| Summary: | 碩士 === 臺灣大學 === 藥理學研究所 === 95 === Levetiracetam (LEV, (-)-(S)-α-ethyl-2-oxo-1-pyrrolidine acetamide), a novel antiepileptic drug, is effective in the treatment of partial-onset epileptic seizures. It is reported that LEV exerts its antiepileptic effect by modulating voltage-dependent potassium channels to lower action potential frequency. Potassium channels regulate neuronal excitability, and serve as target of anticonvulsant. ROMK1 channels, a member of inwardly rectifying potassium channels, are abundant in hippocampus which is highly related to the generation of seizure activities. The effect of LEV on ROMK1 was studied by inside-out patch-clamp recording in xenopus oocyte expression system. LEV dose-dependently enhanced ROMK current with EC¬50¬ of 362.6μM. LEV also enhanced ROMK1 current in mutations of PIP¬2¬ binding sites, R188Q and K218A, indicating that PIP¬2 is not involved in the interaction between LEV and ROMK1 channels. Substitution of PKC phosphorylation sites, Ser183, The191, Thr193, Ser201¬¬, Thr234 for alanine did not abolish the effect of LEV, meaning that LEV does not activate ROMK1 through PKC phosphorylation-dependent pathway. K80M is pH-insensitive, and LEV enhanced K80M currents, indicating that intracellular pH is not involved in the process of LEV enhancing ROMK1 current. Otherwise, PKA inhibitor H89 partially reduced the effect of LEV, and LEV failed to enhance the current expressed by S219A and S313A, mutations of PKA phosphorylation sites, suggesting that LEV may enhance ROMK currents through PKA-related pathway. Furthermore, we substitute PKA phosphorylation sites for asparatate to investigate whether negative charge is required for LEV to enhance ROMK1 currents. LEV had no effect on S219D and S313D, indicating that negative charges are not necessary for LEV to activate ROMK1 channels. We speculate that PKA phosphorylation may offer an appropriate conformation for LEV. LEV applied to the intracellular surface of the excised patches is able to activate ROMK1 channels, suggesting that its binding site could be on the intracellular leaflet, and it may interact with phosphorylated ROMK1 channels and therefore amplify the currents. This study suggests that enhacing ROMK1 currents is one of the underlying mechanisms of LEV in the reduction of neuronal excitability.
|
|---|
