Oxidative modulation of the transient potassium current I_A by intracellular arachidonic acid in rat CA1 pyramidal neurons

Introduction

There has been continuously growing interest in oxidative stress and its roles in the physiology and pathophysiology of immune defense, ischemia and neurodegenerative disease, such as Parkinson's disease (e.g. Barkats et al., 2006), Alzheimer's dementia (e.g. Behl et al., 1994) and multiple sclerosis (e.g. Ferretti et al., 2006). Reactive oxygen species modify membrane lipids as well as a variety of proteins, including membrane channels, affecting cellular functions in both detrimental and protective ways (Kourie, 1998).

Voltage-gated K⁺ currents were among the first ion channels to be recognized and investigated for their important roles in neuronal signaling (Hodgkin & Huxley, 1952). In particular, the voltage-gated transient K⁺ current (I_A) contributes to the resting membrane potential by the so-called window current and, because of its fast activation, is primarily responsible for repolarization and the duration of the action potential, and repetitive firing (Connor & Stevens, 1971b,a). Via their effect on resting membrane potential, I_A channels exert a strong influence on steady-state inactivation of Na⁺ channels and dendritic back-propagation of action potentials, underlying synaptic plasticity (Hoffman et al., 1997). Via action potential duration, I_A channels strongly affect presynaptic Ca²⁺ influx and transmitter release (Pongs, 1999), affecting synaptic and network excitability (cf. Müller & Misgeld, 1990, 1991).

Ruppersberg et al. (1991) demonstrated that inactivation of A-type currents through Kv1.4 channels is dependent on the cellular redox state in a cysteine-dependent manner. Oxidation decreases the conductance of Kv1.3, Kv1.4, Kv1.5, Kv3.4 (Duprat et al., 1995) and Kv3.3 channels (Vega-Saenz de Miera & Rudy, 1992), while it enhances K⁺ currents through human ether-a-gogo-related gene K⁺ channels (Taglialatela et al., 1997). High concentrations of arachidonic acid (AA) (≥ 1 µm) directly modulate I_A in CA1 hippocampal neurons (Keros & McBain, 1997; Colbert & Pan, 1999) and facilitate induction of synaptic long-term plasticity (Ramakers & Storm, 2002). We have demonstrated specific oxidative modulation of voltage-activated K⁺ currents in cultured hippocampal neurons by H₂O₂ and 1 pm AA (Bittner & Müller, 1999; Müller & Bittner, 2002).

Arachidonic acid is an important second messenger in a wide variety of cell types. In neurons, a receptor-dependent event (leading to Ca²⁺ influx or release of Ca²⁺ from intracellular stores) is required for phospholipase A2, phospholipase C or diacyl-glycerol lipase to release arachidonate from the phospholipids in the cell membrane. Metabolism of AA occurs extremely rapidly by lipoxygenase, cyclo-oxygenase or cytochrome P450 and can produce around 20 distinct, short-lived but extremely potent intermediates (e.g. prostaglandins, prostacyclins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic acid and hydroperoxyeicosatetraenoic acid). For example, prostaglandin E2 has been shown to increase membrane excitability by inhibition of K⁺ currents (Chen & Bazan, 2005). AA is at the same time a short-lived free radical that contributes to oxidative stress and is a potent modulator of K⁺ channels in hippocampal culture (Bittner & Müller, 2002). Some of the effects of AA are mimicked by H₂O₂ but only at much higher concentrations (Müller & Bittner, 2002).

However, neuronal cultures are criticized for abnormal neuronal behavior that may result from developmental maturation under artificial conditions, leading many to favor acute brain slice preparations or in vivo recording. For example, inhibitory synapses in dissociated neuronal cultures exhibit abnormal physiology from postnatal day 6–15, continuing to show properties of early (postnatal day 1–5) postnatal synapses even after 13–21 days in vitro (Henneberger et al., 2005). Cortical oscillatory behavior in vivo does not require GABA_A-ergic transmission and disappears at postnatal day 6–7 (Garaschuk et al., 2000) but develops much more slowly in cortical cultures only after days 9–15 in vitro and requires GABA_A receptor activation (Opitz et al., 2002).

