INTRODUCTION

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Oxidative stress has been implicated in a variety of physiological and pathophysiological processes such as immune defense, ischemia, or neurodegenerative diseases, e.g., Alzheimer's disease. In addition to basal oxidative stress from energy metabolism, activity-dependent increase in demand for energy results in increased oxidative stress by means of [Ca²⁺] increases in the cytosol and subsequent uptake into mitochondria. Reactive oxygen species are known to affect the function of a variety of proteins, including membrane channels, in both detrimental and protective ways (Kourie 1998). Oxygen radicals are converted by superoxide dismutase into still reactive and very membrane permeable hydrogen peroxide (H₂O₂). H₂O₂ can be detoxified by two parallel pathways: catalase mediates the transformation of H₂O₂ to H₂O and O₂ and glutathione peroxidase catalyzes the reaction of H₂O₂ with glutathione (GSH) to H₂O and GSSG. Another Ca²⁺-dependent source of reactive radicals is liberation of arachidonic acid (AA) from membrane lipids by phospholipase A₂.

Voltage-gated potassium currents play crucial roles in modifying neuronal cellular and network excitability and activity (Müller and Misgeld 1990, 1991). Potassium currents control action potential duration, release of neurotransmitters and hormones, Ca²⁺-dependent synaptic plasticity and epileptiform burst activity (Müller and Connor 1991a; Müller et al. 1988; Pongs 1999). Many types of cloned K⁺ channels have been shown to be modulated by reactive oxygen species. Oxidation has been shown to decrease the conductance of Kv1.3, Kv1.4, Kv1.5, Kv3.4 (Duprat et al. 1995), and Kv3.3 channels (Vega-Saenz de Miera and Rudy 1992), while K⁺ currents through human ether-a-gogo-related gene (HERG) K⁺ channels are enhanced by oxidation (Taglialatela et al. 1997). Inactivation of A-type currents through Kv1.4 channels is dependent on the cellular redox state in a cysteine-dependent manner (Ruppersberg et al. 1991).

Enhanced excitability via downmodulation of K⁺ channels, or changes in the biophysical properties such as inactivation kinetics and voltage dependence, could all result in enhanced Ca²⁺ responses to firing activity (Pongs 1999). This additional intracellular free Ca²⁺ will be taken up, in turn, by mitochondria and can stimulate oxidative phosphorylation as well as generation of reactive oxygen species (ROS). By acting back again on the K⁺ channels, ROS may form part of a circulus vitiosus. On the other hand, decrease of excitability could protect neurons by reducing action potential firing and evoked increases in intracellular Ca²⁺. This would reduce oxidative bursts generated secondary to Ca²⁺-driven processes. In light of these connections, surprisingly few studies have addressed oxidative modulation of K⁺ currents in central neurons.

We have shown previously that AA selectively inhibits the transient A-type K⁺ current in hippocampal neurons, an effect that depends on an oxidative mechanism (Bittner and Müller 1999). In the present study, we demonstrate a high degree of specificity of AA versus H₂O₂ with respect to biophysical alterations of I_A as well as of I_K(V), suggesting different access of these membrane permeable molecules to redox amino acid residues on these channel proteins.

	METHODS

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Primary rat hippocampal cultures were prepared according to standard methods as described previously (Bittner and Müller 1999). Currents were recorded at room temperature (22-24°C) from neurons cultured for 2-3 wk, using whole cell patch-clamp recording techniques (SEC-05 L, npi electronics, Tamm, Germany) (Misgeld et al. 1989; Müller and Swandulla 1995) and filtered at 1.3 kHz. The internal solution contained (in mM) 120 KCl, 1 CaCl₂, 2 MgCl₂, 11 EGTA, 10 HEPES, and 20 D-glucose; pH 7.3. For intracellular application, the tip of the pipette was filled with 2-5 µl of this solution and backfilled with AA-containing solution. Neurons were continuously superfused (1 ml/min) with external saline containing (in mM) 130 NaCl, 5.4 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 25 D-glucose, and 0.001 TTX. Stock solutions of AA (100 mM) in DMSO were stored at 18°C. Experimental solutions were prepared immediately before use. Control recordings with the same concentration of DMSO had effects neither on I_A nor on I_K(V). H₂O₂ was added to the extracellular saline immediately prior to application from a 30% stock. AA, GSH, and TTX were obtained from Sigma. Averages are given as means ± SE; 2-tailed unpaired Student's t-test was used for statistical analysis.

