiation of the activation of hypoxia-inducible factor-1a, the inhibition of the Qi site of the complex III during hypoxia may also induce an alteration in neuronal excitability via modulation of sodium channels, which may constitute another important pathway for hypoxia-induced functional responses in neurons because neuronal excitability is an important factor in the setting of oxygen consumption and in adaptation to hypoxia (Xia and Haddad, 1999). To test our hypothesis, we used the Qi site inhibitor antimycin A to mimic the inhibition of the Qi site and studied the effects and mechanism of the inhibition of this site on persistent sodium currents, transient sodium currents, and neuronal excitability in rat hippocampal CA1 cells with whole cell patch-clamp methods in slices and isolated cells. These investigations provide evidence for revealing the signaling pathways and functional consequences of the inhibition of the Qi site of the complex III during hypoxia.

Materials and methods

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the cutting stage of a vibratome (VT1000M/E, Leica, Wetzlar, Germany). Transverse slices (400 Am) were cut and transferred to an incubating chamber (30 – 32-C) where they stayed for at least 1 h before recordings were begun. Visualization of pyramidal cells To visualize the cell, the slice was placed in a recording chamber and viewed with a fixed stage, upright microscope equipped with Nomarski optics and a 40 long working distance, water immersion objective (3 mm, N.A:0.7, Olympus, Tokyo, Japan). To increase the clarity of the image, infrared light was used to illuminate the slice. The resultant infrared differential interference contrast (DIC) images were visualized on a black –white TV monitor through the use of a low light sensitive CCD camera. Recordings were made from pyramidal cells located in the CA1 region of the hippocampus. Pyramidal cells were identified by their pyramidal shape and presence of apical dendrites. Acute dissociation of hippocampal CA1 neurons Isolated hippocampal CA1 neurons were prepared according to procedures described by Xu et al. (2003). Briefly, following slice preparation, slices were gently transferred a digestion solution containing: 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 20 mM piperazineN,NV-bis(2-ethanesulfonic acid), 25 mM glucose, 10 mM sucrose and protease (1 mg/ml, Type XIV), pH 7.4, at 30-C for 60 min. The solution was continuously bubbled with 100% O2 to ensure adequate oxygenation of slices. After enzyme treatment, tissue pieces were washed and neurons were dissociated using fire-polished Pasteur pipette. The cell suspension was placed in an open perfusion chamber mounted on the stage of the upright microscope. The cells were allowed to settle for 10 –15 min and the chamber was perfused with recording solution.

Preparation of hippocampal CA1 slices Whole cell recording in slices and isolated cells 14- to 21-day-old Sprague – Dawley rats were anesthetized with chloral hydrate (400 mg/kg, i.p.). All experimental procedures conformed to Fudan University as well as international guidelines on the ethical use of animals and all efforts were made to minimize the number of animals used and their suffering. Slices were prepared according to procedures described previously (Wang and Zheng, 2001). Briefly, following decapitation, the brains were quickly removed and submerged in ice-cold artificial cerebrospinal fluid (ACSF) containing 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, 10 mM sucrose, and saturated with 95% O2/5% CO2. A block of tissue containing the hippocampal CA1 region was cut and placed on a layer of moistened filter paper glued to

The slice was continuously perfused with the ACSF. Electrodes were pulled from glass capillaries using a Narishige micropipetter puller (model PB-7; Narishige, Japan). They had a resistance of 4– 6 MV when filled with the patch pipette solution. To pharmacologically isolate persistent sodium currents, the patch pipette solution was slightly modified from that used by Fleidervish et al. (1996). The internal pipette solution contained 140 mM CsCl, 0.1 mM CaCl2, 2 mM MgCl2, 1 mM EGTA, 2 mM ATP.K2, 0.1 mM GTP.Na3, and 10 mM HEPES, adjusted to pH 7.25 by CsOH. In addition, 200 AM Cd2+ was added to the ACSF to block Ca2+ currents. To achieve adequate space clamp of persistent sodium currents, Cs+ was used as a main cation to block most of the K+ currents and to make the neuron

