4 RESULTS

↓41

4.1 Oxidative modulation of transient and delayed rectifier potassium currents in CA1 pyramidal neurons

↓42

In whole-cell voltage-clamp experiments of CA1 pyramidal neurons, total outward potassium currents were evoked after a 800 ms hyperpolarizing prepulse to –110 mV (to remove inactivation) during subsequent step depolarization of the membrane to potentials between –80 and +50 mV (increments of 10 mV, 200 ms duration). When the prepulse was followed by an additional 50 ms step at –20 mV, IA inactivated completely and isolated delayed rectifier currents (IK(V)) were recorded during subsequent voltage steps (Fig.12 A,B, pulse protocols see inset Fig.12). By subtracting delayed rectifier from mixed currents (IK(V))+IA), IA was isolated, as shown in Fig. 12 C. 

4.1.1 Potassium currents in untreated CA1 pyramidal neurons

In control conditions, the evoked transient potassium current IA 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. IA inactivated completely with a time constant of 7.8 ± 1.5 ms (Fig. 12 C, Table 5). The maximal amplitude of IA was stable over 15 min of whole-cell recording (-10.9 ± 4.6 %, Fig.12 C, Table 5).

Fig. 12 Native IA  and IK(V) in control condition in CA1 pyramidal neurons.

A,B Original traces showing total outward potassium(IK(V) + IA) and delayed rectifier (IK(V))currents at 0 s (A) and 900 s (B) after reaching whole-cell configuration. C Subtracted transient potassium current (IA) recorded at +30 mV over time. D Delayed rectifier current at +30 mV over time.

↓43

Like IA, IK(V) was stable under control conditions with a slight decrease of the current amplitude over 15 min (-12.8 ± 2.3 %, Fig.12 D, Table 6). That minimal reduction of both currents in control condition most probably could be due to a ‘run-down’ of these currents, suggesting that important cytoplasmic factor(s) are subject to wash-out.

4.1.2 Modulation of transient IA and delayed rectifier currents IK(V) by arachidonic acid

When 1 pM arachidonic acid (AA) was included in the patch pipette, IA decreased over time, starting within 6 min after obtaining the whole-cell configuration. After 15 min of intracellular AA application through the patch pipette IA was reduced by -41.8 ± 5.5 % (n=9; p<0.01, Fig.13 B, C). Inclusion of AA in the patch pipette did not affect the inactivation time constant of IA (Fig.13 D, Table 5).

Fig. 13 Suppression of IA by arachidonic acid (AA) in CA1 pyramidal neurons.

A: control; B, C: Intracellular application of 1 pM AA reduces the maximal transient potassium current (IA) over time. The non-metabolizable AA analogue ETYA shows similar effect on IA at 100-fold higher concentration (C). D, E: Raw current traces for combined IA and Ik(v) and for delayed rectifier currents (pulse protocols: inset in E).Currents are shown for control immediately after obtaining whole cell configuration (D, 0 s AA), after and after 900 s of recording with 1 pM of AA in the patch pipette (E).

↓44

In order to test whether the effects of applied AA on IA were due to AA itself or one of its many bioactive metabolites, the non-metabolizable AA analogue eicosatetraynoic acid (ETYA) was employed. ETYA is neither hydrolyzed by cyclooxygenase, lipoxygenase, nor by cytochrome P450, but furthermore blocks the activities of these enzymes. Intracellular 1 pM ETYA was ineffective in reducing IA. Intracellular application of a 100-fold higher concentration of ETYA (100 pM) reduced IA to a similar extent as 1 pM AA (-47.1 ± 8.7 %, n=7, Fig.13 C). Like AA, ETYA did not affect maximum amplitude of Ik(v) (not shown).

Fig.14 shows that IK(V) did not change its maximal amplitude in the presence of 1 pM intracellular arachidonic acid (-15.2 ± 1.8 %, n=9) in comparison to control (-12.8 ± 2.3 %, n=9).

Fig. 14 The delayed rectifier current Ik(v) is not affected by AA in CA1 pyramidal neurons. 

A, B: Maximal Ik(v) recorded at 0 s and at 900 s of whole cell recording shows a 13% decrease over time in control (A) as well as with 1 pM AA in the patch pipette (B). C: Plot of Ik(v) amplitude over time of whole cell recording for control and for AA. D, E: Original current recordings of Ik(v) for several voltage steps (see inset in C) for control (D) and for AA at 0 s (D1, E1) and 900 s (D2, E2) of whole cell recording.

↓45

The conductance–voltage plot of Fig.15 A, left curves, illustrates that AA shifted steady-state of 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 of the transient potassium current (Fig.15 A, curves on the right side).

Fig.15 Intracellular AA (1pM) shifts steady-state inactivation, but not voltage dependence of activation of IA in CA1 pyramidal neurons.

A: Conductance-voltage relations and Boltzmann fits for inactivation (left curves) and activation (right curves) of IA. Inset: Pulse protocols for determining voltage dependence of steady state inactivation (left) and of activation (right). Red part shows length of the traces below. B  and  C: Representative recordings for inactivation (B) and activation (C).D: voltage-conductance dependence of IK(V) and pulse protocols for activation of IK(V) (inset). E: Original recordings of IK(V) activation in control conditions and in the presence of 1 pM AA.

4.1.3 Oxidative mechanism in reduction of the transient potassium current IA by arachidonic acid

AA and ETYA may affect protein function by binding-induced conformational changes. Alternatively, both molecules are free radicals that can oxidize certain amino acids. To test for an involvement of oxidative effects of AA was used together with antioxidants, glutathione (GSH), ascorbic acid and Trolox (water-soluble analogue of α-Tocopherol) either alone or in different combinations.

↓46

Influence of   glutathione on   arachidonic acid-mediated effects  

Figure 16 B illustrates the surprising finding that when GSH (20mM) was included in the patch pipette, the AA effect on the maximal IA was not inhibited, but actually enhanced (-61.6 ± 5.8 % at 15min). In addition, reduction of IA was accelerated, as visible by significant reduction of IA after only 3 min of whole-cell recording (*, -44.1 ± 9.3 %, n=6 for AA + GSHin; as compared to -2.4 ± 4.8 %, n=9 for AA alone, Fig. 16 B) and inactivation was significantly slowed (p<0.01, Fig. 16 C inset). GSH itself had neither effect on IA nor on IK(V) maximal conductance(n=6, Tables 5 and 6; Fig.20 A, D). The augmentation of the AA effect by GSH suggests that redox reactions may be responsible for reduction of IA by AA and by GSH.

Fig. 16 Interaction of AA and antioxidants with IA. in CA1 pyramidal neurons 

A, B: GSHi enhances the AA effect on IA (A1, B: blue line), particularly early after begin of recording/application (*), while ascorbic acid blocks the AA effect (A2, B: green line). C, D: GSHi and ascorbic acido do not prevent the AA-mediated shift in steady-state inactivation (C, D, control=0 s vs. 900 s, left curves) but ascorbic acid slightly reduces it and shifts as well the activation curve to more negative potential (D, right curves). AA+GSHi (but not AA, only) slow inactivation of IA, as demonstrated by an overlay of traces after scaling to the same amplitude (C, inset, trace=40 ms).

↓47

I nfluence of glutathione an d ascorbate on arachidonic acid –mediated effects

In contrast to GSH, ascorbic acid (0.4 mM), an antioxidant that apparently crosses cell membranes easily, blocked reduction of IA when applied by the bath in addition to AA + GSHin (Fig. 16 A2, B-green line). In slices superfused with ascorbic acid only, IA decreased during whole cell recording over 15 min steadily to 40.9 ± 6.7 % and IK(V) to 60.3 ± 7.3 % of initial current amplitude (n=5). Under this conditions, tau of IA inactivation was also significantly slowed (p<0.01, Table 5), further supporting that ascorbic acid can catalyze oxidative reactions with these channels, depending on the oxidative stress in the vicinity of the membrane.

Moreover, combination of GSHin and ascorbic acid did not prevent the AA-mediated shift in steady-state inactivation (Fig. 16 D), further suggesting that this shift is caused by a mechanism independent of reduction of maximal conductance. GSHin plus AA had, like AA alone, no effect on voltage dependence of activation. Combination of GSH and ascorbic acid plus AA caused a 6 mV shift of activation to more negative potentials that is statistically significant with 95 % confidence (Fig. 16 D, right curves).

↓48

Influence of Trolox on arachidonic acid-mediated effects  

Despite its water solubility Trolox, like tocopherol, is assumed to primarily protect membrane lipids and transmembrane protein segments from oxidation. In slices superfused with Trolox (100 µM), both IA and IK(V) were completely stable during 15 min whole cell recording.

The inactivation time constant of IA was progressively slowed over 15 min during whole cell recording from 4.5 ms to 9.9 ms (p<0.01, Table 5). Figure 17 A shows that intracellular application of AA together with Trolox, that is applied from both sides of the cell membrane, did also enhance the early effect of AA on IA (Fig. 17 B, *). With prolonged recording time in the presence of Troloxin/out IA showed significant recovery (Fig. 17 B). Again, voltage dependent inactivation showed responses independent from modulation of maximal conductance. Troloxin inhibited the AA shift of steady-state inactivation (Fig. 17 C).

↓49

Fig. 17 Interaction of AA and antioxidants with IA in CA1 pyramidal neurons.

A, B: Intracellular application of the vitamin E analogue Trolox (10 µM) with AA does not prevent reduction of IA (A1, B: blue line), but Trolox, applied from both sides of the cell membrane, does significantly recover IA with prolonged recording time (A2, B: green line). Intracellular application of 10 µM Trolox by itself reduces IA similar to 1 pM AA (B: black line). C: Troloxi inhibits the AA shift of steady-state inactivation (left curves). There is no shift in voltage dependence of activation (right curves, control=0 s vs. 900 s). D: In the presence of Trolox on both sides of the membrane plus AAi, steady state inactivation shifts over 900 s recording time by -12mV to more negative potential, similar to the effect of AAi or of Troloxi.

