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Divergent Voltage-dependent Gating Properties of REag1 and REag2 Potassium Channels
No full text電位控制開關的鉀離子通道(voltage-gated potassium channel)是一種陽離子通道，可以感應膜電位的變化選擇性的讓鉀離子通透。大鼠的ether à gogo（rEag） 鉀離子通道是屬於EAG家族(EAG family)的一種，有兩個子類型分別為rEag1和rEag2。rEag主要分布在中樞神經系統當中，對於調控神經細胞的興奮性和神經物傳導物質的釋放扮演重要角色。和其他鉀離子通道比較，rEag不同的地方除了在選擇性濾孔（selective filter）的指紋(signature)為GFG之外，還包括Ｎ端有PAS domain(Per-Arnt-Srm)，C端有環核苷酸結合區（cyclic-nucleotide binding domain）。
Eag鉀離子通道的開啟速度(activation kinetic)會受到不同的前置過極化負電位(hyperpolarized prepulse)所影響，越hyperpolarized的電位除了會造成activation delay之外還會讓eag的activation kinetics變慢。和其他鉀離子通道比較，前置過極化負電位只會延緩鉀離子通道的開啟時間。Eag對前置過極化負電位特殊的反應暗示了eag在activation之前可能會經過一個以上rate-limiting close state的transition。除了前置過極化電位之外，鎂離子和pH值也會改變通道開啟的速度。
本論文的研究目的是探討rEag1和rEag2電位活化機制之差異為何，並進一步了解產生這些差異的潛在分子結構基礎。我們的假設是造成rEag1和rEag2有不相同的gating property的主因可能為兩者在C端的差異。實驗時使用雙電極膜電位箝制(two-electrode voltage clamp)的電生理紀錄方法，首先比較rEag1和rEag2的gating propetty。之後並利用PCR的方法配合適當的引子(primer)以及利用兩次PCR( first run and second run)的技術，將rEag1和rEag2上相對應的C端互相置換，來探討不同的C端片段與兩者電位活化機制差異的可能關係。本實驗一共選擇了六個不同片段長度的C端段落置換，總計得到12個突變(chimera)。之後將這些突變轉型（transform）到pcDNA3的vector上，經由轉錄後（transcription）將cRNA注射到Xenopus oocytes 內表達(express)，最後測試這十二個突變對於前置過極化電位的反應以及其他的電位活化特性。
我們實驗的結果發現，rEag1和rEag2兩者在gating kinetics上存有先天明顯的差異。另外經由分析chimeras gating property的差異，我們首先觀察到rEag的C端會影響rEag1和rEag2 voltage-dependent gating property，但是不會改變rEag1和rEag2 steady-state voltage dependence。Voltage-gated potassium channels are cation channels which upon membrane potential change, undergo significant comformational change, leading to the opening of potassium-selective pores. Rat ether à gogo（rEag）potassium channels, which belongs to EAG family, specifically express in the central nervous system and are thought to plays an important roles in the modulation neuron of excitability and neurontransmeter release. Unlike other potassium channels, the signature sequence in the selective filter of rEag potassium channel is GYG. Furthermore, all EAG channels contain the PAS (Per-Arnt-Srm) domain and cyclic-nucleotide binging domain in the amino (N) and carboxyl-(C) terminus, respectively.
The activation kinetics of rEag potassium channel is significantly slower in the presence of hyperpolarizing prepulses, which is also known as the non-superimposable Cole -Moore shift. This phenomenon is consistent with the idea that prior to pore opening, ion channel must go through several voltage-dependent conformation changes. In addition, Mg2+ and pH can also modulate the activation kinetics of rEag potassium channels. There are two rEag potassium channels subtypes: rEag1 and rEag2. The two isofoms share 70% indntity in amino acid sequence, which encompasses the six transmembrane segments. Their activation kinetics, however, displays distinct features.
