Der neuronale Zelluntergang bei einer Vielzahl von Krankheiten des ZNS, wie z.B. Morbus Alzheimer (AD) und Temporallappenepilepsie (TLE), wird mit oxidativem Stress sowie Fehlfunktionen von Kaliumkanälen in Verbindung gebracht. In dieser Studie soll die selektive neuronale Sensitivität auf oxidativen Stress durch die Messung der oxidativen Modulation von Kaliumströmen untersucht werden. Dabei werden sternförmige Neuronen der zweiten Schicht des entorhinalen Kortex (EC) (bei AD bereits früh geschädigt) mit pyramidalen Neuronen der dritten Schicht des EC (früh geschädigt bei TLE) sowie hippocampalen pyramidalen Neuronen der CA1 Region (bei AD und TLE erst spät geschädigt) miteinander verglichen.

Mittels patch-clamp Ganzzellmessung zeigt diese Studie die differentielle Hemmung spannungsabhängiger transienter (I_A) und „delayed-rectifier“ K⁺-Ströme (I_K(V)) durch Arachidonsäure (AA) und Wasserstoffperoxid (H₂O₂). Die intrazelluläre Applikation von AA (1 pM) reduzierte I_A in Neuronen des entorhinalen Kortex signifikant stärker verglichen mit Neuronen des CA1.

ETYA imitiert diesen Effekt, dies schliesst die Metabolite der AA als Mediatoren des Effekts auf Kaliumkanäle aus. Weder AA noch ETYA reduzierten I_K(V). Im Gegensatz dazu reduzierte H₂O₂ I_A in Neuronen des CA1 effektiver als in Neuronen der Schichten II und III des entorhinalen Kortex. Die Reduktion des I_A, vermittelt durch AA, wurde durch Radikalfänger (Glutathion, Ascorbinsäure, Vitamin E Analogon Trolox) blockiert. Dabei verstärkten manche dieser Antioxidantien den Effekt der AA, dies legt eine komplexere Modulation dieser Ströme in Schnitten verglichen mit Kulturen nahe. Dies sollte bei der Entwicklung antioxidativer Therapien von AD und TLE berücksichtigt werden.

Bei der heterologer Expression von Kv1.4 und Kv4.2 in HEK-293 Zellen wurden funktionelle Kanäle gebildet und A-Typ Ströme ausgelöst. Diese Ströme wurden nach der Applikation von 1 pM AA stark reduziert.

ROS scheinen neben ihrer zellschädigenden Wirkung physiologische Prozesse zu regulieren, indem sie eine Reihe von Signalwegen beeinflussen. Da spannungsabhängige Kaliumkanäle vielen wichtigen zellulären Funktionen zugrundeliegen, könnte die Modulation dieser Kanäle durch ROS einen Mechanismus für die Feinabstimmung zellulärer Prozesse darstellen.

Inhaltsverzeichnis

• 1 INTRODUCTION

  • 1.1 Voltage-gated potassium currents

    • 1.1.1 Molecular basis of voltage-gated potassium currents 

    • 1.1.2 Principal subunits

    • 1.1.3 Regulatory subunits of Kv4.2 and Kv1.4

    • 1.1.4 Molecular basis of I_K(V)

    • 1.1.5 Structure of a Kv channel

  • 1.2 Arachidonic Acid

    • 1.2.1 Arachidonic acid structure

    • 1.2.2 Arachidonic acid release 

    • 1.2.3 Arachidonic acid metabolism 

    • 1.2.4 Modulation of voltage-gated potassium channels by arachidonic acid

  • 1.3 Oxidative Stress

    • 1.3.1 Antioxidants

    • 1.3.2 Modulation of ion channels by ROS

  • 1.4 Hippocampal Formation

    • 1.4.1 Hippocampal formation damage in neurological diseases

• 2 AIM OF THE STUDY

• 3 MATERIALS AND METHODS

  • 3.1 Brain slices 

  • 3.2 HEK 293 cells

  • 3.3 Electrophysiological recordings

    • 3.3.1 Patch pipettes

    • 3.3.2 Intrapipette solution (IPS)

    • 3.3.3 Pharmaka 

    • 3.3.4 Voltage-clamp and discontinuous amplifier 

    • 3.3.5 Recording electrode

    • 3.3.6 Experimental setup 

    • 3.3.7 Experimental protocols

  • 3.4 Data and statistical analysis

    • 3.4.1 Separation of fast and persistent K⁺-currents using prepulse inactivation