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electronically more compact to minimize ‘‘space-clamp’’ error (Gorelova and Yang, 2000). Under these recording conditions, a depolarizing voltage step (from 70 to 40 mV, 1-s duration) at the holding potential of 70 mV was applied to activate persistent sodium currents. Persistent sodium current was demonstrated by the experiment that TTX (0.5 AM) could completely abolish this current. The current– voltage relationship of persistent sodium currents was determined in the range from 70 to 20 mV using step pulse commands (1-s duration) with 10-mV increments from a holding potential of 70 mV. It was similar to those reported by other authors (Alzheimer et al., 1993; French et al., 1990). There was significant persistent sodium current at 60 mV, which increased in amplitude with further depolarization to reach a maximum at 40 mV. However, as described by Alzheimer (1994) and confirmed by our experiment, even in the presence of Cs+, an outward current is still present at potentials more positive than 20 mV, which not only influences the reversal potential of the persistent sodium currents, but also complicates the effect at these potentials, so in the present study, when constructing the current – voltage curve, we discard the currents at potentials more positive than 20 mV. In addition, we monitored the access resistance and rejected all recordings with access resistance >20 MV. The current signals were recorded with an Axopatch 200 B amplifier (Axon, Union City, USA) connected to a Digidata1200 interface (Axon, Union City, USA). The data were digitized and stored on disks using pClamp (v.6; Axon, Union City, USA). All experiments were conducted at room temperature (21 – 24-C). The amplitude of persistent sodium currents was measured at the end of a 1-s voltage pulse. Mean current– voltage relationships were calculated by averaging the individual current –voltage relationship obtained from each cell in the study. When recording transient sodium currents, we used isolated cells because they could enable a better space clamp due to the lack of an extensive dendritic arborization (Parri and Crunelli, 1998). Neurons that were phrase-bright with distinct outlines and dendrites without rounding up and clubbing of dendrites were chosen for recording. Whole-cell currents were recorded using electrodes (2– 4 MV) filled with a solution containing: 150 mM CsF, 2 mM MgSO4I7H2O, 0.1 mM CaCl2, 1 mM EGTA, 2 mM K2ATP, 0.1 mM Na3GTP, 10 mM HEPES. pH was adjusted to 7.4 with CsOH. Currents were recorded under whole-cell voltage clamp with an Axopatch 200 B amplifier (Axon, Union City, USA) connected to Digidata1200 interface (Axon, Union City, USA). A 70– 90% series resistance compensation was routinely used. The cells were allowed to stabilize for 3 –5 min after establishing the whole cell recording configuration. In order to isolate sodium currents, reduce current amplitudes, and decrease series-resistance voltage-clamp errors, the cells were bath perfused with a recording solution containing: 15 mM NaCl, 123 mM choline chloride, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2,

10 mM sucrose, 10 mM glucose, 0.4 mM CdCl2, 0.3 mM NiCl2, and 10 mM HEPES. pH was adjusted to 7.3 by NaOH. The solution was bubbled with pure oxygen. In voltage clamp mode, transient sodium currents were evoked using a depolarizing voltage step (from the holding potential of 70 mV to the test potential of 30 mV, 20-ms duration). The transient sodium currents were demonstrated by the experiment that TTX (0.5 AM) could completely abolish this current. The current –voltage relationship of transient sodium currents was determined in the range from 70 to +60 mV using step pulse commands (20-ms duration) with 10-mV increments from a holding potential of 70 mV. Mean current – voltage relationships were calculated by averaging the individual current – voltage relationship obtained from each cell in the study. The current –voltage relationship of the transient sodium currents was similar to those reported by other authors (Cantrell et al., 1997; Jeub et al., 2002). Data analysis Statistical significance between two groups was evaluated by Student’s two-tailed t test for paired data. The significance of the differences between multiple groups at different time-points was evaluated by one-way ANOVA with a post hoc Tukey test. All values were expressed as means T standard errors of means (SEM) and the number of cells (n) in each group was given. Drugs Antimycin A, ATP.K2, GTP.Na3, CsCl, CsF, CsOH, CdCl2, NiCl2, 1,10 phenanthroline (PHEN), and 2-mercaptopropionylglycine (MPG), stigmatellin, chelerythrine, and N-[2-( p-bromocinnamylamino)-ethyl]-5-isoquinoline-sulfonamide 2HCl (H89) were purchased from Sigma. Tetrodotoxin (TTX) was made in the Research Institute of the Aquatic Products of Hebie. Other reagents in AR grades were products of Shanghai Chemical Plant. Antimycin A, PHEN, MPG, TTX, H2O2, stigmatellin, chelerythrine, and H89 were applied by bath perfusion.