Brain slices are usually maintained in a 95% O₂ atmosphere in order to avoid O₂-dependent cell damage at a depth of 90–150 µm below both surfaces of the slice, as observed for an interface slice configuration at 20% O₂ with histology (Bingmann & Kolde, 1982). Actually, with an O₂ partial pressure of 600 mmHg (79%) in the perfusion, microelectrode measurements have shown steep gradients of O₂ partial pressure in the bath adjacent to the slice surface, with an O₂ pressure gradient inside brain tissue of 400-µm slices at 24 °C from 60 to 15 mmHg, i.e. in a normal physiological range for neurons only 50 µm below the surface and a hypoxic range in the center of the slice, with observations of vacuolization of cytoplasm even in 300-µm-thick slices (Bingmann & Kolde, 1982). The work of Bingmann & Kolde (1982), comprising data from 300–1000-µm-thick slices at temperatures from 24 to 35 °C, shows somewhat lower O₂ pressure at the submerged side of slices in comparison to the gas interface side, indicating even less efficient O₂ supply in the submerged configuration. In order to avoid tissue hypoxia, recordings from submerged slices are usually performed at temperatures of 24–32 °C, significantly below 37 °C (cf. Colbert & Pan, 1999; Garaschuk et al., 2000; Henneberger et al., 2005). This appears to be essential for healthy brain slice electrophysiology and histology, typically showing excellent agreement with in vivo findings.

In addition, in brain slice we can unambiguously identify CA1 pyramidal neurons. For all these reasons, here we investigated in rat hippocampal slice the modulation of voltage-dependent K⁺ currents by AA and H₂O₂ in CA1 pyramidal neurons. We report strong selective suppression of the transient A-current of CA1 neurons by AA at a very low concentration (1 pm) that is mimicked by H₂O₂ (80 µm). Surprisingly, unlike the situation in neuronal culture, antioxidants do not simply block the effects of AA and H₂O₂ on K⁺ channels but often enhance their effects.

Materials and methods

Horizontal hippocampus/entorhinal cortex slices (300 µm) were prepared from ether-anesthetized 14–21-day-old Wistar rats, using standard procedures. Tissue was sliced at ∼4 °C in artificial cerebrospinal fluid (in mm: NaCl, 129; NaH₂PO₄, 1.25; glucose, 10; MgSO₄, 1.8; CaCl₂, 1.6; KCl, 3; NaHCO₃, 26, pH 7.4) continuously bubbled with 95% O₂/5% CO₂. Slices were then maintained in oxygenated artificial cerebrospinal fluid at room temperature (24 °C) for at least 1 h before recording.

Whole-cell patch-clamp recordings were obtained from CA1 pyramidal neurons at 24 °C in submerged acute brain slices under an upright microscope (Olympus BX 51) under visual guidance using infrared (IR) video microscopy (Alix et al., 2003). Borosilicate pipettes filled with intrapipette solution (in mm: KCl, 120; CaCl₂, 1; HEPES, 10; EGTA, 11; MgCl₂, 2; glucose, 20, pH 7.3) had initial open-pipette resistances of 3–6 mΩ. The unphysiologically high intracellular conentration of Cl^– causes a depolarizing shift of the reversal potential of Cl^– currents that reduces Cl^– leak currents in the voltage range of activation of voltage-dependent K⁺ currents. Eicosatetraynoic acid (ETYA), ascorbic acid, glutathione (GSH) and Trolox were applied intracellularly, diluted in intrapipette solution. In some experiments ascorbic acid, Trolox or GSH were applied by bath perfusion. The latter compounds were added to freshly-made oxygenated artificial cerebrospinal fluid, supplemented with 0.001 mm tetrodotoxin. All drugs were from Sigma (Deisenhofen, Germany). AA and ETYA were stored as 1 m stock solutions in dimethylsulfoxide at −20 °C. Experimental solutions were prepared immediately before use. Control recordings with the same concentration of dimethylsulfoxide had no effects on either I_A or on voltage-gated delayed rectifier K⁺ current [I_K(V)]. Ascorbic acid was used from freshly made stock when needed and kept in a dark container for up to 5 days. GSH was stored as a 20 mm stock in intrapipette solution when used for intracellular application. During experimentation solutions were kept on ice in darkness. Voltage clamp was maintained in whole-cell mode with an EPC-9 amplifier controlled by pulse software (both HEKA Electronik, Lambrecht, Pfalz, Germany) and currents filtered at 1.3 kHz. Subtraction and curve fitting of K⁺ currents were performed by IgorPro (WaveMetrics Inc.). Data are given as means ± SEM in absolute values or percentage of control. Statistical analyses were performed with prism (GraphPad Software Inc.) using two-tailed unpaired Student's t-test. A probability of P < 0.05 was considered to be significant.