The voltage dependence of activation of IA was unchanged. Surprisingly, AAi in the presence of Trolox on both sides of the membrane shifted steady state inactivation by -12 mV to more negative potentials (over 15 min), similar to the effect of AAi or of Troloxin alone. For the triple combination (AAi + Troloxin,out) there was no shift of voltage dependence of activation of the transient potassium current (Fig. 17 D, curves on right side).

4.1.4 Effects of H2O2 on outward potassium currents

To evaluate direct effects of oxidative modulation of IA (in the absence of AA), the non-lipid oxidizing compound hydrogen peroxide (H2O2) was applied via the patch pipette. Generally, 80 µM H2O2 mimicked the effect of 1 pM AA on the maximal amplitude of IA  (-79.6 ± 1.9 %, p<0.01, n=6, Fig. 18A). Like AA, it also shifted the voltage dependence of inactivation to more negative potentials (-7mV, Fig. 18 B, left curves). Unlike AA, H2O2 in addition shifted the voltage dependence of activation to more negative potentials (-6 mV, Fig. 18 B, right curves) and slowed inactivation of IA (p<0.05, Table 5). The effects of H2O2 further support the idea that modulation of IA by AA is mediated primarily by oxidative reactions. Therefore it was tested for effects of antioxidants on the effects of H2O2, expecting complex changes as described above for AA.

↓50

Influence of glutathione on H 2 O 2 –mediated effects on potassium currents

The physiological antioxidant GSHin (20 mM) strongly reduced inhibition of maximal IA by H2O2 (-34.8 ± 7.2 %, n=4, Fig.18 A) and eliminated the H2O2 mediated shift of steady state inactivation (Fig. 18 C). While H2O2 shifted the voltage dependence of activation to the left, H2O2 in the presence of GSHin caused a shift to the right (+7 mV, Fig. 18 C). 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 plus GSH) and slowing of IA inactivation was enhanced (p<0.001, Table 5).

Fig. 18 Effects of intracellular H2O2 (80 µM) and antioxidants on IA in CA1 pyramidal neurons. 

A: H2O2 mimics the effect of AA on maximal IA (red line). This effect is not enhanced but inhibited by GSHi or GSHi+ascorbic acido (green and blue line, respectively). B: H2O2 shifts voltage conductance relations of inactivation (left curves) and activation (right curves) to more negative potentials. C: GSHi eliminates the H2O2 mediated shift in inactivation (left curves), but for activation causes a shift to more positive potential and change in slope (right curve). D: In the presence of ascorbic acido, GSHi + H2O2 shift inactivation and activation of IA by -22 mV to more negative potentials. E: Representative current traces of IA for determination of the voltage dependence of activation for control immediately after obtaining whole cell configuration (E1) and after 900 s of recording with H2O2 in the patch pipette (E2).

↓51

Influence of glutathione and ascorbate on H 2 O 2 –mediated effects on potassium currents

Addition of ascorbic acid to the ACSF (0.4 mM), appeared to strengthen inhibition of the H2O2 effect by GSHin on maximal IA amplitude (-31.5 ± 4.9 %, n=4, Fig. 18 A). In contrast, ascorbic acid did not support GSHin inhibition of the shifts of voltage dependence of inactivation and of activation, but even enhanced shifts caused by H2O2 by more than 150% (Fig. 18 D, Table 5). Unlike with H2O2+GSHin there was no significant change of slopes, neither of voltage dependence of activation, nor of inactivation (Fig. 18 D, Table 5). IA 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 + GSHin (p<0.01, Table 5).

In agreement with results reported for neurons in primary cultures, H2O2 also suppressed the delayed rectifier potassium current IK(V) in CA1 pyramidal neurons in brain slices (-63.2 ± 5.1 %, n=6, Fig.19 A, C, Table 6). H2O2 shifted the voltage dependence of activation to more negative potentials (from 1.4 ± 1.5 mV to -5.7 ± 1.5 mV, n=7, Fig.19 B). GSHin and also GSHin plus ascorbic acid inhibited reduction of I k(v) by H2O2, (-21.0 ± 5.3 %, n= 4 and –23.0 ± 3.9 %, n=4, respectively, Fig.19 A).

↓52

Fig. 19 Effects of intracellular H2O2 (80 µM) and antioxidants on IK(V) in CA1 pyramidal neurons. 

A: H2O2 suppresses the delayed rectifier potassium current IK(V), and this effect is inhibited by GSHi or GSHi+ascorbic acido (+GSHi and +GSHi,asc.acido, respectively). B: H2O2 shifts voltage dependence of activation of IK(V) to a more negative potential. C, D: Representative traces of IK(V) for determination of voltage dependence of activation for control immediately after obtaining whole cell configuration (C1, D1, 0 s) and after 900 s recording with H2O2 (C2) or H2O2 + GSHi in the patch pipette (D2). Recordings of D have been obtained in the presence of ascorbic acido.

4.1.5 Control antioxidant studies

Due to unexpected behavior of antioxidants in this set of experiments with arachidonic acid, control (only in the presence of antioxidants) studies on IA and IK(V), were performed in CA1 neurons in order to investigate potential K+-current susceptibility to antioxidant exposure.

Intracellular GSH

↓53

Application of 20 µM GSH to the intrapipette solution for 15 min resulted in a significant leftward shift of steady-state inactivation of IA compared to that observed in controls (-54.6 ± 0.3 mV and -65.1 ± 0.5 mV, respectively). Glutathione by itself reduced the rate of inactivation of IA within 15 min. (τ=8.5 ± 1.3 vs. τ=12.3 ± 2.0). Under GSHin conditions, the maximal conductances of IA and IK(V) were reduced to ~20 % (Fig.20 A, D).

Fig. 20 Effects of GSH and ascorbate on K+ currents behavior in CA1 pyramidal neurons. 

A,   D :  Effects on the maximal amplitude of IA (A) and IK(V) (D). B,   C :  Steady-state inactivation and activation curves for control (black line) and GSH (blue) and ascorbic acid (green) conditions. E, F: Kinetics of activation of IK(V) in the presence of GSH (E) and ascorbate (F).

Extracellular ascorbic acid 

↓54

The perfusion of the slices with 0.4 mM ascorbate reduced significantly the maximal amplitude of IA to -59.1 ± 6.7% as well as the maximal amplitude of IK(V) to -39.7 ± 7.3% (Fig.20, A and D). Ascorbic acid applied on the outer side of the membrane did not affect the kinetics of activation and steady-state inactivation of IA. 0.4 mM ascorbate, applied trough the perfusion, did not influence the activation of IK(V) as well (Fig.20 E, F).

Intracellular Trolox

Inclusion of 10 µM Trolox to the patch pipette led to significant decrease of the peak current amplitude of IA to -61.1 ± 2.7 % and those of IK(V) to -39.4 ± 10.8 % (Fig.21 A, D-blue line). Intracellular Trolox shifted voltage dependence of activation (from -21.3 ± 1.2 mV to -13.4 ± 1.0 mV) and inactivation (from -44.8± 0.4mV to -54.0 ± 0.1 mV) of IA Troloxin increased the IA time constant of inactivation (τ = 4.5 ± 0.6 at 0s and τ = 9.9 ± 1.4 at 900 s).

↓55

Fig. 21 Effects of Trolox on K+ currents behavior in CA1 pyramidal neurons. 

A,   D :  Effects on the peak amplitude of IA (A) and IK(V) (D). B,   C :  Steady-state inactivation and activation curves of IA in control (black line) and after intracellular (blue) and extracellular (green) application of Trolox. E, F: Activation kinetics of IK(V) in the presence of Trolox in(E) and Trolox out  (F).

On Fig.21 E, IK(V) voltage-conductance relation of activation in the presence of Trolox in the patch pipette showed a small shift (from 0.5 ± 0.9 mV to -4.5 ± 0.7 mV, n=5) to more hyperpolarizing values.

Ex tracellular Trolox

↓56

Bath application of 100 µM Trolox did not influence IA or IK(V) in any way. There were neither changes in maximal amplitude nor in the kinetic of activation and steady-state inactivation of these currents (Fig.21 A, C, D, F-green line, Table 5). Trolox out did not affect the rate of inactivation of IA as well.

Table 5 Overview of effects of oxidants and antioxidants on behavior parameters of IA in CA1 pyramidal neurons.

I A CA1

n

max amplitude,

% of control

V 50a

0 min mV

V 50a

15 min mV

k activation

0 min

k a

15 min

V 50i  

0 min
mV

V 50i  

15 min
mV

k ina ctivation

0 min

k i

15 min

τ i 0 min ms

τ i  

15 min ms

control

9

-10.9   ± 4.6

-14.3 ± 0.3

-18.1 ± 0.5

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

+ 1pM 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 GSH in

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+GSH in

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.acid out

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+GSH in /

asc.acid out

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

c ontrol Trolox in

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+Trolox in

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 Trolox out

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+Trolox in , 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

+80µM H 2 O 2

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

H 2 O 2   +GSH in

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

H 2 O 2   +GSH in /

asc.acid out

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

Table 6 Overview of effects of oxidants and antioxidants on behavior parameters of IK(V).in CA1 pyramidal neurons.