Results Antimycin A decreases the amplitude of persistent sodium currents The effect of antimycin A on persistent sodium currents was examined in hippocampal CA1 slices by a depolarizing voltage step from the holding potential of 70 mV to the test potential of 40 mV of 1-s duration with a frequency of 0.03 Hz. The results in a typical cell (Fig. 1A) and in a group of cells (n = 8, Fig. 1B) before and 4 min after application of antimycin A (3 AM) showed that antimycin A induced a significant decrease in the amplitude of persistent

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Fig. 1. Effect of antimycin A on persistent sodium currents in hippocampal CA1 slices. (A) Raw data before and 4 min after application of antimycin A 3 AM. Persistent sodium currents were evoked by a depolarizing voltage step from the holding potential of 70 mV to the test potential of 40 mV of 1-s duration with a frequency of 0.03 Hz. (B) The averaged amplitude of persistent sodium currents before and 4 min after antimycin A. n = 8, *P < 0.05 vs. control before antimycin A. (C) The time course of the antimycin A effect. *P < 0.05, n = 8.

sodium currents. The averaged amplitude of persistent sodium currents was reduced from 73.2 T 5.2 pA to 45.0 T 4.8 pA at 4 min after antimycin A (Fig. 1B, P < 0.05). The time course showed that the effect of antimycin A began to appear at 3 min ( P < 0.05) and persisted throughout the rest of the experiment (Fig. 1C). The effect of antimycin A remained unchanged after 10-min washout. The averaged percentage of inhibition before and after washout was 40.5% and 50.8% of control (n = 4). Effect of antimycin A on persistent sodium currents is canceled by MPG or PHEN We next studied whether reactive oxygen species (ROS) was involved in the effect of antimycin A on persistent sodium currents. Two different antioxidants 1,10 phenanthroline (PHEN, an inhibitor of hydroxyl radical formation) and 2-mercaptopropionylglycine (MPG, a scavenger of H2O2) were used. Cells were treated with PHEN or MPG during preincubation and antimycin A exposure. Fig. 2 was the effect of antimycin A (3 AM) on the amplitude of persistent sodium currents in the presence of PHEN 10 AM (Fig. 2A) or MPG 300 AM (Fig. 2B). It showed that PHEN or MPG could completely inhibit the effect of antimycin A (n = 6). Interestingly, PHEN and MPG alone had no significant effect

on persistent sodium currents (n = 4), but addition of PHEN and MPG at 4 min after antimycin A could restore the inhibited sodium currents from 51.9 T 20.3% to 75.2 T 20.7% of control ( P < 0.05, compared to antimycin A before PHEN and MPG, n = 4). These results suggest that ROS (hydroxyl radical and H2O2) plays a key role in the inhibitory effect of antimycin A on persistent sodium currents. Antimycin A decreases the amplitude of transient sodium currents The effect of antimycin A on transient sodium currents was examined in acutely isolated hippocampal neurons by a depolarizing voltage step from the holding potential of 70 mV to the test potential of 30 mV of 20-ms duration with a frequency of 0.03 Hz. As shown in Fig. 3, the addition of antimycin A (3 AM) produced a significant inhibition of the amplitude of transient sodium currents (n = 6). The averaged amplitude of transient sodium currents was decreased from 1957.4 T 289.1 pA to 753.6 T 220.6 pA (Fig. 3B, P < 0.05) at 4 min after antimycin A. The time course showed that the effect of antimycin A began to appear at 2 min ( P < 0.05) and persisted throughout the rest of the experiment (Fig. 3C). The effect of antimycin A was irreversible after 10-min washout. The averaged percentage