Currents at each test potential were converted to conductances using g = I/(V_i − E_k) and voltage–conductance relations for activation and inactivation were plotted and fitted with these Boltzmann equations for activation and inactivation, respectively

For effects on the maximum amplitude, currents for all recording times, measured at +30 mV, were normalized to the current at time 0 (immediately after obtaining whole-cell configuration) and plotted against time.

Results

Modulation of voltage-gated transient K⁺ current by arachidonic acid

In whole-cell patch-clamp recording from CA1 pyramidal neurons, total outward K⁺ currents were recorded after an 800-ms hyperpolarizing prepulse to −110 mV (to remove inactivation) and subsequent depolarization of the membrane to potentials between −80 and +50 mV in increments of 10 mV. When the prepulse was followed by a 50-ms interval at −20 mV, I_A inactivated completely and isolated I_K(V) were recorded during subsequent voltage steps (Fig. 1D, 2, for pulse protocols see insets in C). I_A was isolated by subtracting delayed rectifier from mixed currents (Fig. 1D, 1), as shown in 1, 3. I_A had a maximum amplitude of 0.5–4 nA at +30 mV that was reached within 4 ms after the beginning of the voltage pulse. I_A inactivated completely with a time constant of 7.8 ± 1.5 ms (Fig. 1A, Table 1). The maximum amplitude of I_A was quite stable over 15 min of whole-cell recording (Fig. 1C, Table 1). When 1 pm AA was included in the patch pipette, I_A decreased over time, starting within 6 min after obtaining whole-cell configuration. After 15 min of intracellular AA application through the patch pipette I_A was reduced by −41.8 ± 5.5% (n = 9; P < 0.01, Fig. 1B and C). Inclusion of AA in the patch pipette did not affect the inactivation time constant of I_A (Table 1).

Table 1. Overview of effects of oxidants and antioxidants on behavior parameters of voltage-gated transient K⁺ current (I_A)