Curve fitting of K⁺ currents and data and statistical analyses were performed by IGOR and the Jandel Sigma Plot 2.0 software (Jandel GmbH, Erkrath, Germany).

Currents at each test potential were converted to conductances (g) using the following formula

(1)

where E_K is the equilibrium potential (82 mV) confirmed experimentally. Because [K⁺]_i is clamped by perfusion of the cell through the patch pipette, this method does not allow assessment of possible effects of oxidants on E_K. The peak conductance value for each test potential was normalized to g_max (maximal g for the cell) and plotted against the test potential to produce a voltage-conductance relationship curve.

The current activation and inactivation kinetics were fitted using the following Boltzmann equations (Hlubek and Cobbett 1997), respectively

Activation

(2)

Inactivation

(3)

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

H₂O₂ and AA both reduce I_A but only H₂O₂ reduces I_K(V)

In whole cell patch-clamp recording, delayed rectifier (I_K(V)) and A currents (I_A) were recorded using an 800-ms hyperpolarizing prepulse to 110 mV to remove inactivation of I_A (Fig. 1, left inset). Hippocampal neurons were then step depolarized to potentials between 60 and +40 mV in increments of 10 mV. When the prepulse was followed by a 50-ms interval at 45 mV (Fig. 1, right inset), I_A inactivated completely (Connor and Stevens 1971b; Klee et al. 1995), and pure recordings of I_K(V) were obtained as shown in Fig. 1B. I_K(V) had an amplitude of 0.5-5 nA and activated with a time constant of 2.6 ± 0.4 ms (Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Hydrogen peroxide (H2O2) suppresses voltage-dependent K+ currents. A and B: superfusion of 0.027% H2O2 (8 mM) reduces both, the A current (IA, A) and the delayed rectifier current (IK(V), B), by 60 and 20%, respectively, without affecting the current kinetics. All currents were recorded at +30 mV. Insets (bottom):the pulse protocols for recording both currents together (left) and for recording IK(V) in isolation after inactivation of IA by a 50-ms interval at 45 mV. A-current recordings were obtained by subtracting delayed rectifier currents from combined currents.

By subtracting I_K(V) from mixed currents, I_A was isolated. I_A had an amplitude of 0.5-2 nA and decayed with a time constant of 15 ms (Fig. 1A, Table 1). Figure 1A demonstrates reduction of I_A by H₂O₂. Extracellular perfusion of 0.00027-0.027% H₂O₂ (80-8000 µM) reduced the A current by >50% over a time course of 2-10 min but did not affect the rates of activation and inactivation (Figs. 1 and 2A). These effects are in complete agreement with the effects of intracellular (1 pM) or extracellular (1 µM) perfusion of AA reported previously (Bittner and Müller 1999). While AA did not affect the delayed rectifier current I_K(V), 0.0027% H₂O₂ (800 µM) decreased I_K(V) by 22%, without altering the time constant of activation (Fig. 1B, Table 1). Figure 2B shows the dose-response relation for inhibition of I_A in comparison to intracellular and extracellular application of AA (Bittner and Müller 1999). While with intracellular application of AA, the incredible low concentrations of 1 and 10 pM both reduced I_A by some 55% within 2-3 min (54 ± 7 and 53 ± 8%), extracellular application of AA evoked equivalent effects on I_A only at concentrations of 1 µM over a time course of 10-15 min. Extracellular application of H₂O₂ significantly reduced I_A only at concentrations >80 µM over a time course of 3-6 min (Fig. 2). H₂O₂ (800 µM) reduced I_A by not more than 25%, whereas at 8 mM, it reduced I_A by 68 ± 7% (Fig. 2B, Table 1).

View larger version (30K): [in this window] [in a new window]   Fig. 2. Time courses and dose dependence of inhibition of IA by H2O2 in comparison to arachidonic acid (AA). A: superfusion of H2O2 results within 3-6 min in a stable inhibition of IA while the effect of superfusion of AA (1 µM, [AA]o) develops slowly. Intracellular application of an extreme low concentration of AA (1 pM, [AA]i), is acting rapidly and effectively (AA data taken from Bittner and Müller 1999). B: especially intracellular but also extracellular application of AA requires much lower concentrations than extracellular application of H2O2 for inhibition of IA.