Fig. 2. Influence of MPG or PHEN on the effect of antimycin A on persistent sodium currents. (A) The effect of antimycin A 3 AM on the amplitude of persistent sodium currents in the presence of PHEN 10 AM. n = 6, P > 0.05, PHEN + antimycin A vs. PHEN. (B) The effect of antimycin A 3 AM on the amplitude of persistent sodium currents in the presence of MPG 300 AM. n = 6, P > 0.05, MPG + antimycin A vs. MPG.

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Fig. 3. Effect of antimycin A on transient sodium currents in acutely isolated hippocampal CA1 neurons. (A) Raw data before and 4 min after application of antimycin A 3 AM. Transient sodium currents were evoked by using a depolarizing voltage step from the holding potential of 70 mV to the test potential of 30 mV of 20-ms duration with a frequency of 0.03 Hz in acutely isolated hippocampal neurons. (B) The averaged amplitude of transient sodium currents before and 4 min after antimycin A. n = 6, *P < 0.05. (C) The time course of the antimycin A effect. n = 6, *P < 0.05, vs. control before antimycin A.

of inhibition before and after washout was 39.6% and 38.9% of control (n = 4). Effect of antimycin A on transient sodium currents is also canceled by MPG or PHEN We also tested whether ROS was involved in the effect of antimycin A on transient sodium currents. Fig. 4 was the effect of antimycin A (3 AM) on the amplitude of transient sodium currents in the presence of the antioxidant PHEN 10 AM (Fig. 4A) or MPG 300 AM (Fig. 4B). It could be seen that PHEN or MPG could completely inhibit the effect of antimycin A (n = 4). Moreover, PHEN and MPG alone had no significant effect on transient sodium currents (n = 4), but application of PHEN and MPG at 4 min after antimycin A could restore the inhibited sodium currents from 37.8 T 12.7% to 66.5 T 25.0% of control ( P < 0.05, compared to antimycin A before PHEN and MPG, n = 4). These results suggest that ROS (hydroxyl radical and H2O2) also plays a key role in the inhibitory effect of antimycin A on transient sodium currents. Effect of antimycin A on persistent and transient sodium currents is canceled by pretreatment with stigmatellin To test if the antimycin A-induced ROS is produced from the quinol oxidation (Qo) site of the complex III, we

observed the influence of the Qo site inhibitor stigmatellin on the effect of antimycin A. The result showed that stigmatellin (1 AM) alone had no effects on both persistent and transient sodium currents (n = 6), but in the presence of stigmatellin the effect of antimycin A (3 AM) on both persistent and transient sodium currents disappeared. The averaged persistent sodium currents before and after antimycin A in the presence of stigmatellin were 258.2 T 79.5 pA and 245.9 T 67.5 pA ( P > 0.05, n = 4); the averaged transient sodium currents before and after antimycin A in the presence of stigmatellin were 2305.7 T 692.1 pA and 2307.7 T 729.3 pA. These results suggest that the antimycin A-induced ROS is produced from the Qo site of the complex III. The voltage dependence of the effect of antimycin A on persistent sodium currents is similar to that on transient sodium currents The voltage dependence of the effect of antimycin A on persistent and transient sodium currents was analyzed by constructing the current –voltage relationship and the change percentage of persistent and transient sodium currents at different potentials before and after antimycin A. As shown in Fig. 5A, antimycin A (3 AM) decreased the amplitude of both persistent and transient sodium currents only at more depolarized membrane potentials ( 40 to 20 mV for

Fig. 4. Influence of the antioxidants MPG or PHEN on the effect of antimycin A on transient sodium currents. (A) The effect of antimycin A 3 AM on the amplitude of transient sodium currents in the presence of PHEN 10 AM. n = 4, P > 0.05, PHEN + antimycin A vs. PHEN. (B) The effect of antimycin A 3 AM on the amplitude of transient sodium currents in the presence of MPG 300 AM. n = 4, P > 0.05, MPG + antimycin A vs. MPG.