IA CA1	n	Maximum amplitude (Δ% of control)	V50a (mV)		kactivation		V50i		kinactivation		τi
			At 0 min	At 15 min	At 0 min	At 15 min	At 0 min	At 15 min	At 0 min	At 15 min	At 0 min	At 15 min
Control	9	−10.9 ± 4.6	−14.3 ± 0.3	−18.1 ± 0.4	7.9 ± 0.3	7.8 ± 0.4	−38.1 ± 0.7	−36.7 ± 0.6	9.2 ± 0.5	9.3 ± 0.5	7.8 ± 1.5	8.5 ± 1.4
+1 pm AA	9	−41.8 ± 5.5	−18.1 ± 0.4	−19.2 ± 0.9	7.8 ± 0.4	8.1 ± 0.8	−41.4 ± 0.4	−53.6 ± 0.6	10.2 ± 0.3	11.3 ± 0.6	8.1 ± 1.3	10.4 ± 1.7
Control GSHin	8	−15.5 ± 7.2	−20.1 ± 0.7	−25.0 ± 1.2	11.5 ± 0.7	13.5 ± 1.1	−54.6 ± 0.3	−65.1 ± 0.5	6.4 ± 0.3	5.9 ± 0.4	8.5 ± 1.3	12.3 ± 2.0
AA + GSHin	6	−61.6 ± 5.8	−29.2 ± 0.9	−21.5 ± 1.0	13.0 ± 0.9	12.3 ± 0.9	−43.0 ± 0.8	−58.7 ± 0.7	10.1 ± 0.7	12.3 ± 0.6	11.9 ± 2.5	21.3 ± 3.6
Control asc.acidout	5	−59.1 ± 6.7	−22.4 ± 0.9	−26.6 ± 1.6	12.9 ± 0.8	16.3 ± 1.5	−49.5 ± 0.3	−56.5 ± 0.8	9.8 ± 0.3	9.5 ± 0.7	9.9 ± 1.0	15.8 ± 2.3
AA + GSHin/asc.acidout	4	−23.6 ± 11.8	−10.9 ± 0.8	−18.9 ± 0.9	13.2 ± 0.7	13.8 ± 0.8	−44.7 ± 0.6	−56.0 ± 0.9	10.5 ± 0.5	10.7 ± 0.8	12.9 ± 4.9	18.3 ± 5.1
Control Troloxin	5	−61.1 ± 2.7	−21.3 ± 1.2	−13.4 ± 1.0	13.5 ± 1.0	14.2 ± 0.9	−44.8 ± 0.4	−54.0 ± 0.1	9.9 ± 0.4	9.0 ± 0.7	4.5 ± 0.6	9.9 ± 1.4
AA + Troloxin	7	−47.6 ± 12.2	−19.5 ± 0.8	−18.2 ± 1.0	11.7 ± 0.7	13.1 ± 0.9	−50.2 ± 0.5	−50.3 ± 0.7	11.3 ± 0.5	11.8 ± 0.7	8.4 ± 3.1	13.3 ± 3.7
Control Troloxout	4	−6.6 ± 9.1	−28.1 ± 1.4	−32.5 ± 1.3	11.3 ± 1.3	12.8 ± 1.2	−68.3 ± 0.8	−68.4 ± 0.8	6.7 ± 0.7	8.5 ± 0.7	6.1 ± 0.8	7.5 ± 1.2
AA + Troloxin,out	4	−26.8 ± 1.1	−20.3 ± 2.1	−25.5 ± 1.0	14.1 ± 1.8	13.2 ± 0.8	−45.6 ± 0.6	−58.0 ± 0.7	9.5 ± 0.5	12.0 ± 0.6	8.9 ± 0.9	10.7 ± 1.1
+80 µm H2O2	7	−79.7 ± 1.9	−12.9 ± 0.8	−20.8 ± 0.9	12.2 ± 0.7	14.9 ± 0.8	−55.0 ± 0.6	−63.6 ± 0.5	8.7 ± 0.5	9.0 ± 0.5	7.9 ± 1.6	13.5 ± 1.5
H2O2 + GSHin	4	−34.8 ± 7.2	−26.1 ± 1.2	−17.2 ± 2.0	11.5 ± 1.1	16.6 ± 1.6	−47.4 ± 0.4	−50.2 ± 0.8	10.6 ± 0.4	11.6 ± 0.7	10.0 ± 3.3	19.5 ± 2.6
H2O2 + GSHin/asc.acidout	4	−31.5 ± 4.9	−11.8 ± 0.4	−34.3 ± 1.4	10.5 ± 0.3	12.8 ± 1.2	−44.9 ± 0.5	−67.8 ± 0.6	9.1 ± 0.4	8.4 ± 0.5	5.7 ± 1.0	9.8 ± 1.0

• AA, arachidonic acid; GSH, glutathione. time = 0 (immediately after obtaining whole-cell configuration.

Like I_A, I_K(V) was quite stable under control conditions with a slight decrease of current amplitude over 15 min (−12.8 ± 2.3%, Fig. 2, cf. Table 2). AA in the patch pipette did not affect the delayed rectifier current I_K(V) in any way (Fig. 2E, Table 2).

Table 2. Overview of effects of oxidants and antioxidants on behavior parameters of voltage-gated delayed rectifier K⁺ current [I_K(V)]

