辨認KcsA鉀通道內側入口處支鏈指向孔洞之胺基酸

Tunghai University Institutional Repository > 理學院 > 生命科學系所 > 碩博士論文 > Item 310901/31558

Please use this identifier to cite or link to this item: http://140.128.103.80:8080/handle/310901/31558

Title:	辨認KcsA鉀通道內側入口處支鏈指向孔洞之胺基酸
Other Titles:	Identification of Pore-facing Residues at the Internal Entrance of KcsA Potassium Channel
Authors:	葉松翰 YEH, SONG-HAN
Contributors:	陳仁祥 CHEN, REN-SHIANG 生命科學系
Keywords:	鉀離子通道;導電度 Conductance;Potassium channel;KcsA
Date:	2018
Issue Date:	2019-12-16T02:59:52Z (UTC)
Abstract:	鉀離子通道的電流大致上決定了細胞膜的靜止電位。在鉀離子通道中，負責篩選鉀離子進出的結構稱作選擇性過濾器(selective filer)，其中GYG序列在所有鉀通道的演化中極度保守。雖然已知的鉀通道(MthK、KvAP、Kv1.2和KcsA)選擇性過濾器具有幾乎一致的結構，但是鉀通道的導電度卻有相當大的差異。最近的研究顯示BK通道在其孔洞內側入口處的兩個帶負電的胺基酸(E321和E324)能夠吸引鉀離子並有效提升該處的鉀離子濃度，因而提高穿過BK通道之電流。同樣地，當KcsA通道內側入口處的A108突變為帶負電的aspartate時，KcsA的導電度也會顯著提高。Furini等人在2007年運用Poission-Nernst-Planck (PNP)理論模型模擬KcsA A108E、T112E的點突變，獲得導電度會上升的結論。然而Furini等人的預測結果，以及KcsA通道中相對於BK通道E324之位點是A111或T112仍未經實驗所證實。本研究中，我們在KcsA通道內側入口處進行A108E、A111E和T112E之負電荷點突變，並以脂雙層記錄術(planar lipid bilayer technique)記錄不同膜電位下的單通道電流大小。我們觀察到A108E會導致通道導電度上升，此結果與先前文獻結果相符。此外，在細胞內溶液為pH 4和pH 5時，我們記錄到KcsA A111E的導電度高於控制組，而T112E的導電度與控制組相似，這暗示了A111(而非T112)之支鏈指向通道孔洞，幫助吸引鉀離子。我們的結果顯示BK通道於內側入口處的E321和E324在KcsA通道上相對應之位置為A108和A111。根據本研究之結果，我們可以在此二位點上放置不同體積大小之胺基酸支鏈，進一步探討改變內側入口大小對鉀離子通道導電度之影響。 The efflux of potassium ions (K+) essentially determines resting membrane potentials in virtually all cells. The selective filter of potassium channels, with its GYG sequence being extremely conserved during evolution, allows the K+ to pass the membrane. Although the selective filter structure is almost identical in all crystallized potassium channels (such as MthK, KvAP, Kv1.2, and KcsA), the conductance of these channels vary widely. Recent researches showed that the two negatively charged amino acids (E321 and E324) at the internal pore entrance of BK channels attract potassium ions, leading to increased potassium concentration at the pore entrance and elevated channel conductance. Similarly, when the A108 at the internal entrance of KcsA channel is mutated to aspartate, the conductance of KcsA channel was significantly increased. Furini and colleagues (2007) suggested that A108E and T112E mutations in KcsA channel can increase channel conductance in their simulation with the Poisson-Nernst-Planck (PNP) theory. However, Furini and colleagues’ prediction, as well as whether the corresponding position to BK channel’s E324 in KcsA channel is A111 or T112, have not been experimentally confirmed. In this study, we created mutant KcsA channels by introducing negative charges at the A108, A111 and T112 positions, followed by recording their single channel currents at various membrane potentials with the planar lipid bilayer technique. The A108E mutation caused increased channel conductance over the control channel, consistent with results in the literature. Furthermore, the channels with A111E, but not T112E, displayed increased conductance in both intracellular pH 4 and pH 5, indicating that the carboxyl group of the A111E side-chain is pointing to the channel pore and helps attracting K+ ions. Our results indicated that the A108/A111 positions in the KcsA channel correspond to the E321/E324 positions in the BK channel. Based on these results, we can introduce side-chains of various size to further investigate the influence of changing the dimension of internal entrance on the conductance of potassium channels.
Appears in Collections:	[生命科學系所] 碩博士論文

Files in This Item:

File	Size	Format
106THU00112009-001.pdf	2334Kb	Adobe PDF	44	View/Open

Loading...