    • 3.4.2 Data analysis

• 4 RESULTS

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

    • 4.1.1 Potassium currents in untreated CA1 pyramidal neurons

    • 4.1.2 Modulation of transient I_A and delayed rectifier currents I_K(V) by arachidonic acid

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

    • 4.1.4 Effects of H₂O₂ on outward potassium currents

    • 4.1.5 Control antioxidant studies

  • 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

    • 4.2.2 Modulation of transient I_A  and delayed rectifier currents I_K(V) by arachidonic acid

    • 4.2.3 Effects of H₂O₂ on outward potassium currents

  • 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

    • 4.3.2 Modulation of transient I_A  and delayed rectifier currents I_K(V) by arachidonic acid

    • 4.3.3 Effects of H₂O₂ on outward potassium currents

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

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

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

• 5 DISCUSSION

  • 5.1 Effect of arachidonic acid on the transient potassium current

  • 5.2 Oxidation is involved in arachidonic acid-mediated modulation of the A-current

  • 5.3 H₂O₂ as a model oxidative modulation of ion channels

    • 5.3.1 Culture vs. Slices 

    • 5.3.2 Role of membrane fluidity

    • 5.3.3 Arachidonic acid as a retrograde messenger

    • 5.3.4 Significance of potassium channel modulation by ROS

    • 5.3.5 Arachidonic acid, oxidative stress and neurodegenerative diseases

    • 5.3.6 Mechanism of arachidonic acid modulation of Kv channels still remain to be discovered

• 6 CONCLUSION

  • 6.1 Novel findings

  • 6.2 Significance

  • 6.3 Outlook

• Literaturverzeichnis

• List of Abbrevations

• ERKLÄRUNG

• DANKSAGUNG

• EIGENE PUBLIKATIONEN

• Konferenzbeiträge

• Table 1 Content of acute brain slice preparation and perfusion solutions.

• Table 2 Content of HEK-293 cell line culturing media

• Table 3 Content of HEK-293 transfection solutions and buffers.

• Table 4 Pharmaka

• Table 5 Overview of effects of oxidants and antioxidants on behavior parameters of I_A in CA1 pyramidal neurons_.

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

• Table 7 Overview of effects of oxidants and antioxidants on behavior parameters of I_A in ECLIII pyramidal neurons_.

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

• Table 9 Overview of effects of oxidants and antioxidants on behavior parameters of I_A in ECLII stellate neurons_.

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

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

• Fig. 1 Membrane topology of a voltage gated potassium channel.

• Fig. 2 Structure of arachidonic acid and ETYA

• Fig. 3 Simplified scheme of arachidonic acid release events.

• Fig. 4 Simplified scheme of arachidonic acid metabolism. 

• Fig. 5 Chemical structure of glutathione 

• Fig. 6 Chemical structure of ascorbic acid.

• Fig. 7 Chemical structure of α-tocopherol and Trolox C

• Fig. 8 Schematic drawing of a section through rodent entorhinal cortex (modified after Lorente de Nó, 1934).

• Fig. 9 Preparation of horizontal hippocampal slices [Witter, 00].

• Fig. 10 Test potential protocols for activation and inactivation of total outward and delayed rectifier potassium currents.

• Fig. 11 Procedure for potassium current separation

• Fig. 12 Native I_A  and I_K(V) in control condition in CA1 pyramidal neurons.

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

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

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

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

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

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

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

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

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

• Fig. 22 Native I_A and I_K(V) in control condition in ECLIII pyramidal neurons.

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

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

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

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

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

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

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

• Fig. 30 Native I_A and I_K(V) in control condition in ECLII stellate neurons. 

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

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

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

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

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

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

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

• 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).

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

• Fig. 40 Suppression of Kv1.4- I_A by arachidonic acid.

• Fig. 41 Interaction of AA and Trolox_in with I_A in Kv1.4-transfected HEK-293 cells. 

• Fig. 42 Interaction of AA and Trolox_out with I_A in Kv1.4-transfected HEK-293 cells. 

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

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

• Fig. 45 Interaction of AA and Trolox_in with I_A in Kv4.2-transfected HEK-293 cells.

• Fig. 46 Interaction of AA and Trolox_out with I_A in Kv4.2-transfected HEK-293 cells.