H₂O₂ shifts inactivation and activation of I_A and activation of I_K(V) to more negative potentials, while AA shifts inactivation of I_A only

Voltage dependence of activation was determined by voltage commands from a prepulse to 110 mV for 800 ms to test potentials between 60 and +40 mV in increments of 10 mV (see Fig. 3 middle and right inset). Voltage dependence of steady-state inactivation was determined by 800-ms hyperpolarizing pulses to potentials between 120 and 50 mV in increments of 10 mV, followed by the test pulse to 20 mV (see Fig. 3, left inset). For inactivation of I_A, a 50-ms interval at 45 mV was inserted between the two pulses. AA shifted the voltage dependence of inactivation in parallel from 75 to 85 mV, without affecting the I-V relation for activation (Bittner and Müller 1999). In contrast to the effect of AA, H₂O₂ shifted the voltage dependence of both inactivation and activation to more negative potentials and affected the steepness of both relations. Showing the conductance-voltage (g-V) relation for inactivation and activation of I_A and fits to these data using the Boltzmann equations (Eqs. 2 and 3) in control, during perfusion of H₂O₂ and during wash of H₂O₂, Fig. 3 demonstrates that the voltage of half-maximal conductance for steady-state inactivation, V_0.5i was shifted from 62 to 74 mV during perfusion of H₂O₂ and to 76 mV during washout of H₂O₂ (Table 1). For activation, H₂O₂ shifted V_0.5a from 37 over 44 mV during perfusion of H₂O₂ to 55 mV during wash of H₂O₂. H₂O₂ increased the inverse steepness factor k of the Boltzmann equation for activation from 8.9 in control to 13.8, and for inactivation, it decreased it from 9.8 to 6.5 (Fig. 3A, Table 1).

View larger version (27K): [in this window] [in a new window]   Fig. 3. Effects of H2O2 on the voltage dependence of inactivation and activation of IA. A: superfusion of 0.0027% H2O2 (800 µM) causes a significant shift of steady-state inactivation and activation to more negative potentials. The slope of the relation for activation and inactivation is reduced and enhanced, respectively, by H2O2. The effect of H2O2 even continues to grow during wash for 3 min before degradation of the seal. B: current recordings for activation before (B1), during superfusion (B2), and during wash (B3) of H2O2 (800 µM). Insets: the pulse protocols for recording steady-state inactivation (left), for recording activation (middle), and for recording activation of IK(V) in isolation after inactivation of IA by a 50-ms interval at 45 mV (right).

As already mentioned, H₂O₂ (800 µM) reduced the delayed rectifier current I_K(V) to a similar degree as the transient current I_A, i.e., the delayed rectifier current I_K(V) was reduced by 22% (Fig. 4B, Table 1), whereas AA had no effect on I_K(V) (Bittner and Müller 1999). Figure 4 demonstrates that H₂O₂ shifted, in addition, activation to more negative potentials (pulse protocol: see inset). Fit of the Boltzmann equation to the conductance-voltage relation data gave a 9.4 mV shift of V_0.5 to a more negative potential, i.e., from 6.8 to 16.2 mV for 0.0027% H₂O₂ (800 µM, Table 1). The steepness of the relation was not changed in H₂O₂ (n = 7). H₂O₂ did not affect significantly the time constant of activation of I_K(V) at a membrane potential of 0 mV (unpaired t-test, Table 1). The effects of H₂O₂ were only partially reversible during the remaining recording time.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effects of H2O2 on the voltage dependence of activation of IK(V). A: superfusion of 0.0027% H2O2 (800 µM) causes a significant shift of activation to more negative potentials. The slope of the relation for activation is not affected. The effect is partially reversible within 8 min wash of H2O2. Inset: the pulse protocol with the 50-ms interval at 45 mV for inactivation of IA. B: current recordings for activation before (B1), during superfusion (B2), and during wash (B3) of H2O2 (800 µM).