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Fig. 5. The voltage-dependence of the effect of antimycin A on persistent and transient sodium currents. (A) The current – voltage relationship of persistent and transient sodium currents before and 4 min after antimycin A 3 AM. n = 6. (B) The voltage-dependence of the effect of antimycin A 3 AM on persistent and transient sodium currents. n = 6.

persistent sodium currents, n = 6; 30 to 40 mV for transient sodium currents, n = 6). The decrease percentage of both persistent and transient sodium currents after antimycin A at potentials above 50 mV increased with the increase in potentials toward more depolarized direction (Fig. 5B). This result suggests that the effect of antimycin A on persistent and transient sodium currents has similar voltage-dependence. H2O2 inhibits the amplitude of both persistent and transient sodium currents The effect of H2O2 on persistent and transient sodium currents was tested. As shown in Fig. 6A, H2O2 (100 AM) significantly decreased the amplitude of persistent sodium currents. The averaged amplitude of persistent sodium currents was decreased from 169.1 T 30.1 pA to 86.8 T 34.8 pA (the right panel of Fig. 6A, n = 4, P < 0.05) at 4 min after H2O2. Similarly, the amplitude of transient sodium currents was also inhibited by H2O2 (100 AM) (Fig. 6B). The averaged amplitude of transient sodium currents was decreased from 2269.1 T 806.6 pA to 1069.3 T 334.5 pA (the right panel of Fig. 6B, n = 4, P < 0.05) at 4 min after H2O2. We also tested the effect of 1 mM H2O2 on the persistent and transient sodium currents and obtained a similar result to those of 100 AM H2O2 (data not shown

here). In addition, application of PHEN (10 AM) and MPG (300 AM) at 4 min after antimycin A could restore the inhibited persistent sodium currents by antimycin A from 43.8 T 20.2% to 73.0 T 32.3% of control ( P < 0.05, compared to H2O2 before PHEN and MPG, n = 4) and the inhibited transient sodium currents from 52.0 T 14.9% to 83.3 T 29.5% of control ( P < 0.05, compared to H2O2 before PHEN and MPG, n = 4). Effect of antimycin A on persistent and transient sodium currents is canceled by chelerythrine, but not by H89 To test the possible involvement of protein kinase C (PKC) in the effect of antimycin A on sodium currents, we observed the influence of the PKC inhibitor chelerythrine on the effect of antimycin A. The results showed that in the presence of chelerythrine (2.5 AM) the effect of antimycin A (3 AM) on both the persistent sodium currents (the left panel of Fig. 7A) and the transient sodium currents (the right panel of Fig. 7A) disappeared. The averaged persistent sodium currents before and after antimycin A in the presence of chelerythrine were 288.1 T 74.6 pA and 294.7 T 59.7 pA ( P > 0.05, n = 4); the averaged transient sodium currents before and after antimycin A in the presence of chelerythrine were 2919.3 T 526.1 pA and 2842.4 T 513.7 pA ( P > 0.05, n = 4).

