磷酸化對於人類KCNQ4鉀離子通道異源性表達於爪蟾卵母細胞功能性之研究

本實驗之目的在於研究磷酸化 (phosphorylation)對於KCNQ4鉀離子通道 (potassium ion channel)功能的調節。我們將人類KCNQ4基因製備成KCNQ4 cRNA，藉由顯微注射 (microinjection)的方式打入非洲爪蟾卵母細胞 (Xenopus laevis oocytes)中，使其異源性 (heterologous)表達KCNQ4鉀離子通道。並以雙電極電位箝定技術 (Two-Electrode Voltage-Clamp，TEVC)來進行功能上的研究，利用與磷酸化相關的藥劑進行處理，觀察磷酸化對KCNQ4鉀離子通道調節的作用，與鉀離子電流大小的改變，以及KCNQ4鉀離子通道活化曲線 (activation curve)偏移程度之間的相關性。首先比較對非洲爪蟾卵母細胞進行無任何注射、注射RNase-Free water，與注射KCNQ4 cRNA，所記錄到的電流大小，結果顯示僅有注射KCNQ4 cRNA才能記錄到明顯的電流。並且以KCNQ4抑制劑linopirdine處理後，發現表達的鉀離子電流被抑制到接近無任何注射時的背景電流。證實能以非洲爪蟾卵母細胞異源性表達人類KCNQ4鉀離子通道，且其特性亦符合已發表文獻的結果，屬於一種低閾值 (low-threshold)、非不活化性 (non-inactivating)、延遲性 (delayed)，膜電位依賴型 (voltage-gated)的鉀離子電流。接著實驗以phorbol 12-myristate 13-acetate (PMA)進行處理，發現鉀離子電流會被抑制，而且KCNQ4鉀離子通道活化曲線會往去極化 (depolarization)方向偏移。並以PMA的不活化型同分異構物 (inactive isomer) 4 alpha-phorbol 12-myristate 13-acetate (4α-PMA)處理作對照，發現以4α-PMA處理僅會引起KCNQ4鉀離子通道功能上些微的改變，證實以PMA處理確實能調節KCNQ4鉀離子通道。而以protein kinase C (PKC)抑制劑bisindolylmaleimide I (BIMI)前處理實驗中，發現前處理BIMI會減緩PMA所引發對KCNQ4鉀離子通道的效應。證實以PMA處理後所引發的反應是經由活化PKC的方式，並引起KCNQ4鉀離子通道磷酸化，進而調節其功能與特性。最後實驗以PP1/PP2A型去磷酸化酶抑制劑calyculin A與okadaic acid前處理，結果發現兩者皆會引起KCNQ4鉀離子通道功能上些微的改變，但再以PMA處理後發現，KCNQ4鉀離子通道活化曲線並不會發生偏移。這暗示著調節PKC所引起的磷酸化至少可能會經由PP1/PP2A型去磷酸化酶來進行。我們的實驗結果證實了活化PKC所引起的磷酸化會調節KCNQ4鉀離子通道，導致鉀離子電流被抑制，以及KCNQ4鉀離子通道活化曲線往去極化方向偏移。而此磷酸化現象至少可能會經由PP1/PP2A型去磷酸化酶來調節。

The purpose of this study was to investigate the effect of phosphorylation on KCNQ4 potassium ion channel. The human KCNQ4 cRNA was purified from HpaⅠlinearized plasmid DNA, then inject it into the Xenopus laevis oocytes by microinjection to heterologous express the KCNQ4 potassium ion channel on cell membrane. Two-Electrode Voltage-Clamp (TEVC) technique was used for the measurement of function of KCNQ4. The treatment of protein phosphorylation modulators was utilized to observe the changes of electrical parameters of KCNQ4 channel including the ion current amplitude and the activation curve of KCNQ4 channel.A significant ion current can be observed in oocytes injected with KCNQ4 cRNA as compared with control groups including those had no injection or injected with RNase-Free water. Futhermore these KCNQ4 currents are sensitive to KCNQ4 blocker (M-like current blocker) linopirdine. This result demonstrates that human KCNQ4 channels expressed in Xenopus laevis oocytes are low-threshold, non-activating, delayed, and voltage-dependent potassium channels silimilar to previous reports.Treatment of protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) causes a significant inhibitory effect of KCNQ4 ion current and shifting to the right of the activation curve. However, 4 alpha-phorbol 12-myristate 13-acetate (4α-PMA), an inactive isomer of PMA, causes only slightly changes in the properties of KCNQ4 ion channel. Furthermore, the pretreatment of PKC inhibitor, bisindolymaleimide I (BIMI) attenuates the effect on KCNQ4 induced by PMA. The result indicates that PKC plays a role in regulating the function of KCNQ4 channels.Finally, the PP1/PP2A phosphatase inhibitors, calyculin A and okadaic acid were used in this experiment. Calyculin A or okadaic acid alone causes a slight change in current properties of KCNQ4 channel. Pretreatment of calyculin A or okadaic acid did not shift the activation curve of KCNQ4 induced by PMA. This result suggests that the phosphorylation by PKC may, at least involve the activity of PP1/PP2A phosphatase.In summary, our experimental results demonstrate that PKC phosphorylates KCNQ4 and thus results in the reduction of the current amplitude of KCNQ4 and shift to the right of the voltage-dependence activation curve. In addition, the PP1/PP2A phosphatase may play a role, at least in part, in mediating the dephosphorylation of KCNQ4.