Effects of H₂O₂ are mediated by both intracellular and extracellular sites of action

To address a mediation of H₂O₂ effects by intracellular oxidative modifications, we included the physiological antioxidant GSH (2 mM) into the intracellular recording saline. Figure 5 shows that this antioxidative agent completely blocked the effect of H₂O₂ on the maximal conductance of I_A and on the steepness of the conductance voltage relation of activation while the shift of V_0.5a to more negative potentials was not affected by intracellular GSH (Fig. 5A, Table 1). The effects of H₂O₂ were only partially reversible during the remaining recording time.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Effects of H2O2 during intracellular application of glutathione (GSH, 5 mM) on the voltage dependence of inactivation and activation of IA. A: superfusion of 0.0027% H2O2 (800 µM), during intracellular application of GSH still causes a significant shift of steady-state inactivation to more negative potentials. In contrast to recording with GSH-free patch pipettes, now H2O2 significantly reduces the slope of the relation. With GSH, the slope of the conductance-voltage relation for activation is no longer changed by H2O2. B: current recordings before (B1), during superfusion (B2) and during wash (B3) of H2O2 (800 µM) from a GSH-filled neuron demonstrate lack of effect on the maximal current amplitude.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Effects of H2O2 during intracellular application of GSH (5 mM) on the voltage dependence of activation of IK(V). A: superfusion of 0.0027% H2O2 (800 µM) during intracellular application of GSH still causes a significant shift of activation to more negative potentials. The slope of the relation for activation is marginally increased. The effect was not reversible during wash for 6 min. B: current recordings for activation before (B1), during superfusion (B2), and during wash (B3) of H2O2 (800 µM) reveal enhanced inhibition of the current amplitudes, i.e., the effect of H2O2 is enhanced during intracellular application of GSH (cf. Fig. 4).

Figure 5A, left, demonstrates that the H₂O₂-evoked increase of the steepness of steady state inactivation was reversed to a decrease in the presence of GSH (Table 1), while the effect on V_0.5i was not significantly changed by GSH (V_0.5i = 13.5 mV in control vs. V_0.5i = 14.2 mV in the presence of GSH). The effects of H₂O₂ were hardly reversible during washout of H₂O₂ during the remaining recording time (3-10 min).

Intracellular application of GSH (5 mM) significantly reduced the delayed rectifier conductance by 23% (n = 6, P < 0,05, Table 1). Figure 6 illustrates that during intracellular application of GSH (2 mM), H₂O₂ reduced the maximal conductance of I_K(V) by 39 ± 9% (P < 0.01) instead of by 22 ± 3% in the absence of GSH in the patch pipette (Table 1). The shift of activation to more negative potentials was not affected by [GSH]_i (Fig. 6; Table 1). Table 1 gives an overview of all effects by H₂O₂ and AA on the biophysical parameters of I_A and I_K(V).

	DISCUSSION

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Our experimental data characterize oxidative modulation of voltage-activated K⁺ currents in hippocampal neurons by H₂O₂ in comparison to AA. Like AA, H₂O₂ significantly reduces g_max of the transient voltage-activated K⁺ current I_A and causes a shift of steady-state inactivation to more negative membrane potentials (Figs. 1 and 3). However, the fast time course of the effect of H₂O₂, compared with the effect of extracellular application of AA, indicates a much better membrane permeability for H₂O₂ over AA (Fig. 2). A limited exchange of AA across the membrane was already suggested by the enormous ratio of extracellular to intracellular effective concentrations of AA (>10⁶:1) in our previous work (Bittner and Müller 1999).

Inclusion of the antioxidant GSH in the patch pipette blocks all effects of very low intracellular concentrations of AA (1 pM), and the reduction of g_max by superfusion of H₂O₂ (Fig. 5). These results support the conclusion that these effects of AA and H₂O₂ appear to be mediated by oxidation of intracellular amino acid residues, most likely of A-current K⁺-channel subunits.

H₂O₂ not only mimics the effects of AA, albeit at much higher concentrations (>800 vs. 1 µM, respectively, with extracellular application), but has additional effects on the transient A current in hippocampal neurons. The slope of voltage dependence of steady state inactivation is significantly enhanced by H₂O₂ (Fig. 3). This effect is probably due to oxidation of amino acid residue(s) facing the intracellular space because this effect is blocked by intracellular application of GSH (Fig. 5). However, in the intracellular presence of GSH, the reversed effect is observed, i.e., H₂O₂ causes a reduction in the slope of voltage dependence of steady-state inactivation. This suggests that oxidation of an extracellular site or a site facing the lipid bilayer of the cell membrane causes this reduction in the voltage dependence of steady-state inactivation (Fig. 5). Hence the important increase in this voltage dependence in control conditions appears to be even an underestimate of the intracellular effect because of partial balance by the opposite effect, occurring beyond the intracellular face of the channel protein.