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Fig. 6. Effect of H2O2 on the amplitude of persistent and transient sodium currents. (A) Effect of H2O2 on the amplitude of persistent sodium currents in hippocampal CA1 slices. Left panel: raw data before and 4 min after application of H2O2 100 AM. Persistent sodium currents were evoked by a depolarizing voltage step from the holding potential of 70 mV to the test potential of 40 mV of 1-s duration with a frequency of 0.03 Hz. Right panel: the averaged amplitude of persistent sodium currents before and 4 min after H2O2. n = 4, *P < 0.05 vs. control before H2O2. (B) Effect of H2O2 on the amplitude of transient sodium currents in isolated hippocampal CA1 cells. Left panel: raw data before and 4 min after application of H2O2 100 AM. Transient sodium currents were evoked by using a depolarizing voltage step from the holding potential of 70 mV to the test potential of 30 mV of 20-ms duration with a frequency of 0.03 Hz. Right panel: the averaged amplitude of transient sodium currents before and 4 min after H2O2. n = 4, *P < 0.05 vs. control before H2O2.

This result suggests that PKC may be involved in the effect of antimycin A on the persistent and transient sodium currents. We also investigated the role of protein kinase A (PKA) in the effect of antimycin A on sodium currents by observing the influence of the PKA inhibitor H89 on the effect of antimycin A. The results showed that in the presence of H89 (1 AM) antimycin A (3 AM) still had an inhibitory effect on both the persistent sodium currents (the left panel of Fig. 7B) and the transient sodium currents (the right panel of Fig. 7B). The averaged persistent sodium currents before and after antimycin A in the presence of H89 were 230.2 T 55.0 pA and 119.9 T 26.6 pA ( P < 0.05, n = 4); the averaged transient sodium currents before and after antimycin A in the presence of H89 were 2733.1 T 486.4 pA and 1668.6 T 470.8 pA ( P < 0.05, n = 4). This result suggests that PKA may not be involved in the effect of antimycin A on the persistent and transient sodium currents.

changes in spike number in response to a 1-s-long depolarizing current injection. The amplitude of the injected current (50 –200 pA) was adjusted to evoke approximately six action potentials. After 2 – 5 min of baseline measurements, antimycin (3 AM) was bath applied. The result showed that antimycin A had no significant effect on resting membrane potentials (data not shown here), but apparently decreased the number of spikes (Fig. 8A). The time course showed that the effect of antimycin A began to appear at 3 min ( P < 0.05) and persisted throughout the rest of the experiment (Fig. 8B, n = 8). In addition, the amount of current required to trigger spikes was increased and the number of spikes produced by varying levels of currents was decreased by antimycin A (Fig. 8C, n = 6). These results suggest that antimycin A significantly decrease neuronal excitability.

Antimycin A decreases neuronal excitability

Discussion

The effect of antimycin A on the excitability of hippocampal CA1 neurons in slices was studied by measuring

The main findings of this study are that antimycin A decreases the amplitude of both persistent and transient

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Fig. 7. Influence of the PKC inhibitor chelerythrine and the PKA inhibitor H89 on the effect of antimycin A on persistent and transient sodium currents. (A) The influence of the PKC inhibitor chelerythrine (2.5 AM) on the effect of antimycin A (3 AM) on the persistent sodium currents (the left panel) and the transient sodium currents (the right panel). n = 4, P > 0.05 vs. control and chelerythrine before antimycin A. (B) The influence of the PKA inhibitor H89 (1 AM) on the effect of antimycin A (3 AM) on the persistent sodium currents (the left panel) and the transient sodium currents (the right panel). n = 4, P > 0.05 vs. control, *P < 0.05 vs. H89 before antimycin A.

sodium currents; the effect of antimycin A on persistent and transient sodium currents is abolished by MPG or PHEN; the complex III Qo site inhibitor stigmatellin and the PKC inhibitor chelerythrine, but not the PKA inhibitor H89, cancel the effect of antimycin A; the voltage-dependence of the effect of antimycin A on persistent sodium currents is similar to that on transient sodium currents; H2O2 inhibits the amplitude of both persistent and transient sodium currents; antimycin A decreases neuronal excitability. Antimycin A is a highly selective inhibitor of complex III. Here, to confirm that antimycin A could inhibit complex III in our conditions, we used the method described by