IK(V) CA1	n	Max. amplitude (Δ% of control)	V50activation (0 min)	V50activation (15 min)	kactivation (0 min)	kactivation (15 min)
Control (2–6.8 nA)	9	−12.8 ± 2.3%	6.3 ± 1.1	0.1 ± 1.0	18.5 ± 1.0	18.8 ± 0.9
+1 pm AA	9	−15.2 ± 1.8%	5.3 ± 0.9	−2.8 ± 1.0	17.3 ± 0.8	18.6 ± 0.9
Control GSHin	8	−20.8 ± 5.7	3.8 ± 0.8	1.2 ± 0.7	16.4 ± 0.7	16.2 ± 0.6
AA + GSHin	6	−13.5 ± 4.6%	0.7 ± 1.3	−1.9 ± 0.9	19.1 ± 1.1	17.3 ± 0.8
Control asc.acidout	5	−39.73 ± 7.3	3.0 ± 0.8	0.7 ± 0.7	16.4 ± 0.8	17.0 ± 0.8
AA + GSHin/asc.acidout	4	−17.1 ± 1.3%	4.4 ± 1.1	2.8 ± 1.0	18.4 ± 1.0	17.8 ± 0.9
Control Troloxin	5	−39.4 ± 10.8	0.5 ± 0.9	−4.5 ± 0.7	18.0 ± 0.8	17.1 ± 0.6
AA + Troloxin	7	−20.8 ± 8.5%	−3.2 ± 0.9	−1.2 ± 1.0	18.2 ± 0.8	19.4 ± 0.9
Control Troloxout	4	−1.0 ± 5.7	−2.3 ± 0.7	−6.7 ± 0.8	17.7 ± 0.6	17.9 ± 0.7
AA + Troloxin/Troloxout	4	−18.4 ± 10.6%	−3.3 ± 1.1	−1.0 ± 1.0	19.3 ± 1.0	16.7 ± 0.9
+80 µm H2O2	7	−63.2 ± 5.1%	1.4 ± 1.5	−5.7 ± 1.5	11.3 ± 1.4	12.5 ± 1.3
H2O2 + GSHin	4	−21.1 ± 5.3%	−3.3 ± 1.1	−6.7 ± 1.2	19.2 ± 1.0	17.6 ± 1.0
H2O2 + GSHin/asc.acidout	4	−23.0 ± 3.9%	3.5 ± 0.8	−8.2 ± 0.9	17.7 ± 0.7	17.7 ± 0.8

• AA, arachidonic acid; GSH, glutathione.

In order to test whether the effects of applied AA on I_A were due to AA itself or one of its many bioactive metabolites, we employed the non-metabolizable analog ETYA. ETYA is not hydrolysed by cyclo-oxygenase, lipoxygenase or cytochrome P450 but it also blocks the activities of these enzymes. Intracellular ETYA (1 pm) was ineffective in reducing I_A. Intracellular application of a 100-fold higher concentration of ETYA (100 pm) reduced I_A to a similar extent as 1 pm AA (−47.1 ± 8.7%, n = 7, Fig. 1C). Like AA, ETYA did not affect I_K(V) in any way (not shown).

In dissociated hippocampal cultures AA (10 pm intracellular) has also been shown to shift the voltage dependence of inactivation of I_A (Bittner & Müller, 1999). The conductance–voltage plot of Fig. 3A (left curves) illustrates that AA shifted voltage dependence of steady-state inactivation to the left by 11 mV in CA1 pyramidal neurons in rat brain slice, in addition to the reduction of maximal conductance described above. AA did not affect the voltage dependence of activation (Fig. 3, curves on the right side).

Oxidative mechanisms in reduction of voltage-gated transient K⁺ current by arachidonic acid

Arachidonic acid and ETYA may affect protein function by binding-induced conformational changes. Alternatively, as both molecules can act as free radicals, they may oxidize certain amino acids. To test for an involvement of oxidative effects of AA we used three antioxidants, GSH, ascorbic acid and Trolox (a water-soluble analog of vitamin E or α-tocopherol), either alone or in different combinations. Figure 4B illustrates the somewhat surprising finding that, when GSH (20 mm) was included in the patch pipette, the AA effect on the maximal I_A was not inhibited but actually enhanced (−61.6 ± 5.8% at 15 min). In addition, the reduction of I_A was accelerated, as shown by the significant reduction of I_A after only 3 min of whole-cell recording (*, −44.1 ± 9.3%, n = 6 for AA + GSH_i as compared with −2.4 ± 4.8%, n = 9 for AA alone, 4, 1) and inactivation was significantly slowed (P < 0.01, Fig. 4C inset). GSH itself had no effect on I_A or I_K(V) (n = 6, Tables 1 and 2). The augmentation of the AA effect by GSH could be mediated by oxidation of GSH to oxidized glutathione (GSSG) and, more effectively, oxidation of the channel protein by GSSG, i.e. a catalytic effect of GSH (Nelson & Cox, 2005).

