內整流型鉀離子通道表現在非洲爪蟾卵細胞之基本特性研究

The basic property of inward rectifier potassium channel expressed in Xenopus laevis oocytes

蔡榮隆(Jung-Lung Tsai)

指導教授 ： 蔡明正

國立臺灣大學/醫學院/藥理學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.01889

摘要

1.本實驗以非洲爪蟾(Xenopus laevis) 卵細胞為表現載體，以雙電極膜電位箝制(two-electrode voltage clamp)技術，探討Kir6.2ΔC26鉀離子通道之一般藥理學和電生理學基本特性，以及其抑制劑對鉀離子通道之影響。

2.在實驗當中的Kir6.2/pET20b+ 基因序列是第一次表現在非洲爪蟾(Xenopus laevis)卵細胞，為了再次確定其Kir6.2基因序列的正確性，在第一次體外轉錄(in vitro transcription) mRNA之前，先將Kir6.2/pET20b+ DNA拿至臺大醫院第二共研作DNA序列比對，結果顯示基因序列確實屬於老鼠內整流型鉀離子通道基因。

3.Sodium azide是一種代謝性抑制劑，會使細胞內的ATP降低，相對上提高MgADP，由實驗結果發現，細胞外給與sodium azide到表現有Kir6.2ΔC26鉀離子通道的非洲爪蟾卵細胞，會使細胞內的ATP降低，進一步活化通道，不過活化的程度小於KATP通道，兩者的差別在於KATP通道含有SUR單元，這暗示著當細胞內ATP的含量減少時，容易活化鉀離子通道，而SUR單元的存在會加強鉀離子通道的活化，顯然，細胞內的MgADP相對量提高時會結合到SUR單元，而加強通道的活化。

4.為了確定表現在非洲爪蟾卵細胞的Kir6.2ΔC26鉀離子通道其功能性，在實驗當中以細胞外給予BaCl2來證明Kir6.2ΔC26鉀離子通道受到抑制作用，同時也發現將膜電位固定在-100mV時，BaCl2抑制Kir6.2ΔC26鉀離子通道電流的IC50大於抑制胰臟β細胞KATP通道電流的IC50，顯然BaCl2對於Kir6.2ΔC26鉀離子通道的抑制程度小於β細胞KATP通道。

5.在本實驗室先前的研究顯示，procaine(10mM)會在非洲大蝸牛(Achatina fulica)RP1神經元引起猝發性(burst firing)動作電位，其主要原因之一是抑制steady-state鉀離子通道，而在本實驗當中則是以初濃度1mM procaine細胞外給予至表現Kir6.2ΔC26鉀離子通道的非洲爪蟾卵細胞，當procaine濃度提升至30mM對於鉀離子通道並沒有顯著的抑制作用，因此推論procaine引起RP1神經元引起猝發性(burst firing)動作電位，可能不是抑制KATP鉀離子通道所引起。

6.在之前的研究顯示d-amphetamine會在非洲大蝸牛(Achatina fulica)RP4神經元引起猝發現象，為探討d-amphetamine引起猝發現象是否因為抑制KATP鉀離子通道而產生，本實驗以細胞外給予d-amphetamine至表現Kir6.2ΔC26鉀離子通道的非洲爪蟾卵細胞，發現d-amphetamine當細胞外給予300μM並沒有顯著的抑制鉀離子通道電流，當d-amphetamine提高至5mM以上，鉀離子通道電流才有顯著的抑制作用，以d-amphetamine(IC50約83.89mM)與BaCl2(IC50約242.2μM)兩者作比較，顯然d-amphetamine對於Kir6.2ΔC26鉀離子通道屬於作用較弱的抑制劑。

關鍵字

非洲爪蟾 ； 內整流型 ； 鉀離子通道 ； 代謝性抑制劑 ； 猝發現象

並列摘要

Inward rectifier potassium channels (Kir6.2ΔC26 channel) were expressed in Xenopus laevis oocytes. The basic properties of Kir6.2ΔC26 channels were studied by using two-electrode voltage clamp.
The Kir6.2/pET20b+ cDNA which we owned was expressed in Xenopus laevis oocytes for the first time. In order to confirm the accuracy of Kir6.2 sequence once again , the Kir6.2 sequence of the selected plasmids was confirmed by DNA sequencing (Core Facility, National Taiwan University).The result revealed that this sequence belongd to mouse musculus potassium inwardly rectifying channel and this amino acid alignments lacked the C-terminal 26 amino acid residues.
Sodium azide was a kind of metabolic inhibitors which could decrease intracellular adenosine triphosphate (ATP).Extracellular application of sodium azide activated Kir6.2ΔC26 potassium channel, but the activated extent was smaller than that of KATP (Kir6.2+SUR) channel. The existence of SUR enhanced the activation of potassium channel. Extracellular application of BaCl2 inhibited Kir6.2ΔC26 potassium channel. When the membrance potential was hold at -100mV, the IC50 for BaCl2 block of Kir6.2ΔC26 potassium channel was smaller than that of pancreatic β cell KATP channel. In the previous study, Extra-cellular application of procaine (10 mM) or d-amphetamine (270μM) reversibly elicited bursts of potential on the central neuron of giant African snails. One of the reasons was that procaine or d-amphetamine decreased the delayed outward K+ current. Extra-cellular application of procaine had on effect on Kir6.2ΔC26 channel，but d-amphetamine inhibited Kir6.2ΔC26 channel current. The IC50(83.89mM) for d-amphetamine block of Kir6.2ΔC26 potassium channel was smaller than that of BaCl2(IC50=242.2μM).Apparently, compared with BaCl2, d-amphetamine was a weak inhibitor of Kir6.2ΔC26 potassium channel.

並列關鍵字

sodium azide ； procaine ； d-amphetamine ； Kir6.2 ； Xenopus laevis

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