Balijepalli et al. (1999) to check the antimycin A-induced inhibition of complex III activity. The result showed that the activity of complex III was reduced by 20.0 T 4.6% in response to 3 AM antimycin A. This result is consistent with those reported for antimycin A-induced complex III inhibition (32.2 T 6.8%) (Janssens et al., 2000) and ischemia-induced complex III inhibition (22%) (Petrosillo et al., 2003). Interestingly, all the above-mentioned inhibition involves partial, rather than complete, blockade of complex III. In addition, we also checked if antimycin A had an inhibitory effect on complex I using the method by Ben-Shachar et al., 2004). The result showed that antimycin

Fig. 8. The effect of antimycin A on neuron excitability in hippocampal CA1 slices. (A) Typical records of action potentials evoked by a 1-s-long depolarizing current injection before and 4 min after antimycin A 3 AM. (B) The time course of the effect of antimycin A on the number of spikes. n = 8, *P < 0.05 vs. control before antimycin A. (C) The effect of antimycin 3 AM on the number of spikes produced by varying levels of currents. n = 6.

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A had no effects on the activity of complex I, eliminating the possibility of a secondary response-induced complex I defect. Within the complex III, the binding site of antimycin A has been known to be at Qi site and defined by X-ray crystallography (Jeub et al., 2002; Kim et al., 1998). The action of antimycin A at the QI site is to inhibit electron transfer from semiquinone to the QI site and increase the semiquinone formation which can result in ROS generation (Chen et al., 2003). Therefore, it is most likely that antimycin A produces its effect on sodium currents by ROS. This hypothesis is confirmed by the present results that the antioxidant PHEN or MPG can completely abolish the effect of antimycin A on sodium currents. Moreover, since semiquinone can be generated in the Qo site of complex III (Junemann et al., 1998), it is possible that pretreatment of preparation with the Qo site inhibitor can block the effect of antimycin A. To test this hypothesis, we observed the influence of the Qo site inhibitor stigmatellin on the effect of antimycin A. The result showed that in the presence of stigmatellin the effect of antimycin A on both persistent and transient sodium currents disappeared. This result provides strong evidence that the antimycin Ainduced ROS is produced from the Qo site and excludes non-specific effects of respiratory inhibition on the sodium current response. ROS is a collective term including free radicals containing oxygen atom(s) and highly reactive non-radical oxygenated compounds (Castagne et al., 1999). Increasingly complex behavior of ROS, including cellular damage (Fridovich, 1978; Halliwell, 1992) and signaling (Finkel, 1998), is noted within biological systems, especially under ischemia and hypoxia. Previous studies showed that significant release of ROS in the brain was detected during ischemia and hypoxia (Li et al., 1999). The roles of released ROS during hypoxia have been found to influence gene activation (Chandel et al., 1998; Cimino et al., 1997; Merrill and Murphy, 1997) and subsequent events leading to ischemic cell death (Clemens, 2000) or cell survival (Chandel et al., 2000). In addition, a number of studies have examined possible roles of ROS in modulating synaptic transmission and spike generation (Avshalumov et al., 2000; Pellmar, 1995). However, to date, few studies have been performed to examine the actions of ROS produced from the inhibition of the Qi site of the mitochondrial complex III under hypoxia on the function of sodium channels. To our knowledge, the present paper is the first study that reports a pathway that connects the inhibition of the Qi site of the mitochondrial complex III with the function of sodium channels via ROS. ROS is initially generated as superoxide, which is subsequently converted to H2O2 (Turrens et al., 1985). A number of studies have suggested that H2O2 is important during hypoxia (Arthur et al., 2004) and antimycin Ainduced inhibition of the Qi site (Chen et al., 2003). Therefore, we hypothesize that H2O2 may play an important