In contrast to AA, H₂O₂ also affects the voltage dependence of activation of I_A. H₂O₂ causes a significant shift of activation to more negative potentials and reduces the dependence of activation on the membrane potential (Fig. 3). The latter effect is sensitive to intracellular application of GSH (Fig. 5) and, hence, may be mediated by oxidation of a cytosolic domain of the K⁺ channel that, again, is not readily oxidized by AA. In contrast, the shift of activation to more negative potentials may be associated with a site not facing the intracellular space because it is not prevented in the intracellular presence of GSH (Fig. 5).

Whereas AA does not affect the delayed rectifier current, H₂O₂ causes a decrease of the maximal conductance that was enhanced with intracellular perfusion of GSH (Figs. 4 and 6). This can be explained by H₂O₂ causing a decrease of I_K(V) by oxidation of an extracellular site or a site within the cell membrane. The enhancement of this decrease of I_K(V) with intracellular perfusion of GSH points to oxidation of an intracellular amino acid residue causing an increase of I_K(V) and prevention of this effect by GSH. In agreement with this conclusion, intracellular perfusion with 5 mM GSH decreases g_max of the delayed rectifier current by 23% even during basal oxidative stress (Table 1). The intracellular oxidative increase of I_K(V) apparently compensates, in part, the reduction of I_K(V) in control conditions. In addition, H₂O₂ causes a left shift of activation of I_K(V) that is not sensitive to intracellular supply of GSH (Figs. 4 and 6).

Oxidative modulation of cloned K⁺ channels in expression systems has been addressed in several previous studies. In contrast to slowing or removal of inactivation of A-type K⁺ channels and an increase in conductance (Ruppersberg et al. 1991; Serodio et al. 1996; Vega-Saenz de Miera and Rudy 1992), these effects were not observed in hippocampal neurons. In hippocampal neurons, the respective Kv1.4 K⁺-channel subunits (Maletic-Savatic et al. 1995; Veh et al. 1995) appear to be segregated to axons and presynaptic terminals while postsynaptic I_A and I_K(V) is most likely mediated by Kv4.2 and Kv2.1 channels, respectively (Murakoshi and Trimmer 1999; Sheng et al. 1992). Similar to our findings, 20 µM AA strongly reduced the A current in Kv4.2-expressing oocytes (Villarroel and Schwarz 1996), although clear discrepancies in the effects on the voltage dependence of inactivation and activation point toward involvement of additional molecules and mechanisms in the oxidative modulation of K⁺ currents in hippocampal neurons. Interestingly, coexpression of Kv1.2 with Kv4.2 confers sensitivity of the current to oxidizing and reducing agents. In disagreement with our findings, oxidation increases and reduction decreases this current (Perez-Garcia et al. 1999). Kv2.1-mediated currents are reduced in the presence of H₂O₂ only after a site directed mutation (Zhang et al. 1996). Hence the molecular components and mechanisms in the oxidative inhibition of both currents in hippocampal neurons, I_A and I_K(V), remain to be clarified.

Delayed rectifier currents and A-type currents contribute to action potential repolarization while the A current plays, in addition, an important role in repetitive firing and backpropagation of action potentials into dendrites (Connor and Stevens 1971a; Hoffman et al. 1997). Indeed, Colbert and Pan demonstrated in hippocampal CA1 neurons a strong enhancement of dendritic action potentials during inhibition of I_A by AA (Colbert and Pan 1999). Oxidative inhibition of these K⁺ currents and augmentation of postsynaptic Ca²⁺ responses to excitatory input result in enhanced oxidative stress in a positive feedback loop. Changes in Ca²⁺ signals as well as oxidative stress can impair synaptic plasticity and memory (Auerbach and Segal 1997; Giese et al. 1998) and may contribute to slow and fast neurodegeneration in a variety of neurological diseases. The high potency of AA supports an involvement of oxidative modulation of A-channel activity in physiological signal transduction, assuming that rather high levels of AA will be reached at hot spots of intracellular free Ca²⁺ (Dumuis et al. 1988; Müller and Connor 1991b) and will easily overcome protection by GSH.

Because of the nature of oxidative reactions, long time exposure of ion channels to low levels of H₂O₂ is expected to exert effects in a slowly developing fashion. High concentrations of H₂O₂ and increased oxidative stress have been demonstrated in brain slices from aged rats (Auerbach and Segal 1997). Therefore the oxidative effects of H₂O₂ on K currents should become even more important with aging.