In contrast to GSH, ascorbic acid (0.4 mm), an antioxidant that apparently crosses cell membranes easily, blocked reduction of I_A when bath-applied in addition to AA + GSH_i (Fig. 4A, 2, and B). In slices superfused with ascorbic acid only, I_A steadily decreased during whole-cell recording over 15 min to 40.9 ± 6.7% and I_K(V) to 60.3 ± 7.3% of initial current amplitude (n = 5), and tau of I_A inactivation was also significantly slowed (P < 0.01, Table 1) indicating that ascorbic acid may also catalyse oxidative reactions with these channels, depending on the oxidative stress in the vicinity of the membrane.

The combination of GSH_i and ascorbic acid did not prevent the AA-mediated shift in steady-state inactivation (Fig. 4C and D), suggesting that this shift is caused by a mechanism independent of reduction of maximal conductance. GSH_i + AA, like AA alone, had no effect on voltage dependence of activation (Fig. 4C). The combination of GSH and ascorbic acid + AA caused a 6-mV shift of activation to more negative potentials that is statistically significant with 95% confidence (Fig. 4D).

Despite its water solubility Trolox, like its relative tocopherol, is assumed to primarily protect membrane lipids and transmembrane protein segments of oxidation. In slices superfused with Trolox (100 µm), both I_A and I_K(V) were completely stable during 15 min whole-cell recording (Tables 1 and 2). Intracellular application of Trolox strongly reduced I_A by −61.1 ± 2.7%(Fig. 5A, 1, and B) and I_K(V) by −39.4 ± 10.8%, and shifted steady-state inactivation of I_A by −9 mV and activation of I_A by +8 mV and of I_K(V) by −4 mV (Tables 1 and 2). The inactivation time constant of I_A was progressively slowed over 15 min during whole-cell recording from 4.5 to 9.9 ms (P < 0.01, Table 1). Figure 5B shows that intracellular application of AA together with Trolox, applied from both sides of the cell membrane or only from the intracellular side, did not enhance the early effect of Trolox_i on I_A (Fig. 5B, *). I_A showed significant recovery (Fig. 5B) with prolonged recording time in the presence of Trolox_i/o.

Again, voltage-dependent inactivation showed responses independent of modulation of maximal conductance. Trolox_i inhibited the AA shift of steady-state inactivation and vice versa (Fig. 5C). The voltage dependence of activation of I_A was unchanged. Further, AA_i in the presence of Trolox on both sides of the membrane shifted steady-state inactivation by −12 mV to a more negative potential (over 15 min), similar to the effect of AA_i or of Trolox_i alone. For the triple combination (AA_i + Trolox_i,o) there was no shift of voltage dependence of activation (Fig. 5D, curves on right side).

To evaluate the direct effects of oxidative modulation of I_A (in the absence of AA), we applied the non-lipid oxidizing compound H₂O₂ via the patch pipette. Generally, 80 µm H₂O₂ mimicked the effect of 1 pm AA on the maximal amplitude of I_A. The H₂O₂ effect was even stronger than that of AA (−79.6 ± 1.9%, P < 0.01, n = 6, Fig. 6A and E). Like AA, it also shifted the voltage dependence of inactivation to more negative potentials (−7 mV, Fig. 6B). Unlike AA, H₂O₂ also shifted the voltage dependence of activation to more negative potentials (−6 mV, Fig. 6B) and slowed inactivation of I_A (P < 0.05, Table 1). The effects of H₂O₂ further support the proposal that modification of I_A by AA is mediated primarily by oxidative reactions. We therefore also tested for effects of antioxidants on the effects of H₂O₂, expecting complex changes as described above for AA. The physiological antioxidant GSH_i (20 mm) strongly reduced inhibition of maximal I_A by H₂O₂ (−34.8 ± 7.2%, n = 4, Fig. 6A) and eliminated the H₂O₂-mediated shift of steady-state inactivation (Fig. 6C). While H₂O₂ shifted the voltage dependence of activation to the left, H₂O₂ in the presence of GSH_i caused a shift to the right (+7 mV, Fig. 6C right curves). In addition, the slope of the conductance–voltage relationship was decreased (k = 14.9 ± 0.8 for H₂O₂ and k = 11.6 ± 0.7 for H₂O₂ + GSH_i) and slowing of I_A inactivation was enhanced (P < 0.001, Table 1).