I k(v)   CA1

n

max amplitude

% of control

V 50activation  

0 min

V 50a  

15 min

k activation  

0 min

k a

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

+ 1pM AA

9

-15.2 ± 1.8%

5.3 ± 0.9

-2.8 ± 1.0

17.3 ± 0.8

18.6 ± 0.9

control GSH in

8

-20.8±5.7

3.8 ± 0.8

1.2 ± 0.7

16.4 ± 0.7

16.2 ± 0.6

AA+GSH in

6

-13.5 ± 4.6%

0.7 ± 1.3

-1.9 ± 0.9

19.1 ± 1.1

17.3 ± 0.8

control asc.acid out

5

-39.7±7.3

3.0 ± 0.8

0.7 ± 0.7

16.4 ± 0.8

17.0 ± 0.8

AA+GSH in /

asc.acid out

4

-17.1 ± 1.3%

4.4 ± 1.1

2.8 ± 1.0

18.4 ± 1.0

17.8 ± 0.9

c ontrol Trolox in

5

-39.4±10.8

0.5 ± 0.9

-4.5 ± 0.7

18.0 ± 0.8

17.1 ± 0.6

AA+Trolox in

7

-20.8 ± 8.5%

-3.2 ± 0.9

-1.2 ± 1.0

18.2 ± 0.8

19.4 ± 0.9

control Trolox out

4

-1.0±5.7

-2.3 ± 0.7

-6.7 ± 0.8

17.7 ± 0.6

17.9 ± 0.7

AA+Trolox in /

Trolox out

4

-18.4 ± 10.6%

-3.3 ± 1.1

-1.0 ± 1.0

19.3 ± 1.0

16.7 ± 0.9

+80µM H 2 O 2

7

-63.2 ± 5.1%

1.4 ± 1.5

-5.7 ± 1.5

11.3 ± 1.4

12.5 ± 1.3

H 2 O 2 +GSH in

4

-21.1 ± 5.3%

-3.3 ± 1.1

-6.7 ± 1.2

19.2 ± 1.0

17.6 ± 1.0

H 2 O 2 +GSH in /

asc.acid out

4

-23.0 ± 3.9%

3.5 ± 0.8

-8.2 ± 0.9

17.7 ± 0.7

17.7 ± 0.8

4.2 Oxidative modulation of transient and delayed rectifier potassium currents in ECLIII pyramidal neurons

4.2.1 Potassium currents in untreated ECLIII pyramidal neurons

↓57

In whole-cell patch–clamp experiments with pyramidal neurons in layer III from medial entorhinal cortex the membrane potential of the cells were hyperpolarized to -110 mV for 800 ms from holding potential of -80 mV (for protocols see insets Fig.22). Depolarizing command pulses of 200 ms duration between -80 and + 50 mV (every 10 mV) elicited families of potassium outward currents, that are depicted here on Fig.22 A and B. With the above described subtracting procedure (see p. 35), the transient and delayed rectifier potassium currents were further separated. The isolated transient potassium current IA had an amplitude of 0.5-4 nA and inactivated fast with a time constant of 13.0 ± 3.7 ms. IA maximal amplitude showed no changes within 15 min. after opening the cells (-1.2 ± 7.2 %, n=9, Fig.22 C and Fig.23 A, black line). The time constant of inactivation of IA was diminished after 15 min to 9.0 ± 3.5 ms (Fig.22 C, Table 7) however this difference was not statistically significant.

Fig. 22 Native IA and IK(V) in control condition in ECLIII pyramidal neurons.

 A,   B Original traces showing total outward potassium (IK(V)+IA) and delayed rectifier (IK(V)) currents at 0 s (A) and 900 s (B) after reaching whole-cell configuration. C Subtracted transient potassium current (IA) recorded at +30 mV over time. D, Delayed rectifier current at +30 mV over time.

As shown on Fig.23 D and Fig.24 A and C (black line) and D, IK(V) had maximal amplitude of 2 to 9 nA in control conditions which remained relatively stable (-10.5 ± 4.3 %, n=9) during an experiment of 15 min duration.

4.2.2 Modulation of transient IA  and delayed rectifier currents IK(V) by arachidonic acid

↓58

Application of 1 pM intracellular arachidonic acid to ECLIII pyramidal neurons strongly reduced the transient potassium current amplitude from -1.2 ± 7.2% in control to -57.9 ± 7.2 % after 15 min with AA in the pipette (Fig.23 A, red line; C). Arachidonic acid insignificantly slowed down IA inactivation kinetics from 8.8 ± 1.6 to 12.9 ± 1.9 ms (Table 7).

Fig. 23 Suppression of IA by arachidonic acid (AA) in ECLIII pyramidal neurons.

 A: control; B, C: Intracellular application of 1 pM AA reduces the maximal transient potassium current (IA) over time. The non-metabolizable AA analogue ETYA shows similar effect on IA at 100-fold higher concentration (C). D, E: Raw current traces for combined IA and I k(v) and for delayed rectifier currents (pulse protocols: inset in E). Currents are shown for control immediately after obtaining whole cell configuration (D, 0 s AA), after and after 900 s of recording with 1 pM of AA in the patch pipette (E).

100 pM intracellularly applied ETYA mimicked the effect of 1 pM arachidonic acid, as shown on Fig.23 A, green line. This fact qualifies the effect of AA in ECLIII pyramidal neurons as direct, as ETYA blocks the enzymes from the arachidonic acid metabolitic pathway. This effect was already shown for CA1 pyramidal neurons (see p.40).

↓59

In contrast to IA, IK(V) was found to be less sensitive to 1 pM AA (Fig.24). Its maximal amplitude in ECLIII pyramidal neurons was reduced by 19.7 ± 8.7 % (n=9).

Fig. 24 The delayed rectifier current I k(v) is not affected by AA in ECLIII pyramidal neurons. 

A, B: Maximal Ik(v)  recorded at 0 s and at 900 s of whole cell recording shows a ~ 10% decrease over time in control (A) and ~ 20% decrease with 1 pM AA in the patch pipette (B). C: Plot of Ik(v) amplitude over time of whole cell recording for control and for AA. D, E: Original current recordings of Ik(v) for several voltage steps (see inset in C) for control (D) and for AA at 0 s (D1, E1) and 900 s (D2, E2) of whole cell recording.

In ECLIII, as well as in CA1 pyramidal neurons, the activation and steady-state inactivation kinetics of the isolated IA and IK(V) were best fitted with Boltzmann equations. Resulting curves revealed that IA in control conditions is activated at ~ -60 mV and saturated at potentials more positive than +20 mV. IA in control had a V50 of -18.2 ± 1.3mV and slope factor (k) of 16.0 ± 1.1. Inclusion of 1 pM AA to the patch pipette led to insignificant change of V50 and the slope factor of activation kinetic of IA (V50 = -21.4 ± 1.0 mV and k= 8.6 ± 0.9). In contrast, the steady-state inactivation of IA was shifted with ~11 mV to hyperpolarizing direction (Fig.25 A) after application of 1pM AA (Table 7). AA also significantly shifted the activation half-maximal voltage V50 of IK(V) from 5.7 ± 0.3 mV to -1.9 ± 0.5 mV (Fig.25 C).

↓60

Fig. 25 Intracellular AA shifts steady-state inactivation, but does not affect voltage dependence of activation of IA in ECLIII pyramidal neurons. 

A: Conductance-voltage relations and Boltzmann fits for inactivation (left curves) and activation (right curves) of IA. Inset: Pulse protocols for determining voltage dependence of steady state inactivation (left) and of activation (right). Red part shows length of the traces below. B and C: Representative recordings for inactivation (B) and activation (C).D: voltage-conductance dependence of IK (V) and pulse protocols for activation of IK(V) (inset). E: Original recordings of IK(V) activation in control conditions and in the presence of 1 pM AA.

Influence of glutathione on arachidonic acid-mediated effects

Intracellular GSH (20 mM), applied together with 1 pM AA further decreased IA in ECLIII pyramidal neurons (Fig. 26 A, blue line), as reported for CA1 neurons. In agreement with this data, incubation of the slices for 2 hours prior to experiment with NAC ( N-acetyl-L-cysteine, precursor of GSH) could not prevent the effect of arachidonic acid on IA in ECLIII pyramidal neurons (data not shown). Additionally, GSH slowed down the inactivation of IA from 10.3 ± 3.4 to 17.3 ± 2.8 ms, as seen on Fig.26 B1.

↓61

Fig. 26 Interaction of AA, GSH and ascorbic acid with IA in ECLIII pyramidal neurons. 

A, B: GSHi enhances the AA effect on IA (A: blue line; B1), particularly early after begin of application (*). As well, ascorbic acid could not block the AA effect (A: green line; B2). C, D: GSHi and ascorbic acido both prevent the AA-mediated shift in steady-state inactivation (C, D, control=0 s vs. 900 s, left curves) but GSH slightly shifts the activation curve to more positive potential (D, right curves).

Influence of glutathione and ascorbic acid on arachidonic acid-mediated effects

Yet, addition of ascorbic acid at concentrations up to 0.4 mM to the bath failed to remove effects of AA on the maximal IA amplitude (Fig.26 A, green line), which is in contrast to the results obtained for CA1 pyramidal neurons, although it prevented GSH-mediated slow-down of this current (Fig.26 B2).

↓62

However, reduced glutathione, applied intracellularly, did significantly block the AA-induced shift of steady-state inactivation (Fig.26 D). Moreover, it shifted the activation V50 of IA from -15.5 ± 0.7 mV to -25.8 ± 0.9 mV, as the slope factor k remained unaffected (Table 7). The triple combination treatment of AA and the antioxidants ascorbic acid and GSH, resulted again in blocked arachidonic acid effect on the steady-state inactivation of the transient potassium channels (Fig.26 C). IK(V) in the presence of AA+GSHin reached 99.3 ± 9.7 % of control amplitude. Further perfusion of the slice with ascorbic acid led to IK(V) with maximal amplitude of 76.8 ± 6.6% and shifted IK(V) voltage–conductance relation of activation towards more negative potentials by ~15 mV (from -2.2 ± 0.8 mV to -17.6 ± 1.3 mV, n=6).

