2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38710The purpose of the studies is to investigate the phosphorylation site of KCNQ4 channels by protein kinase C. We use the KinasePhos and NetPhosK 2.0 server to predict the phosphorylation sites of KCNQ4, twenty-five possible phosphorylation sites in the C-terminus and three in the intracellular S4-S5 segment are chosen to be mutated in this investigation (see experimental design and hypothesis in C-012-2, page 7~8:). KCNQ4 channels are low-threshold non-inactivating voltage-gated K+ channels, which have been linked to inherited hearing loss. Protein Kinase C has been thought to play a role in M-current (KCNQ) inhibition, it is still not clear the phosphorylation site of KCNQ4. Our preliminary data showed that, the protein kinase C activator can induce an inhibition of conductance of KCNQ4 (Fig.1, see attached file in C-012-2, page 28). In this study KCNQ4 and mutant channels were expressed in Xenopus oocyte. Microinjection of wild or mutant type of KCNQ4 cRNA into the Xenopus oocyte, the KCNQ4 protein will insert into the oocyte membrane about after 2-4 days. Changes in KCNQ4 current induced by protein kinase C activators were investigated in the oocyte using the two-electrode voltage clamp. The possible phosphorylation sites of KCNQ4 by protein kinase are using the site-directed mutagenesis to identification. Substitutions of serine (or threonine) residues at various positions in C- terminus and the region of S4-S5 are expected to obtain the difference of channel response to protein kinase C activators. These results therefore will indicate to the phosphorylation sites of KCNQ4 by protein kinase C. We have completed some experiments using two-electrode voltage clamp to measure over 200 oocytes of the normal control and wild-type KCNQ4 currents, and the result are stable and repeatable. The accomplishment of the studies will provide the important information of phosphorylation sites of KCNQ4 by protein kinase and further, to understand the relationship between protein structure and hearing function, and signal linkage between channel and cellular messengers. These data may provide important information on the new drug development in the future.本實驗研究之目的是探討KCNQ4 蛋白受到激酶磷酸化功能改變其可能的磷酸化 位點(phosphorylation sites)。以KinasePhos 與NetPhosK 2.0 server 預測磷酸化位 點，交叉比對尋找可能的位置進行突變研究，預測出可能的位置共有28 個，分別 有25 個位於C-端3 個位於S4-S5 區(見計畫內容第7-8 頁:實驗理論與設計) 。 KCNQ4 管道是一種低閾值，沒有不活化性質的電位依賴型鉀管道，它可能與某些 遺傳性聽障有關。又蛋白激酶丙被認為在M-電流(KCNQ 家族) 抑制調控扮演重要 的角色，然而KCNQ4 受到磷酸化的位點並不清楚。我們的初步實驗已證實蛋白激 酶活化劑的確會去抑制KCNQ4 蛋白的離子傳導度(Fig.1, see attached file in C-012-2, 第28 頁)，但它的磷酸化位點是過去研究尚未了解的。在本計畫中我們表 現KCNQ4 與它的點突變cRNA 在爪蟾卵母細胞中。微注射KCNQ4 野生型與變 異型cRNA 於爪蟾卵母細胞中，KCNQ4 蛋白約2-4 天會表達於卵細胞膜上，其 可測得KCNQ4 鉀電流。我們將使用雙電極電位鉗定技術來測量蛋白激酶丙所引發 的電流變化。KCNQ4 蛋白可能的磷酸化位點則使用位置導向的點突變來觀察它受 到蛋白激酶丙調控的變化。在可能的蛋白C 端或S4-S5 區序列做絲胺酸(或羥丁胺 酸)點突變的取代並尋找對於蛋白激酶丙調控的敏感性產生改變的突變點。這個實 驗的完成將會找出一個或數個受到蛋白激酶丙調控可能的磷酸化位點。先前我們已 完成約200 顆卵的控制組與一般型KCNQ4 電流測量前導實驗，它們的穩定性與再 現性都不錯。完成此計畫將可提供找到KCNQ4 受蛋白激酶丙調控的磷酸化位點， 更進一步可釐清KCNQ4 蛋白結構與聽覺功能傳訊的關聯與及細胞內訊息對於 KCNQ4 蛋白分子序段的調控。這個結果或可提供將來新藥開發設計研發的參考依 據。KCNQ4磷酸化位點蛋白C-端蛋白質結構蛋白激酶丙聽障鉀電流KCNQ4Phosphorylation siteC-terminusProtein structureProtein kinaseChearing lossK+ currentProject