| Abstract: | CLCN1(Chloride channel 1)電位依賴型離子通道在哺乳類動物骨骼肌細胞的膜電位再極化作用中扮演著很重要的角色。如果人類CLCN1發生變異很有可能會造成先天性人類肌強直症。目前己知蛋白質磷酸化酵素C (Protein kinase C，PKC) 可以調控人類CLCN1氯離子通道，但其磷酸化位點之所在還是未知的。本實驗的目的就是要研究人類CLCN1氯離子通道蛋白中可能的磷酸化位點。首先我們使用單點突變的技術針對可能的磷酸化位點，做出了21個CLCN1的突變種，所有突變點點都位於人類CLCN1氯離子通道蛋白的C端位置。再將從 hCLCN1/pTLN質體反轉錄作用而來的CLCN-1 cRNA以顯微注射的方式打入非洲爪蟾卵母細胞 (Xenopus laevis oocyte) 之中，再利用雙電極電位鉗定法 (Two-electrode voltage clamp, TEVC) 來測量人類CLCN1表達在爪蟾卵母細胞膜的離子電流。
可以以一個量化的數值V1/2 (當離子通道開啟機率為0.5時之膜電位)的變化做為觀察離子通道性質變化的標準。其中，野生型的人類CLCN1氯離子通道會受到PMA的抑制，而使其V1/2偏向較正電方向， (由 -42.9 12.5 偏移到 -13.7 5.0, n = 8)。而其它突變種的CLCN1也有類似於野生型的情況，如S682A、S720A、S740A。但是在這之中，S892P和 雙重點突變的S892A/T893A卻在PMA作用後沒出現明顯的改變，而Ser892所在，即Thr890到Ser903之間的這序列充滿了多個可能的磷酸化位點，所以可能有磷酸化位點存在。
因此我們將892突變為 Aspartic acid，用以模擬受磷酸化後性質偏向酸性的情況。而S892D本身表現出來的V1/2相對於野生型更為偏向於正向，但卻和野生型受到PMA作用後的V1/2極為接近。同時，S892D在受到PMA作用後幾乎沒有任何改變。但是突變為Proline或是進行雙重點突變都有可能改變三級結構而造成重大的改變，所以無法確定Ser892 就是磷酸化位點，而S892D的突變也有可能會干擾到該區域的磷酸化作用，所以推測Thr890到Ser903序列之間有磷酸化位點存在。CLCN1 voltage-dependent chloride channel play an important role in membrane potential repolarization of mammalian skeletal muscle. Mutations in human CLCN1 lead to the myotonia congenital which is an inherited human disease. It has been known protein kinase C (PKC) can modulate the CLCN1 channel, however, the phosphorylated sites are still unclear. The purpose of this study was to investigate the possible phosphorylation sites in the human CLCN1 Cl- channel. The human CLCN1 cRNA was in vitro transcribed from linearized hCLCN1/pTLN plasmid, and then inject it into the Xenopus laevis oocytes using a microinjector. Two-electrode voltage clamp (TEVC) technique was used for the measurement of ionic current of human CLCN1. In the present study we construct 21 mutant recombinant plasmids by use the technique of site-directed mutagenesis. All mutants were designed according to the putative phosphorylation sites in C-terminus of CLCN1. Human CLCN1 and mutants were transient expressed in Xenopus oocytes by the microinjection of CLCN1 cRNA. To test whether mutant proteins lacking sensitivity to protein kinase C, an activator of PKC phorbol 12-myristate 13-acetate (PMA, 2 μM) was used in this study.
The functional effects of PMA on wild-type and various mutants were determined by using TEVC recording from Xenopus oocytes. The treatment with PMA causes an inhibiting effect of wild-type CLCN1 current and a shift of the half-maximum activation of open probability (V1/2) to more positive membrane potentials (from -42.9 12.5 to -13.7 5, n = 8). These shifted-effects of most CLCN1 mutants by treated with PMA are similar to the wild-type, including S682A (from -32.9 4.2 to -15.9 10.2, n = 4), S720A (from -26.8 5.7 to -5.2 12.1, n = 4), S740A (from-55.7 13.5 to -24.2 9.1, n = 4), T782A (-40.4 10.3 to -8.1 11.5, n = 4), T891A (from -36.5 9.2 to -14.8 6, n = 6), S892A (from -27.5 6.8 to -1.3 10.4, n = 4) and S896R (from -45.4 13.4 to -13.7 12.9, n = 8). As so far only the S892P (from -39.5 11.2 to -35.7 14, n = 6) and a double mutant S892A/T893A (from -45.2 4.4 to -38.4 4.4, n = 4) were not change V1/2 by treated with PMA.
In order to mimic phosphorylation at position 892, we made a mutant where a serine residue at 892 was substituted by aspartic acid. The result showed that the V1/2 of S892D was more positive than the wild-type, but similar to wild-type in the presence of PMA (-10.2 9.7 vs -13.7 5.0). Furthermore, the treatment of S892D with PMA did not change the value of V1/2 (from -10.2 9.7 to -9.6 6.9, n = 4). These results suggest that sequence including serine 892 has functional phosphorylated site for protein kinase, and therefore to modulate the functional properties of human CLCN1 channels. |