Influence of Trolox on arachidonic acid-mediated effects

Trolox, applied only on the inner side of the cell membrane, blocked completely the reduction of IA by arachidonic acid (Fig.27 A, blue line). Troloxin did fail in preventing AA-mediated shift in inactivation and changed the activation parameters from V50= -27.0 ± 0.4 mV and k=9.7 ± 0.4 to V50= -21.8 ± 0.7 mV and k=12.2 ± 0.6 (Fig.27 C). In comparison, Trolox on both sides of the membrane accelerated the early AA effect, in GSH-like manner. It did not block the leftward shift of the steady-state inactivation curve, caused by 1pM arachidonic acid, but it shifted the activation kinetic by ~10 mV in depolarizing direction as shown on Fig.27 D. No significant changes on the time constant of inactivation of IA in both cases with Trolox were observed (Table 7). On the other hand, Trolox, independently of the site of application, fully inhibited AA effect on the maximal amplitude of IK(V) (Table 8).

↓63

Fig. 27 Interaction of AA and Trolox with IA in ECLIII pyramidal neurons. 

A, B: Intracellular application of the vitamin E analogue Trolox (10 µM) with AA (A1, B: blue line) did significantly recover IA, but Trolox, applied from both sides of the cell membrane, did not prevent reduction of IA (A2, B: green line). C: Troloxi did not inhibit the AA shift of steady-state inactivation (left curves). There is shift in voltage dependence of activation (right curves, control=0 s vs. 900 s). D: In the presence of Trolox on both sides of the membrane plus AAi, steady state inactivation shifts over 900 s recording time by -12mV to more negative potential, as well as activation mV to more positive potential, similar to the effect of Troloxi

4.2.3 Effects of H2O2 on outward potassium currents

As observed for CA1 pyramidal neurons, H2O2 mimics the effect of AA on maximal IA in ECLIII pyramidal neurons (Fig.28 A, red line).

H2O2 did shift voltage conductance relations of inactivation to more negative and activation to more positive potentials (Fig.28 B). Mean parameters were: for activation, V50= -24.9 ± 1.7 mV; k=14.2 ± 1.5 and for inactivation, V50= -53.6 ± 0.6 mV; k=11.3 ± 0.5 (n=7).

↓64

Influence of glutathione and ascorbate on H 2 O 2 –mediated effects on potassium currents

Fig. 28 Effects of intracellular H2O2 (80 µM) and antioxidants on IA in ECLIII pyramidal neurons. 

A: H2O2 mimics the effect of AA on maximal IA. This effect is not inhibited by GSHi or by GSHi+ascorbic acido. B: Voltage conductance relations of inactivation (left curves) and activation (right curves) in the presence of H2O2. C: GSHi does not eliminate the H2O2 mediated shift in inactivation (left curves), but leads to further shift of activation (right curve). D: Steady-state inactivation (left curves) and activation (right curves) kinetics of IA in the presence of ascorbic acido, GSHi and H2O2. E: Representative current traces of IA for determination of the voltage dependence of activation for control immediately after obtaining whole cell configuration (E1) and after 900 s of recording with H2O2 in the patch pipette (E2).

As seen on Fig.28 C, GSHi does not eliminate the H2O2 mediated shift in inactivation, but augments the rightward shift of the steady state activation curve. In the presence of H2O2, GSHi and ascorbic acido, the steady-state inactivation and activation kinetics of IA were not significantly different from those for H2O2 alone (Fig.28 D).

↓65

The delayed rectifier current from ECLIII pyramidal neurons is sensitive to H2O2, as already shown for CA1 pyramidal neurons. H2O2 reduced IK(V) by 26.3 ± 5.5 %, n=7 (Fig.29 A, red line). Intracellular GSH as well as GSH in combination with ascorbic acid, reduced the effect of H2O2 on the maximal amplitude to -3.7 ± 8.6 %, n=8 and -12.7 ± 7.7 %, n=6 (Fig.29 A, blue and green lines), respectively. Although a change in the activation voltage dependence of IK(V) in the presence of H2O2 was not observed (Fig.29 B), application of antioxidants on one or both sides of the membrane shifted it to more negative values. GSHin caused a small, but significant shift from -5.7 ± 0.7 mV to -11.3 ± 1.5 mV, and moreover, changed the slope factor k from 17.8 ± 0.6 to 22.8 ± 1.4, n=8. In the case of GSHin and ascorbic acidout the shift of the steady state activation curve was large, with V50 of about ~20 mV (from -0.1 ± 0.9 mV to -22.0 ± 1.5 mV, n=6), while the slope of the steady-state activation curve remained unaltered (Table 8).

Fig. 29 Effects of intracellular H2O2 (80 µM) and antioxidants on IK(V) in ECLIII pyramidal neurons.

 A: H2O2 suppresses the delayed rectifier potassium current IK(V) (red line), and this effect is inhibited by GSHi or GSHi+ascorbic acido (blue and green lines, respectively). B: H2O2 does not shift voltage dependence of activation of IK(V). C, D: Representative traces of IK(V) for determination of voltage dependence of activation for control immediately after obtaining whole cell configuration (C1, D1, 0 s) and after 900 s recording with H2O2 (C2) or H2O2 + GSHi in the patch pipette (D2). Recordings of D have been obtained in the presence of ascorbic acido.

Table 7 Overview of effects of oxidants and antioxidants on behavior parameters of IA in ECLIII pyramidal neurons.

I A  

ECLIIIpn

n

max amplitude,

% of control

V 50a

0 min mV

V 50a

15 min mV

k activation

0 min

k a

15 min

V 50i  

0 min
mV

V 50i  

15 min
mV

k ina ctivation

0 min

k i

15 min

τ i 0 min ms

τ i 15 min ms

control

9

-1.2 ±7.2 %

-21.0 ±1.5

-18.2 ± 1.3

15.6 ± 1.3

16.0 ± 1.1

- 52.2 ±0.9

-50.8 ±1.0

15.2 ±0.8

15.8 ±1.1

13.0 ±3.7

9.0 ±3.5

+ 1pM AA

9

-57.9 ±7.2 %

-18.7 ±0.7

-21.4 ±1.0

8.9 ±0.7

8.6 ±0.9

-44.4 ±0.8

-53.3 ±1.6

9.8 ±0.7

10.7 ±1.5

8.8 ±1.6

12.9 ±1.9

AA+GSH in

6

-66.3 ±4.4 %

-15.5 ±0.7

-25.8 ±0.9

12.9 ±0.8

12.6 ±0.8

-49.5 ±0.3

-53.6 ±0.6

9.8 ±0.3

11.2 ±0.5

10.3 ±3.4

17.3 ±2.8

control asc.acid in

6

-41.9 ±9.5 %

-18.7 ±1.3

-18.3 ±1.9

15.9 ±1.1

16.4 ±1.7

no data

no data

no data

no data

17.4 ±6.4

17.7 ±7.1

AA+GSH in /

asc.acid out

6

-60.7 ± 10.0 %

-18.1 ± 0.4

-19.2 ± 0.9

7.8 ± 0.4

8.1 ± 0.8

-49.5 ± 0.3

-54.0 ± 0.8

9.8 ± 0.3

11.9 ± 0.7

6.4 ± 1.4

7.1 ± 1.9

AA+Trolox in

7

-16.2 ± 3.9 %

-27.0 ± 0.4

-21.8 ± 0.7

9.7 ± 0.4

12.2 ± 0.6

-45.5 ± 0.5

-54.0 ± 0.8

11.0 ± 0.4

10.7 ± 0.7

7.0 ± 2.5

9.5 ± 2.1

AA+Trolox in , out

6

-56.1 ± 5.4 %

-26.3 ± 0.9

-15.3 ± 0.7

12.9 ± 0.8

12.9 ± 0.6

-46.3 ± 0.8

-57.4 ± 0.7

11.0 ± 0.7

9.0 ± 0.6

3.6 ± 0.4

4.6 ± 0.5

+80µM H 2 O 2

7

-53.0 ± 4.1 %

-31.7 ± 1.0

-24.9 ± 1.7

9.9 ± 0.8

14.2 ± 1.5

-41.4 ± 0.3

-53.6 ± 0.6

10.2 ± 0.3

11.3 ± 0.5

3.3 ± 0.5

3.2 ± 0.5

H 2 O 2   +GSH in

8

-70.7 ± 8.7 %

-29.3 ± 1.0

-20.1 ± 1.5

9.5 ± 0.9

15.9 ± 1.3

-53.1 ± 0.7

-63.6 ±0.6

11.3 ±0.6

11.3 ±0.6

9.9 ±2.7

5.2 ±1.2

H 2 O 2 +GSH in /

asc.acid out

6

-57.3 ±3.7 %

-20.8 ±0.6

-17.1 ±1.0

12.2 ±0.5

17.6 ±0.9

-62.9 ±0.5

-73.5 ± 0.6

11.2 ± 0.4

9.9 ± 0.6

4.9 ± 1.5

9.8 ± 2.8

↓66

Table 8 Overview of effects of oxidants and antioxidants on behavior parameters of IK(V) in ECLIII pyramidal neurons.