Addition of ascorbic acid to the artificial cerebrospinal fluid (0.4 mm) appeared, together with GSH_i, to inhibit the H₂O₂ effect on maximal I_A amplitude (−31.5 ± 4.9%, n = 4, Fig. 6A). In contrast, ascorbic acid did not support GSH_i inhibition of the shifts of voltage dependence of inactivation and activation but even enhanced shifts caused by H₂O₂ by more than 150% (Fig. 6D, Table 1). Unlike with H₂O₂ + GSH_i there was no significant change of slopes of voltage dependence of activation or inactivation (Fig. 6D, Table 1). I_A inactivated much faster in the presence of ascorbic acid but tau still almost doubled from 5.7 to 9.8 ms during 15 min whole-cell recording with H₂O₂ + GSH_i + ascorbic acid_o (P < 0.01, Table 1).

In agreement with the results reported for neurons in primary cultures, H₂O₂ also suppressed the delayed rectifier K⁺ current I_K(V) in CA1 pyramidal neurons in brain slices (−63.2 ± 5.1%, n = 6, Fig. 7A and C, Table 2). H₂O₂ shifted the voltage dependence of activation to more negative potentials (−7 mV, Fig. 7B). GSH_i and also GSH_i + ascorbic acid_o inhibited reduction of I_K(V) by H₂O₂ (−21.0 ± 5.3%, n = 4 and −23.0 ± 3.9%, n = 4, respectively, Fig. 7A and D).

Discussion

Our experimental data characterize oxidative modulation of voltage-activated K⁺ currents in CA1 pyramidal neurons in rat hippocampal brain slice by the free fatty acid AA, H₂O₂ and antioxidants. An ultra low patch pipette concentration of AA (1 pm) consistently inhibits the maximal conductance of the transient K⁺ current I_A by about 50% while not affecting the delayed rectifier current I_K(V) (1, 2). This effect is not mimicked by the non-metabolizable AA analog ETYA (1 pm), except at a 100-fold higher concentration (100 pm). As ETYA inhibits the AA-metabolizing enzymes cyclo-oxygenase, lipoxygenase and cytochrome P450, these results suggest a direct specific effect of AA (1 pm) or ETYA (100 pm) on I_A. This effect develops over 15 min of dialysis in agreement with diffusional exchange between the patch pipette and soma/proximal dendrites of a CA1 pyramidal neuron (cf. Pusch & Neher, 1988) with clear reduction of I_A after 3 min for ETYA and 6 min for AA. The need for a higher concentration of ETYA is probably due to substitution of the four highly reactive carbon double bonds of AA with the much more stable triple bonds of ETYA.

In addition, AA causes a significant shift (−12 mV) of steady-state inactivation to more negative potentials. This AA shift will cause a further reduction of I_A by 10–40% when I_A is activated from a membrane potential of −60 to −50 mV (Fig. 3).

The effects of AA are probably mediated by at least two oxidation sites of the channel protein as the two effects, reduction of maximal conductance and shift of steady-state inactivation, are independently affected by ascorbic acid, trolox and H₂O_2. The oxidation sites appear to be intracellular, as intracellular AA and (membrane-impermeable) GSH have strong effects. Surprisingly, intracellular application of 20 mm GSH did not block the effect of AA in CA1 neurons from brain slice, as reported for hippocampal neurons in primary culture (Bittner & Müller, 1999; Müller & Bittner, 2002), but strongly enhanced it, particularly at 3 min of whole-cell recording (Fig. 4). It is possible that GSH is oxidized to GSSG that, in turn, more effectively oxidizes a cystein in the channel protein that is not accessible to GSSG in cultured neurons because of subunit composition or differential expression of other interacting proteins. In contrast, a combination of GSH_i with extracellular application of ascorbic acid (0.4 mm) significantly inhibited the reduction of I_A by AA. Shift of steady-state inactivation to more negative potentials was not affected by GSH_i but was reduced to −7 mV by the combination of GSH_i and ascorbic acid.