The goal of this thesis is to characterize the divergence voltage-dependent activation of rEag1 and rEag2 K+ channels and to find out the potential underlying structural bases. We applied two-electrode voltage clamp (TEVC) technique to study different rEag1 and rEag2 K+ channels heterologously expressed in Xenopus oocytes. Moreover, N and C termini, have previously been suggested to may a play role in the modulation channel activation. Since the major difference in amino acid sequence between rEag1 and rEag2 K+ channels lies in the C terminals, we aim to test the hypothesis that C terminal may confer the unique channel activation feature of rEag channels by using PCR mutagenesis technique. A total of 12 chimeras, 6 in each rEag backbone, will we constructed and tested for their biophysical properties.
Our date indicate that there is distinct divergence of gating property between rEag1 and rEag2. Besides, instead of steady state voltage dependence,we first report that C terminus will modulate rEag voltage-dependent gating property.目錄
誌謝 ……………………………………………………………………………………I
目錄……………………………………………………………………………………II
圖次……………………………………………………………………………………IV
中文摘要 ……………………………………………………………………………V
英文摘要 …………………………………………………………………………VII
第一章、導論 …………………………………………………………………1
I. EAG鉀離子通道介紹 ………………………………………………………2
A. 分類 ………………………………………………………………………… 2
B. EAG鉀離子通道家族功能 …………………………………………………2
C. EAG鉀離子通道的gating mechanism…………………………………… 3
II. Eag鉀離子通道的N端和C端 ………………………………………… 5
A. 一般結構 ……………………………………………………………… 6
B. N terminus………………………………………………………………… 6
C. C terminus………………………………………………………………… 7
III. 本實驗展望 …………………………………………………………… 9
A. rEag1和rEag2鉀離子通道的差異 …………………………………… 9
B. 實驗假說及實驗步驟 ……………………………………………… 10
第二章、材料與方法 ……………………………………………………………… 12
I. 分子生物技術 ……………………………………………………… 12
A. 置換GFP-rEag1和GFP-rEag2的C端位置，產生突變 ………… 12
B. 將突變從GFP vector換到pcrEag1或是pcrEag2上 ……………… 15
C. Gel extraction ………………………………………………………… 16
D. transformation，inoculation，miniprep …………………………… 16
E. RNA transcription ……………………………………………………… 17
II. 電生理記錄………………………………………………………………… 17
A. Xenopus laevis （南非爪蟾） oocyte取得和處理 ………………… 17
B. oocyte RNA injection ……………………………………………………18
C. 電生理記錄………………………………………………………………… 19
D. 電生理紀錄之分析………………………………………………………… 20
第三章、結果 ……………………………………………………………… 21
I. rEag1和rEag2鉀離子通道的voltage-dependent gating property 21
A. rEag1和rEag2鉀離子通道activation kinetics的異同性 ……… 23
B. rEag1和rEag2鉀離子通道deactivation kinetics的異同性……… 24
C. 前置過極化負電位對rEag1和rEag2鉀離子通道
activation kinetics的影響 …………………………………………26
II. 以rEag1為骨架的chimeras其電生理特性與rEag1鉀離子通道的差別29
A. steady-state voltage dependence………………………………………29
B. activation kinetics of chimeras………………………………………30
C. Deactivation kinetics of chimeras……………………………………30
D. 前置過極化負電位對chimeras的影響……………………………………31
III. 以rEag2為骨架的chimeras其電生理特性與rEag2鉀離子通道的差別32
第四章、討論 …………………………………………………………………………36
I. C端對於rEag1和rEag2 gating property異同性的影響………………37
A. Steady-state voltage dependence………………………………………37
B. Voltage dependent gating kinetics……………………………………39
II. C端在rEag1和rEag2 gating過程中扮演的角色………………………40
III. 相關的實驗討論 ………………………………………………………… 45
參考資料 ………………………………………………………………………… 7
Going Beyond Counting First Authors in Author Co-citation Analysis
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koamabayili/VECTRON-author-checklist: VECTRON author checklist
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Author-wise bibliometric analysis based on entropy.
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Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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