I k(v) ECLIIIpn

n

max amplitude

% of control

V 50activation  

0 min

V 50a  

15 min

k activation  

0 min

k a

15 min

control

9

-10.5 ±4.3 %

-10.7 ±1.3

-12.8 ±1.3

19.5 ±1.5

19.4 ±1.1

+ 1pM AA

9

-19.7 ± 8.7 %

5.7 ± 0.3

-1.9 ± 0.5

18.1 ± 1.1

19.9 ± 0.7

AA+GSH in

6

-0.7 ± 9.7 %

1.5 ± 1.2

-0.9 ±0.6

18.5 ± 0.9

17.2 ±0.6

control asc.acid in

6

-36.3 ±6.5 %

-8.8 ±1.3

-10.4 ±1.3

19.7 ±1.1

19.3 ±1.0

AA+GSH in /

asc.acid out

6

-23.2 ±6.6 %

-2.2 ±0.8

-17.6 ±1.3

18.9 ±0.7

22.1 ±1.2

AA+Trolox in

7

-4.1 ± 10.2 %

1.1 ± 0.8

2.4 ± 0.8

14.3 ± 0.7

15.8 ± 0.7

AA+Trolox in /

Trolox out

6

-7.9 ± 6.5 %

-7.5 ± 1.1

-2.7 ± 0.8

21.1 ± 1.0

17.1 ± 0.7

+80µM H 2 O 2

7

-26.3 ± 5.5 %

-18.1 ± 0.4

-19.2 ± 0.9

16.1 ± 0.6

17.9 ± 0.8

H 2 O 2 +GSH in

8

-3.7 ± 8.6 %

-5.7 ± 0.7

-11.3 ± 1.5

17.8 ± 0.6

22.8 ± 1.4

H 2 O 2 +GSH in /

asc.acid out

6

-12.7 ± 7.7 %

-0.1 ± 0.9

-22.0 ± 1.5

16.3 ± 0.8

19.1 ± 1.3

4.3 Oxidative modulation of transient and delayed rectifier potassium currents in ECLII stellate neurons

4.3.1 Potassium currents in untreated ECLII stellate cells

Step depolarization of the membrane potential (protocols: inset Fig.30 B) of LII stellate neurons from the entorhinal cortex activated large outward potassium currents (0.5-13 nA) that are illustrated in Fig. 30 A. Digital subtraction of the sustain component (IK(V)) revealed robust transient potassium current IA that was stable during the course (15 min.) of the experiment (-11.8 ± 4.7 % from control, n=9, Fig.29 C). Analysis of the delayed rectifier current also showed stability of IK(V) over 15 min. (-12.7 ± 7.6 %, n=9, Fig.30 D).

↓67

Fig. 30 Native IA and IK(V) in control condition in ECLII stellate neurons. 

A,   B Original traces showing total outward potassium (IK(V) + IA) and delayed rectifier (IK(V)) currents at 0 s (A) and 900 s (B) after reaching whole-cell configuration. C Subtracted transient potassium current (IA) recorded at +30 mV over time. D  Delayed rectifier current at +30 mV over time.

4.3.2 Modulation of transient IA  and delayed rectifier currents IK(V) by arachidonic acid

1pM intracellular arachidonic acid reduced the subtracted IA current by -66.3 ± 9.6 %, n=10 within 15 min. (Fig.31 A, red line). ETYA, inhibitor of the enzymes that metabolize AA and triple-bond analogue of arachidonic acid, required higher concentration (100 pM) to mimic the effect of 1 pM AA (Fig.31 A, green line). However, this effect tended to develop faster (in the first 3 min.) which is probably the result of the higher concentration of ETYA.

Fig. 31 Suppression of IA by arachidonic acid (AA) in ECLII stellate cells. 

A  (black line), B: control; A , C: Intracellular application of 1 pM AA reduces the maximal transient potassium current (IA) over time. The non-metabolizable AA analogue ETYA shows similar effect on IA at 100-fold higher concentration. D, E: Raw current traces for combined IA and Ik(v) and for pure delayed rectifier currents (pulse protocols: inset in E).Currents are shown for control immediately after obtaining whole cell configuration (D, 0 s AA) and after 900 s of recording with 1 pM of AA in the patch pipette (E).

↓68

An additional effect of 1 pM AA was an 11 mV leftward shift of the steady-state inactivation curve with a slight change of the slope factor (from V50= -42.8 ± 0.4 mV; k= 9.2 ± 0.6 to V50= -55.1 ± 0.7 mV; k= 12.4 ± 0.6, n=10; Fig.32 A, left curves). AA did not affect the time constant of IA inactivation. The steady state activation curve of IA was also shifted to the left by ~6mV and the slope factor changed from 9.1 ± 0.5 to 13.7 ± 0.7 (Fig.32 A, right curves).

Fig. 32 Intracellular AA shifts steady-state inactivation, but does not affect voltage dependence of activation of IA in ECLII stellate neurons. 

A: Conductance-voltage relations and Boltzmann fits for inactivation (left curves) and activation (right curves) of IA. Inset: Pulse protocols for determining voltage dependence of steady state inactivation (left) and of activation (right). Red part shows length of the traces below. B: Representative recordings of transient potassium current for control (B1) and in the presence of 1 pM AA (B2). C: Voltage-conductance dependence of IK(V) and pulse protocols for activation of IK(V) (inset). D: Original recordings of IK(V) activation in control conditions (D1) and in the presence of 1 pM AA (D2).

Like in ECLIII pyramidal neurons, arachidonic acid had a modest, but significant effect on the delayed rectifier current in ECLII stellate cells (Fig.33 B, E). As in ECLIII pyramidal neurons, 1 pM AA suppressed IK(V) by -29.1 ± 6.7 %, n=10 (Fig.33 C). This suggests that this effect is probably specific to the delayed rectifier current in the entorhinal cortex.

↓69

However, 1 pM arachidonic acid did not alter the voltage dependence of activation of the delayed rectifier current in ECLII stellate neurons (Fig.33 C).

Fig. 33 The delayed rectifier current I k(v) is not affected by AA in ECLII stellate neurons. 

A, B: Maximal Ik(v)  recorded at 0 s and at 900 s of whole cell recording shows a 13% decrease over time in control (A) and 30 % decrease with 1 pM AA in the patch pipette (B). C: Plot of Ik(v) amplitude over time of whole cell recording for control and for AA. D, E: Original current recordings of Ik(v) for several voltage steps (see inset in C) for control (D) and for AA at 0 s (D1, E1) and 900 s (D2, E2) of whole cell recording.

Influence of glutathione on arachidonic acid-mediated effects

↓70

Glutathione (20 mM), added to AA in the patch pipette failed to block the AA-mediated decrease of the maximal amplitude of the transient current (-75.6 ± 9.3 %, n=6 compared to -66.3 ± 9.6 %, n=10). Moreover, GSHin+AA significantly reduced the IA current within first 3 min. of the experiment (Fig.34 A, blue line, *). GSHin did not prevent the leftward shift of voltage dependence of inactivation, caused by 1pM AA (Fig.34 D, left curves), but slowed the kinetic of inactivation of the transient potassium current (Table 9).

Fig. 34 Interaction of AA, GSH and ascorbic acid with IA in ECLII stellate neurons.

A, B: GSHi (A: blue line, B1), as well as ascorbic acid (A: green line, B2) enhanced the AA effect on IA, particularly early after begin of recording/application (*). C, D: GSHi and ascorbic acido do not prevent the AA-mediated shift in steady-state inactivation (C, D, control=0 s vs. 900 s, left curves). Ascorbic acid further shifts the inactivation curve to more negative potential and changes its slope (C, left curves).

Influence of glutathione and ascorbic acid on arachidonic acid-mediated effects

↓71

Addition of 4 mM ascorbic acid to the perfusion solution (AA and GSH in the patch pipette) resulted in effects similar to those of GSHin and AA: slowed inactivation (Fig.34 B2), acceleration of the AA effect on the maximal conductance, no change of the voltage dependence of activation, large (V50= -41.4 ± 0.4 mV vs. -55.4 ± 1.9 mV, n=6) shift of the steady-state inactivation to more negative potentials (Fig.34 A: green line, C).

On one hand, GSH on the inner side of the cell membrane did not result in significant attenuation of the AA effect on the maximal amplitude of the delayed rectifier current (-19.8 ± 8.9 % , n=6 vs. -29.1 ± 6.7 %, n=10). On the other hand, in addition to GSH, ascorbic acid on the outer side of the membrane did reduce the maximal amplitude of IK(V) to -10.0 ± 11.6 %, n=6 and also changed V50 (from -7.9 ± 0.8 mV to -16.4 ± 1.4 mV) and the slope factor (from 16.8 ± 0.7 to 22.0 ± 1.3) of the voltage dependence activation of IK(V) (Table 10).

Influence of Trolox on arachidonic acid-mediated effects

↓72

The vitamin E-based antioxidant Trolox C, on the inner membrane of the ECLII stellate neurons, failed to block the arachidonic acid effect on the maximal amplitude of the transient potassium current, -74.3 ± 0.7 % vs. -66.3 ± 9.6 %, n=6 (Fig.35 A: blue line), but it was successful in blocking the effect of AA on the voltage dependence of steady-state inactivation and activation and their slopes (Fig.35 C). Trolox, applied through the pipette and through the bath, tended to slightly reduce the effect of AA (-60.4 ± 3.5 % vs. -66.3 ± 9.6 %, n=8) at late recording time, however this result is not statistically significant (Fig.35 A: green line). It also blocked the effect of 1 pM AA on the steady-state inactivation kinetic and further shifted it by ~10 mV to more positive potentials (Fig.34 D). Trolox, applied on the both sides of the membrane, block the effect of AA on the voltage-conductance relation of the IA activation, but not the effect on its slope (Fig.35 D). Moreover, it did accelerate the kinetic of inactivation of IA (Fig.35 B2). Interestingly, Trolox, applied both through pipette and perfusion or only through the patch pipette accelerated the effect of 1 pM arachidonic acid on the maximal conductance of IA (Fig.35 A).

