Shaker鉀離子通道第四穿膜區段與N端間之相互作用

The Interaction between S4 and N-terminus in Shaker K+ Channels

林曉君(Hsiao-Chun Lin)

指導教授 ： 郭鐘金

國立臺灣大學/醫學院/生理學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02328

摘要

Shaker鉀離子通道由四個次單元所組成，一個次單元含有六個穿膜區段S1-S6。S1-S4形成電位感應區，負責感應膜兩側電位的變化。S5-S6則是孔洞區，控制鉀離子的通透。細胞膜去極化之後，感應電荷會穿越細胞膜，而感應電荷的移動則會使通道產生形變，導致離子通道的開啟與不活化。N型不活化通常以ball and chain的模型來解釋，離子通道的N端會去堵塞孔洞的內口區，進而阻擋了離子的進出。
在這個研究中，我們在N端G6、L7、Y8和S4上的L366分別做點突變，並利用置換胞內溶液離子成分後，觀察電流的行為變化，以檢視N端不活化球和S4之間的交互作用。當胞內以MTSET灌流時，L7C和L7C L366C會迅速的被MTSET所修飾，不活化行為完全消失，且電流明顯增大，但兩者被MTSET修飾的效果沒有明顯差異。而N端單C突變受到胞內Cd2+的影響，會大幅降低快速不活化的程度，L366C則不受胞內Cd2+的影響。一旦N端單C突變搭配上L366C突變，則快速不活化行為程度的降低會比N端單C突變小，不只電流殘餘較少，不活化速率也較快。最後，利用改變胞內環境的pH值觀察N端單H突變和L366E突變的電流變化，當pH值從7.4降到5.9時，WT和N端單H突變不活化行為的程度都會降低，而且電流峰值會降到原來的一半，但是N端單H突變搭配上L366E突變，改變pH值並不會影響電流的不活化行為，電流峰值也不會降低。
綜合結果，我們認為L366和面臨水洞的N端胺基酸應該是靠得很近，換言之，在休息態時，L366可能處在近於N端胺基酸的位置上，使得N端單C突變和L366C突變大部分可形成雙硫鍵，減輕胞內Cd2+的作用，而且使N端單H突變和L366E突變之間有靜電交互作用。因此，S4的運動和N端之間的交互作用，可能在Shaker鉀離子通道上扮演門閥調控的重要角色。

關鍵字

鉀離子通道 ； 快速不活化 ； N端

並列摘要

The Shaker K+ channel is composed of four subunits, and each subunit has six transmembrane segments S1-S6. S1-S4 presumably form the voltage-sensing domain, and are responsible for sensing changes in transmembrane voltage. S5-S6 contribute to the pore domain which conducts the permeating potassium ions. Upon depolarization, the voltage sensors move across the membrane, and the movement of the gating charges would induce further conformational changes that lead to channel opening and inactivation. N-type inactivation is usually explained with the ‘ball-and-chain’ model, in which the N-terminus of the channel occludes the internal pore mouth and prevents ion flux.
In this study, we made several point mutations on G6、L7、Y8 of N-terminus and S4，and then observed the behavior of these mutant channels in different intracellular solutions in order to examine the interaction between the N-terminus and S4. We found that L7C and L7C L366C mutant channels were modified by MTSET rapidly, with macroscopic inactivation completely abolished and very much increased current amplitude by the modification. But there is no significant difference, including the kinetics of modification, in MTSET modification between L7C and L7C L366C. Intracellular Cd2+ also decreased the extent of inactivation of single cysteine mutants of N-terminus, but has no apparent effect on the L366C mutant channels. However, once these single cysteine mutants of N-terminus was coexpressed with L366C mutant, the attenuation of inactivation became much lighter. The macroscopic inactivation kinetics were faster and the steady-state currents were smaller. Moreover, we examined the possible interactions between single histidine mutants of N-terminus and L366E. When intracellular pH were lowered from 7.4 to5.9, the inactivation of wild-type and single mutants of N-terminus became weaker, with the peak current reduced to half of the original value. However, changes of intracellular pH would have no effect on either fast inactivation or peak current in double mutant channels, involving both histidine replacement in the N-terminus and L366E.
Altogether, these findings suggest that the side chain of residues L366 and L7 are in close proximity, resulting in the formation of the disulfide bond between single mutants of N-terminus and L366C to alleviate the effect of intracellular Cd2+, and the electrostatic interaction between single histidine mutants of N-terminus and L366E. Thus, the possible interaction between the movement of S4 and the N-terminus may play an important role in the gating control of the Shaker K+ channel.

並列關鍵字

Shaker channel ； fast inactivation ； N terminus ； S4

參考文獻

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