A qualitatively different profile of effects was seen with the vitamin E (α-tocopherol) analog Trolox. Like GSH_i, Trolox applied from the inside (10 µm) or from the outside (100 µm) mimicked or enhanced, respectively, the early reduction of I_A by AA but did not enhance the AA effect at later times or even reduced it when bath-applied (Fig. 5A and B). Intracellular but not extracellular application of Trolox by itself reduced I_A (Fig. 5B, Table 1). The AA shift of steady-state inactivation was completely blocked by intracellular application of Trolox (and vice versa) but not by the combination of Trolox_o and Trolox_i. This further supports the reduction of maximal I_A conductance and shift of steady-state inactivation being independent modifications of the channel protein. Two or more oxidation sites for shift of steady-state inactivation may explain the puzzling shift of steady-state inactivation with Trolox application from both sides of the membrane.

Enhancement of the AA effect by some antioxidants may be mediated by catalysing the underlying reactions between AA and other radicals in the slice tissue and channel protein. Antioxidants have some capacity to buffer oxidants but in this process they themselves become oxidants that may, in turn, more effectively oxidize certain targets. For example, GSH reductase becomes oxidized by the reduction of GSSG to GSH and, in turn, is reduced by oxidizing NADP⁺ to NADPH, thereby acting as a catalyst of the reaction between NADP⁺ and GSSG. Reduction of superoxide anions to H₂O₂ by superoxide dismutase gives an electroneutral molecule that much better permeates into lipid membranes (Nelson & Cox, 2005). Oxidation of channel proteins may well be facilitated by antioxidants in similar ways. Such a view is further supported by the effects of H₂O₂ on I_A. Intracellular application of H₂O₂ (80 µm) reduced I_A more effectively than AA and to a similar extent as AA together with GSH_i (Fig. 6A, Table 1). In contrast to the effect of AA, GSH_i effectively inhibited the effect of H₂O₂ on I_A, as did the combination of GSH_i + ascorbic acid.

Independent modulation of maximal conductance and steady-state inactivation of I_A is further supported by a significantly weaker, in comparison to AA, shift of V₅₀ with H₂O₂. Further, in the presence of ascorbic acid in the perfusion, H₂O₂ + GSH, but not AA + GSH, causes an enhanced shift of V₅₀ to a more negative potential for both inactivation and activation of I_A (6, 4).

Unlike AA, H₂O₂ also reduced the maximal conductance of the delayed rectifier current I_K(V) in a GSH_i-sensitive way (Fig. 7), further supporting the unique and highly specific interaction of AA with I_A.

We conclude that reduction of I_A by extremely low concentrations of intracellular AA or by H₂O₂ in hippocampal culture is not a developmental artifact of non-physiological culture conditions but is real in CA1 neurons that have developed in vivo. In view of antioxidants blocking the effects of AA in cultured neurons (Bittner & Müller, 1999; Müller & Bittner, 2002), the enhancing effects of several ‘antioxidative’ agents in brain slice were unexpected and suggest fundamental differences of oxidative regulation of the A-current in cultured hippocampal neurons compared with CA1 pyramidal neurons in brain slice. This may have implications for the relevance of excitotoxicity and oxidative stress research using neuronal cultures. Maintenance of slices with 95% O₂ levels in the perfusion is apparently not a problem, as it results in normal to hypoxic tissue O₂ levels (see Introduction and Bingmann & Kolde, 1982). Clearly, AA as well as H₂O₂, particularly in older age or in neurodegenerative disease (Auerbach & Segal, 1997), will strongly affect K⁺ currents and, via depolarization and inactivation of Na⁺ channels, dendritic excitability and spike-timing-dependent synaptic plasticity/learning (Giese et al., 1998). Antioxidative therapy may well have negative effects in this context.

Acknowledgements

Supported by Deutsche Forschungsgemeinschaft grant Mu 809/7-2 and NIH grant RR15636. We thank Dr C. William Shuttleworth for helpful comments on the manuscript.

Abbreviations