role in the antimycin A-induced inhibition of persistent and transient sodium currents. To test this hypothesis, we observed the influence of the H2O2 scavenger MPG (Vanden Hoek et al., 1997) on the effect of antimycin A. The results showed that MPG could cancel the effect of antimycin A on sodium currents, suggesting that H2O2 might play an important role in the effect of antimycin A. This point was further supported by our results that bath application of H2O2 could mimic the effect of antimycin A. Of course, we did not know if the concentrations of H2O2 we used matched the amount of H2O2 produced by antimycin A, but the evidence that both low and high concentrations of H2O2 could mimic it appeared to support our hypothesis. Interestingly, our result also suggested that hydroxyl radicals, which could be produced from H2O2, might play an important role in the effect of antimycin A because the inhibitor of hydroxyl radical formation PHEN (Vanden Hoek et al., 1997) could cancel the effect of antimycin A. In addition, it has been reported that ROS, particularly H2O2, can activate PKC (Banno and Nozawa, 2003; Jung et al., 2004; Polosukhina et al., 2003), so it is possible that the activation of PKC is involved in the effect of antimycin A. To test this hypothesis, we observed the influence of the PKC inhibitor chelerythrine on the effect of antimycin A. The result showed that in the presence of chelerythrine the effect of antimycin A on both the persistent and transient sodium currents disappeared, suggesting that PKC was involved in the effect of antimycin A on the sodium currents. This is consistent with the report that hypoxia inhibits transient sodium currents in rat hippocampal neurons via protein kinase C (O’Reilly et al., 1997). However, we have not found a significant influence of the PKA inhibitor H89 on the effect of antimycin A, suggesting that PKA may not be involved in the effect of antimycin A on the sodium currents. In the mammalian central nervous system, sodium channels play an essential role in the initiation and propagation of action potentials (Cummins et al., 1994). The energy consumption induced by sodium channel activity is great. A large portion of the energy consumed by neurons is related to the maintenance of sodium ionic gradients across the cellular membrane via Na+/K+-ATPase activity after sodium channel activity (Xia and Haddad, 1999). Therefore, an immediate and effective way of reducing energy demand to protect brain tissue under hypoxia has been proposed to inhibit the activity of sodium channels (Xia and Haddad, 1999). In this aspect, neurons appear to have developed a kind of adaptation response by inhibition of sodium channel activity under hypoxia (O’Reilly et al., 1997). However, how neurons produce this response remains to be studied. The present study suggested that the inhibition of the Qi site of mitochondrial complex III under hypoxia might be one of the causes of the inhibition of sodium currents. This result broadens our understanding of how neurons inhibit sodium currents under hypoxia.

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The inhibition of both transient and persistent sodium currents by the Qi site inhibitor antimycin A is another important finding of the present paper. Although it is unknown if the two currents are generated by distinct populations of voltage-dependent sodium channels with independent kinetics or by a single population of sodium channels that possess multiple gating modes, both two currents play important roles in neuronal functions, especially in determining neuronal excitability and discharge (Catterall, 1993; Crill, 1996). Therefore, as the consequence of the decrease in persistent and transient sodium currents, antimycin A should produce an inhibitory effect on neuronal excitability and discharge. This hypothesis is confirmed by the present results that antimycin A increases the amount of current required to trigger spikes and decreases the number of spikes. This result is also another evidence supporting that the inhibition of the Qi site of the complex III can lead to a reduction of energy demand because neuron depolarization and action potential discharge consume a lot of energy (Xia and Haddad, 1999). In summary, the above results suggest that the inhibition of the Qi site of the complex III, which is a direct response to hypoxia (Chandel et al., 2000), can lead to a decrease in both transient and persistent sodium currents via ROS and PKC, and produce an inhibition of neuronal excitability and discharge. We propose that this response may be a protective response because it can lead to a decrease in ATP utilization and energy consumption that can make neurons match the limitation of ATP synthesis under hypoxia and protect neurons.

Acknowledgments This work was supported by the Shuguang Research Program of Shanghai Education Committee, Project 39970241 of National Natural Science, Foundation of China and Trans Century Training Program Foundation for the Talents by the State Education Commission, Project 04DZ14005 of Shanghai Science and Technology Committee.

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