Troloxin reduced the effect of 1pM AA on the maximal amplitude of the delayed rectifier current from -29.1 ± 6.7 % to -18.9 ± 4.9 % and shifted it activation voltage dependence by ~6 mV more negative potentials (Table 10). No changes of the slope of the voltage dependence of activation were observed. Trolox in/out, did not block the reducing effect of AA on the IK(V) maximal amplitude (-33.7 ± 6.8 % vs. -29.1 ± 6.7 %, n=8). It shifted the voltage dependence of IK(V) activation by ~5 mV to the left, without affecting its slope (Table 10).

Fig. 35 Interaction of AA and Trolox with IA in ECLII stellate neurons. 

A, B: Intracellular application of the vitamin E analogue Trolox (10 µM) with AA does not prevent reduction of IA (A: blue line, B1). Trolox, applied from both sides of the cell membrane, does unsignificantly recover IA with prolonged recording time (A: green line, B2). C: Troloxi inhibits the AA shift of steady-state inactivation (left curves). There is no shift in voltage dependence of activation (right curves, control=0 s vs. 900 s). D: In the presence of Trolox on both sides of the membrane plus AAi, steady state inactivation shifts over 900 s recording time by -10 mV to more positive potential.

4.3.3 Effects of H2O2 on outward potassium currents

↓73

As an oxidative damage model system to compare the effects of AA, the effect of H2O2  on the transient potassium and delayed rectifier channels was used. To obtain an effect, similar to that of 1 pM AA on the maximal amplitude of IA and IK(V), 80 µM H2O2 was required (Fig.36 A: red line).

Fig. 36 Effects of intracellular H2O2 (80 µM) and antioxidants on IA in ECLII stellate neurons.

  A: H2O2 mimics the effect of AA on maximal IA (red line). This effect is enhanced early by GSHi and in the presence of ascorbic acido does not differ from those for H2O2 (blue and green line, respectively). B: H2O2 shifts voltage conductance relations of inactivation (left curves) to more negative potentials as well as changes the slope of activation and inactivation curves. C: GSHi diminishes the H2O2 mediated shift in inactivation (left curves), but for activation causes a slight shift to more positive potential. D: In the presence of ascorbic acido, GSHi + H2O2 shift inactivation and activation of IA back by -12 mV to more negative potentials. E: Representative current traces of IA for determination of the voltage dependence of activation for control immediately after obtaining whole cell configuration (E1) and after 900 s of recording with H2O2 in the patch pipette (E2).

H2O2 reduced the maximal amplitude of IA by -64.4 ± 3.8 %, shifted leftwards the the steady-state inactivation curve leftward by ~12mV, but did not affect significantly the rate of IA inactivation (Fig.36 A, B). Those effects are consistent with the effects of 1 pM arachidonic acid on the transient potassium current characteristics in ECLII stellate neurons (Table 9). In the presence of H2O2, the voltage dependence of activation of IA was did not change, but slope factor was altered from 10.9 ± 0.3 to 13.9 ± 0.9, n=8 (Fig.36 B).

↓74

Influence of glutathione and ascorbic acid on H 2 O 2 -mediated effects

Application of GSH through the patch pipette resulted in stronger attenuation of the IA current during the first 6 min. of the experiment (Fig.36 A: blue line), an effect previously observed in other types of neurons (see p.43 and p. 59). In general, the effect of H2O2 on the maximal amplitude of the transient potassium current could be blocked neither by GSH, nor by addition of ascorbic acid to the bath (Fig.36 A). Intracellular GSH decreased the leftward shift in voltage dependence of inactivation from ~12 mV to ~7 mV (Fig.36 C). GSHin did not block the right shift of voltage dependence of activation, but abolished the slope effect. With GSHin and AA, the time constant of inactivation of IA in ECLII stellate cells was reduced from τi=7.8 ± 1.1 ms to τi=4.8 ± 1.3 ms (n=4). Addition of ascorbic acid to the perfusion solution did not block H2O2-induced ~12mV left shift of the voltage dependence of inactivation of IA (-44.0 ± 0.5 mV vs. -55.7 ± 0.6 mV, n=8), did not alter the voltage-conductance relation for activation and accelerated the rate of IA inactivation kinetics (Fig.36 D, Table 9).

As seen on Fig.37 A, H2O2 suppressed the delayed rectifier current to an extent, which was similar to that of 1 pM arachidonic acid (-22.2 ± 9.1 % and -29.1 ± 6.7 %, respectively). However, 80 µM H2O2 caused a small (~6mV), albeit significant shift of IK(V) voltage dependence of steady-state inactivation to more negative potentials (Fig.37 B). Intracellular GSH blocked the effect of H2O2 on the maximal conductance of IK(V) (-12.3 ± 9.6 %, n=4), as did GSHin, combined with asc.acidout (-14.1 ± 5.1 %, n=8), but these effects seemed not to be statistically significant. Glutathione, applied on the inner side of the neuronal membrane led to a ~3 mV shift to hyperpolarizing direction of the voltage dependence of activation of IK(V) with small change of the slope (from 17.4 ± 0.7 to 19.5 ± 1.0, n=8). Ascorbic acid, in the presence of GSH and AA shifted the voltage dependence of activation of IK(V) from V50= -5.9 ± 1.0 mV to V50= -14.2 ± 1.1 mV, without affecting its slope (Table 10).

↓75

Fig. 37 Effects of intracellular H2O2 (80 µM) and antioxidants on IK(V) in ECLII stellate neurons. 

A: H2O2 suppresses the delayed rectifier potassium current IK(V), and this effect is fully inhibited by GSHi or GSHi+ascorbic acido (blue and green line, respectively). B: H2O2 insignificantly shifts voltage dependence of activation of IK(V) to a more negative potential. C, D: Representative traces of IK(V) for determination of voltage dependence of activation for control immediately after obtaining whole cell configuration (C1, D1, 0 s) and after 900 s recording with H2O2 (C2) or H2O2 + GSHi in the patch pipette (D2). Recordings of D have been obtained in the presence of ascorbic acido.

Table 9 Overview of effects of oxidants and antioxidants on behavior parameters of IA in ECLII stellate neurons.

I A   ECLIIst

n

max amplitude,

% of control

V 50a

0 min mV

V 50a

15 min mV

k activation

0 min

k a

15 min

V 50i  

0 min
mV

V 50i  

15 min
mV

k ina ctivation

0 min

k i

15 min

τ i 0 min ms

τ i   15 min ms

control

9

-11.8 ±4.7 %

-12.2 ± 1.0

-14.3 ± 0.3

9.0 ± 0.9

7.9 ± 0.3

-45.3 ± 0.8

-48.8 ± 0.5

9.5 ± 0.7

7.7 ± 0.4

8.6 ± 2.3

9.2 ± 2.1

+ 1pM AA

10

-66.3 ± 9.6 %

-17.8 ± 0.5

-23.7 ± 1.8

9.1 ± 0.5

13.7 ± 0.7

-42.8 ± 0.4

-55.1 ± 0.7

9.2 ± 0.6

12.4 ± 0.6

6.6 ± 2.7

7.4 ± 2.9

AA+GSH in

6

-75.6 ± 9.3%

-32.1 ± 0.7

-28.5 ± 0.9

12.8 ± 1.6

11.3 ± 1.3

-45.8 ± 0.8

-56.2 ± 0.8

9.7 ± 0.5

10.5 ± 0.7

12.0 ± 2.3

18.6 ± 2.5

AA+GSH in /

asc.acid out

6

-71.3 ±12.1 %

-18.1 ±0.4

-21.5 ± 1.2

7.8 ±0.4

9.5 ± 1.0

-41.4 ± 0.4

-55.4 ± 1.9

10.2 ± 0.5

18.3 ± 1.7

3.6 ± 0.6

6.2 ± 0.5

AA+Trolox in

6

-74.3 ±0.7 %

-22.0 ±0.7

-16.9 ±0.5

10.1 ±0.6

9.6 ± 0.4

-40.7 ±0.8

-46.7 ±1.3

12.5 ±0.7

12.4 ±1.1

9.1 ±5.9

8.4 ±3.0

AA+Trolox in , out

8

-60.4 ±3.5 %

-23.4 ± 0.9

-22.9 ± 1.5

13.5 ± 0.8

17.7 ± 1.3

-58.6 ± 0.9

-48.9 ± 0.9

13.4 ± 0.8

12.1 ± 0.8

3.2 ±0.4

5.3 ±0.9

+80µM H 2 O 2

4

-64.4 ± 3.8 %

-11.2 ± 0.4

-7.4 ± 1.0

10.9 ± 0.3

13.9 ± 0.9

-48.8 ± 0.8

-60.1 ± 0.9

9.4 ± 0.9

11.1 ± 0.8

5.6 ±1.7

5.3 ±1.4

H 2 O 2   +GSH in

4

-72.6±7.6 %

-25.5 ±1.0

-18.4 ±1.3

10.3 ±0.8

10.5 ±0.7

-45.2 ±0.9

-53.8 ±0.9

10.8 ±0.5

11.2 ±0.4

7.8 ±1.1

4.8 ±1.3

H 2 O 2 +GSH in /

asc.acid out

8

-69.3 ±4.6 %

-9.2 ±1.1

-7.6 ± 0.8

14.7 ± 1.0

13.3 ± 0.7

-44.0 ± 0.5

-55.7 ± 0.6

10.5 ± 0.5

11.6 ± 0.5

7.5 ±0.9

5.3 ±0.7

Table 10 Overview of effects of oxidants and antioxidants on behavior parameters of in ECLII stellate neurons IK(V).

I k(v) ECLIIst

n

max amplitude

% of control

V 50activation  

0 min

V 50a  

15 min

k activation  

0 min

k a

15 min

control

9

-12.7 ± 7.6 %

-7.1 ±1.2

-8.2 ±1.5

18.3 ±0.7

18.7 ±0.6

+ 1pM AA

10

-29.1 ±6.7 %

-1.7 ±1.1

-1.5 ±1.1

17.3 ±1.0

16.9 ±1.0

AA+ GSH in

6

-19.8 ±8.9 %

-18.1 ±0.4

-21.5 ±1.2

8.7 ±0.4

9.5 ±1.0

AA+GSH in /asc.acid out

6

-10.0 ±11.6 %

-7.9 ±0.8

-16.4 ±1.4

16.8 ±0.7

22.0 ±1.3

AA+Trolox in

6

-18.9 ±4.9   %

-10.6 ±1.2

-16.7 ±1.4

17.4 ±1.0

17.3 ±1.2

AA+Trolox in /Trolox out

8

-33.7 ± 6.8 %

-22.9 ±1.3

-30.5 ±1.2

21.1 ±1.2

24.8 ±1.1

+80µM H 2 O 2

4

-22.2 ±9.1 %

-1.3 ±0.9

-7.4 ±1.1

16.3 ±0.8

17.6 ±1.0

H 2 O 2 +GSH in

4

-12.3 ±9.6 %

-6.6 ±0.8

-10.0 ±0.9

17.4 ±0.7

19.5 ±1.0

H 2 O 2 +GSH in /asc.acid out

8

-14.1 ± 5.1%

-5.9 ± 1.0

-14.2 ± 1.1

20.3 ± 0.9

20.0 ± 1.0

4.4 Functional expression of Kv1 and Kv4 α-subunit-induced K+- currents in HEK-293 cell line

↓76

Whole-cell voltage-clamp recordings from untransfected or mock-transfected HEK-293 cells revealed endogenous voltage-gated outward potassium currents in response to depolarization to potentials positive to 0 mV. The amplitudes of these currents, however, were very small: (~50 pA), a similar current amplitude was measured at +30 mV in mock-transfected (EGFP) cells further revealing that expression of EGFP alone does not influence functional potassium current densities (Fig.38 A, B). Cell input resistances were in the range of 1-20 GΩ. Leak currents were always ≤ 50 pA at -80 mV and were therefore not corrected.

Large outward potassium currents, however, were seen routinely after transfection of HEK-293 cells with Kv1.4 or Kv4.2, alone or in combination with EGFP (Fig.38 C, D).

Fig. 38 Outward K+-currents in untransfected (A) or mock-transfected (EGFP alone, B), Kv1.4-transfected (C) and Kv4.2-transfected HEK-293 cells (D).

↓77

Voltage-gated potassium channel pore-forming (α) subunits of the Kv1 and Kv4 subfamilies are thought to be the molecular determinants of A-currents in the brain. Heterologous expression of these subunits in human embryonal kidney cell line, however, resulted in transient K+-currents that show quantitatively distinct time- and voltage-dependent properties to those recorded from brain neurons.

Part of the experiments with Kv-transfected HEK-293 cells was performed in cells that were co-transfected with EGFP, and recordings were obtained only from EGFP-positive cells. The transfection efficiency was low (1-5%) and approximately 50% of EGFP-positive cells also expressed Kv1.4 or Kv4.2, as revealed by electrophysiological experiments with those cells.

4.4.1 Modulation of transient potassium current by arachidonic acid and Trolox in heterologously expressed Kv1.4-α in HEK-293 cell line

Transient potassium current in untreated Kv1.4-transfected HEK-293 cells

↓78

Whole-cell outward potassium currents were activated during 200 ms-long depolarization steps to test potentials between -80 and +50 mV from a holding potential of -80 mV (pulse protocols: inset Fig.38). An 800 ms-long prepulse at -110 mV was performed in order to remove inactivation of IA. The mean ± SEM peak outward K+-current amplitudes in Kv-transfected HEK cells at +30 mV were significantly (p<0.001) higher than those in untransfected or mock-transfected HEK-293 cells (Fig.38). For inactivating HEK A-currents, the maximal amplitude of the currents were recorded during the +30 mV steps following each conditioning prepulse from -120 mV to +20 mV (pulse protocols: see inset Fig.39, B2).

As seen on Fig.39 A, B, in HEK-293 cells, transfected with Kv1.4 (and EGFP) large (5.5-11 nA) outward potassium currents were evident. The amplitude of the peak current at +30 mV (maximal amplitude for each cell) was followed over 15 min and evolvement over time was plotted as shown on Fig. 39 C. The maximal amplitude of the transient potassium current in Kv1.4-transfected HEK cells did not change significantly under control conditions during the course of a 15 min-recording (-12.1 ± 6.1 %, n=7). The A-currents from Kv1.4-transfected HEK-293 cells activated and inactivated fast (τi=51.7 ± 2.9 ms), but significantly slower as native IA currents from brain slice neurons (CA1 τi=7.8 ± 1.5 ms; ECLIII τi=13.0 ± 3.7 ms; ECLII τi=8.6 ± 2.3 ms). On Fig.39, A2 and B2, one can clearly see the atypical voltage–conductance dependence for the steady-state inactivation curve of Kv1.4-potassium current in comparison to the native neuronal IA currents (for example Fig. 39 B1). Distorted voltage dependence of inactivation is probably due to lack of supplementary β-subunit and/or other auxiliary Kv-channel subunits and factors in HEK cells that could stabilize inactivation of IA channels.

The amplitude of the peak currents at each test potential were measured and peak outward conductances were then calculated (using the K+ equilibrium potential of -92 mV) and normalized to the peak conductance at +30 mV in the same cell. Mean (± SEM) normalized peak conductances for control conditions were plotted as a function of test potentials in Fig 39 D and best fitted with Boltzman monoexponential equation (see Materials and Methods, p.36). Kv1.4-currents activated at ~ -60 mV and reached steady-state at potentials positive to +30 mV. Although the mean (± SEM) voltages at which half of the channels are activated (V50a) derived from this fits at time=0s (-24.7 ± 2.4 mV) differ from V50a for time = 900 s (-4.8 ± 1.8 mV), those differences are not statistically significant. Voltage-dependence of inactivation and the maximum amplitude in control conditions remain unchanged over recording time of 15 min (Fig.39 C, D; Table 11).

↓79

Fig. 39 Transient potassium current in Kv1.4-transfected HEK-293 cells. 

A, B: Original traces showing IA activation (A) and inactivation (B) at 0 s and 900 s (pulse protocols: inset A2, B2). C: Transient potassium current (IA) recorded at +30 mV over time in control condition. D: Voltage-conductance dependence of activation (right curves) and steady-state inactivation (left curves) of IA.

Modulation of I A by arachidonic acid in Kv1.4-transfected HEK-293 cells

Application of 1 pM arachidonic acid through the patch-pipette decreased the Kv1.4-mediated A-currents, expressed in HEK293 cells by 71.4 ± 6.3 % (n=9; p<0.001). This robust response of Kv1.4 channels to 1pM AA was contradictory with the findings of Villarroel and Schwarz (see Villaroel and Schwarz, 1996).

↓80

Fig. 40 Suppression of Kv1.4- IA by arachidonic acid.

A,   B: Control activation (A1) and inactivation (B1) of IA (pulse protocols: inset A2, B2). Raw current traces of IA are shown for control immediately after obtaining whole cell configuration (A1, B1, 0 s AA) and after 900 s of recording with 1 pM of AA in the patch pipette (A2, B2, 900 s AA). C: Intracellular application of 1 pM AA (red line) reduces the maximal transient potassium current (IA) over time. D: Activation (left curves) and steady-state inactivation (right curves) kinetics in control condition (black symbols) and after application of 1 pM arachidonic acid through the pipette (red symbols).

As seen on Fig. 40 C, AA difused to the cytoplasm within 3 min. and reached full effect on the maximal amplitude of IA within 10 min.

AA did not significantly alter the voltage-dependence of activation (V50a from -9.2 ± 1.4 mV to -5.2 ± 1.6 mV and k from 17.1 ± 1.3 to 19.4 ± 1.5, n=9; Fig.40 D, right curves). AA shifted the voltage dependence of steady-state inactivation of Kv1.4-encoded channels from -66.2 ± 2.6 mV to -75.5 ± 2.1 mV (k from 19.6 ± 2.2 to 22.6 ± 1.9, n=9; Fig.40 D, right curves), but did not affected IA -inactivation kinetic (τi =52.8 ± 2.8ms to 49.4 ± 2.4ms).

↓81

Influence of Trolox on arachidonic acid-mediated effects

To examine possible involvement of free radicals in the AA effect on Kv1.4 α-current, the water-soluble analogue of Vit.E, Trolox C was applied. Co-application of 10 µM Trolox with AA to the pipette solution did not significantly block the effect of 1pM AA on the maximal amplitude of Kv1.4 currents (-64.8 ± 7.9 % vs. -71.4 ± 6.3 %; Fig.41 C). Troloxin+AA caused a large leftward shift of the voltage-dependence of IA activation (V50a from -5.6 ± 1.3 mV to -37.1 ± 1.8 mV, n=9), as well as slope factor change from 18.1 ± 1.2 to 12.8 ± 1.6 (Fig.41 D, right curves). Under these conditions (AA+Troloxin) voltage dependence of the steady-state inactivation remained unchanged (Fig.41 D, left curves). Trolox C, applied intracellularly, slowed down the rate of inactivation of Kv1.4 channels (from τi=34.3 ± 2.9 ms to τi =47.9 ± 3.2 ms).

Fig. 41 Interaction of AA and Troloxin with IA in Kv1.4-transfected HEK-293 cells. 

A, B: Intracellular application of the vitamin E analogue Trolox (10 µM) does not prevent reduction of IA by arachidonic acid. C: Intracellular application of Trolox and AA reduces IA similar to 1 pM AA (blue line). D: In the presence of Trolox on the inner side of the cell membrane, AA shifts activation kinetic of IA by 30 mV to more negative potential over 900 s recording time (right curves). There was no shift in voltage dependence of steady-state inactivation (left curves).

↓82

Fig. 42 Interaction of AA and Troloxout with IA in Kv1.4-transfected HEK-293 cells. 

A, B: Original traces for activation (A) and inactivation (B) of transient potassium current prior to (0 s) and after (900 s) application of AA+Troloxin/out. Protocols: inset in A2, B2. C: Trolox, applied from both sides of the cell membrane (green line), did significantly recover IA from 1 pM AA (red line). D: No shift of activation or steady-state inactivation occurs after intra- and extracellular application of Trolox and AA. 

When applied on the both sides of the HEK cell membrane, Trolox reduced significantly

(-31.2 ± 6.1 %, n=6) the effect of 1 pM arachidonic acid (-71.4 ± 6.3 %, n=9) on the maximal amplitude of the Kv1.4-induced currents (Fig.42 C). Trolox, applied simultaneously through the pipette and the bath solutions, did not alter Kv1.4 channel characteristics as voltage dependences of activation and steady-state inactivation, slope factors or rate of inactivation (Fig.42 D).

4.4.2 Modulation of transient potassium current by arachidonic acid and Trolox in heterologously expressed Kv4.2-α in HEK-293 cell line

↓83

Transient pot assium current in untreated Kv4.2 -transfected HEK-293 cells

Transfection of Kv4.2 α-subunit in HEK-293 cells resulted in expression of robust outward current with amplitude at +30 mV of 2 to 8 nA (Fig.42 A, B). Kv4.2 current characteristics as maximal amplitude voltage dependences of activation and inactivation did not change significantly over the recording time of 15 min. (Fig.43 C, D, Table 11).

Modulation of t ransient potassium current by AA in Kv 4.2 -transfected HEK-293 cells

↓84

Fig.44 shows the effect of 1pM arachidonic acid observed as 54.7 ± 6.9 % (n=12) reduction of the maximum amplitude of the Kv4.2-encoded current, measured at +30 mV. The inhibition of the Kv4.2 currents by AA was accompanied by a modest (~3 mV) shift in depolarizing direction of the voltage-conductance relation of Kv4.2 channel inactivation (Fig.44 D, left curves). No changes in voltage dependence of activation or time course of inactivation of Kv4.2 channels were observed after intracellular application of 1 pM AA (Fig.44 A, D; Table 11).

Fig. 43 Transient potassium current in Kv4.2-transfected HEK-293 cells.

A, B: Original traces showing IA activation (A) and inactivation (B) at 0 s (1) and 900 s (2) of recording. Pulse protocols: inset A2, B2. C: Transient potassium current (IA) recorded at +30 mV over time in control condition. D: Voltage-conductance dependence of activation (right curves) and steady-state inactivation (left curves) of IA at 0 s and 900 s after opening the cell.

Fig. 44 Suppression of Kv4.2- IA by arachidonic acid (AA). 

A, B: Control activation (A1) and inactivation (B1) of IA. Raw current traces of IA are shown for control immediately after obtaining whole cell configuration (A1, B1, 0 s AA) and after 900 s of recording with 1 pM of AA in the patch pipette (A2, B2, 900 s AA). Pulse protocols: inset A2, B2. C: Intracellular application of 1 pM AA (red line) reduces the maximal transient potassium current (IA) over time. D: Activation (left curves) and steady-state inactivation (right curves) kinetics in control condition (black symbols) and after application of 1 pM arachidonic acid through the pipette (red symbols).

↓85

Influence of Trolox on arachidonic acid-mediated effects

Again, Trolox C that showed the greatest potency as antioxidant in experiments with CA1, ECLII and III neurons, was used in Kv4.2–transfected HEK-293 cells to test the hypothesis that AA modulates the A-current through an oxidative mechanism.

Trolox failed to block the effect of 1 pM arachidonic acid on the maximal Kv4.2 current when applied to the pipette solution (-63.3 ± 7.1 %, vs. -54.4 ± 6.9 %; Fig.45 C). Intracellular Trolox had effect neither on the voltage dependence, nor on the time course of Kv4.2-current inactivation (Fig.45 B; D, left curves). In the presence of Trolox inside the cell V50 of Kv4.2-current inactivation was shifted from -82.5 ± 1.7 mV to -87.6 ± 1.0 mV and the slope factor changed from 17.5 ± 1.6 to 12.8 ± 0.9 (Fig.45 D, left curves).

↓86

As observed in Kv1.4-transfected HEK-293 cells, Trolox applied both through the pipette and the bath significantly attenuated the effect of 1 pM AA on the maximal conductance of Kv4.2 channels (-24.8 ± 7.4 % vs. -54.7 ± 6.9 %; Fig.46 C).

Trolox, applied to the both sides of the cell membrane dramatically affected Kv4.2 activation by shifting its voltage dependence to depolarizing direction from -25.7 ± 1.8 mV to 7.2 ± 0.6 mV (n=7) and altering its slope factor from 17.8 ± 1.7 to 13.1 ± 0.5 (Fig.46 D, right curves).

On the other hand, no effects of Trolox in/out +AA on Kv4.2-current time constant or voltage dependence of steady-state inactivation were observed (Fig.46 B, D, left curves, Table 11).

↓87

Fig. 45 Interaction of AA and Troloxin with IA in Kv4.2-transfected HEK-293 cells.

 A, B: Intracellular application of the vitamin E analogue Trolox (10 µM) with AA has similar effect on IA as AA only (red line). Original traces for activation (A1, A2) and inactivation (B1, B2), immediately after obtaining whole-cell configuration (0 s, A1, B1) and after 900 s of recording (A2, B2). Protocols: inset A2 and B2. C: Intracellular Trolox (blue line) did not inhibit the AA-induced reduction of the maximal IA amplitude (red line) over time. D: In the presence of Trolox on the inner side of the membrane plus AAi (blue symbols), no significant changes in activation (right curves) and inactivation (left curves) kinetics occur in comparison to control activation and inactivation kinetics (black symbols).

Fig. 46 Interaction of AA and Troloxout with IA in Kv4.2-transfected HEK-293 cells. 

A, B: Raw current traces for activation (A) and inactivation (B) of transient potassium current prior to (0 s) and after (900 s) application of AA+Troloxin/out. Protocols: inset in A2, B2. C: Trolox, applied from both sides of the cell membrane (green line), did significantly recover IA from 1 pM AA (red line). D: Intra- and extracellular application of Trolox (green symbols), together with AAi, causes a 30 mV shift to more positive direction of IA activation (right curves), but does not affect its steady-state inactivation (left curves).

Table 11 Overview of effects of oxidants and antioxidants on behavior parameters of IA in HEK-293 cells, transfected with Kv1.4 and Kv4.2.

I A Kv1.4

n

max amplitude,

% of control

V 50a

0 min mV

V 50a

15 min mV

k activation

0 min

k a

15 min

V 50i

0 min
mV

V 50i

15 min
mV

k ina ctivation

0 min

k i

15 min

τ i 0 min ms

τ i

15 min

ms

Control

7

-12.1 ±6.1 %

-24.7 ± 2.4

-4.8 ± 1.8

23.8 ± 2.3

20.0 ± 1.6

-70.0 ± 2.1

-68.3 ± 1.7

17.6 ± 1.9

17.0 ± 1.5

51.7 ± 2.9

52.9 ± 2.8

+ 1pM AA

9

-71.4 ± 6.3 %

-9.2 ± 1.4

-5.2 ± 1.6

17.1 ± 1.3

19.4 ± 1.5

-66.2 ± 2.6

-75.5 ± 2.1

19.6 ± 2.2

22.6 ± 1.9

52.8 ± 2.8

49.4 ± 2.4

AA+Troloxin

9

-64.8 ±7.9 %

-5.6 ±1.3

-37.1 ±1.8

18.1 ±1.2

12.8 ± 1.6

-73.1 ±2.6

-75.6 ±2.0

20.6 ±2.4

21.7 ±1.9

34.3 ±2.9

47.9 ±3.2

AA+Troloxin, out

6

-31.2 ±6.1 %

-19.4 ± 1.8

-23.6 ± 2.0

17.2 ± 1.6

17.2 ± 1.8

-62.4 ± 2.2

-60.6 ± 1.8

14.0 ± 1.9

13.5 ± 1.6

67.4 ±4.8

75.9 ±3.9

I A Kv4.2

Control

6

-9.9 ±7.6 %

-42.0 ± 2.9

-42.9 ± 2.4

18.4 ± 2.7

16.6 ± 2.2

-73.6 ± 0.6

-68.8 ± 0.7

7.1 ± 0.5

7.3 ± 0.6

35.6 ± 3.9

32.5 ± 3.6

+ 1pM AA

12

-54.7 ± 6.9 %

5.5 ± 1.1

8.4 ± 1.0

15.9 ± 1.0

15.8 ± 0.9

-62.2 ± 0.8

-56.6 ± 1.4

11.6 ± 0.7

10.5 ± 1.2

27.2 ± 4.1

25.2 ± 3.9

AA+Troloxin

4

-63.3 ±7.1 %

-5.9 ±1.4

-1.3 ±0.8

17.9 ±1.3

17.5 ± 0.7

-82.5 ±1.7

-87.6 ±1.0

17.5 ±1.6

12.8 ±0.9

41.3 ±3.4

37.4 ±3.7

AA+Troloxin, out

7

-24.8 ±7.4 %

-25.7 ± 1.8

7.2 ± 0.6

17.8 ± 1.7

13.1 ± 0.5

-31.5 ± 2.8

-30.6 ± 3.7

19.0 ± 2.5

16.3 ± 3.3

24.5 ±4.8

25.6 ±4.6


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