Ionovation Compact人工脂質(zhì)膜重組電生理分析系統(tǒng)
Ionovation Compact人工脂質(zhì)膜重組電生理分析系統(tǒng)
德國(guó)Ionovation公司生產(chǎn)的Ionovation Compact，是該領(lǐng)域的產(chǎn)品之一，該產(chǎn)品克服了傳統(tǒng)膜片鉗的一系列缺點(diǎn)，無(wú)論從產(chǎn)品的技術(shù)含量還是從產(chǎn)品的應(yīng)用領(lǐng)域上來(lái)看，在電生理分析技術(shù)中始終處于的地位，代表著電生理分析技術(shù)發(fā)展的方向，是國(guó)內(nèi)外細(xì)胞電生理分析實(shí)驗(yàn)室*實(shí)驗(yàn)儀器。
系統(tǒng)介紹
中文名稱(chēng)：Ionovation Compact人工脂質(zhì)膜重組分子自定義環(huán)境電生理分析系統(tǒng)；
Innovation Compact是人工脂質(zhì)膜定義環(huán)境中重組分子進(jìn)行電生理分析的可靠工具。這種高度靈活的桌面檢測(cè)系統(tǒng)可適應(yīng)多種實(shí)驗(yàn)條件，目前已經(jīng)被用于對(duì)細(xì)胞膜離子通道、多種動(dòng)物和植物轉(zhuǎn)運(yùn)蛋白和它們的細(xì)胞器進(jìn)行研究。（詳見(jiàn)參考文獻(xiàn)可案例部分）
產(chǎn)品背景：
細(xì)胞膜離子通道是zui古老的功能蛋白之一，廣泛存在于從細(xì)菌到植物到動(dòng)物包括人類(lèi)在內(nèi)的生物界，是許多基本生物活動(dòng)如電活動(dòng)、離子轉(zhuǎn)運(yùn)和細(xì)胞分泌等的基礎(chǔ)。對(duì)于人類(lèi)而言，由離子通道參與的功能(如電活動(dòng))是神經(jīng)及心血管等系統(tǒng)生理功能的zui基本形式之一。對(duì)離子通道功能調(diào)節(jié)機(jī)制的深入研究是了解其生理學(xué)和生理病理學(xué)意義的關(guān)鍵所在。
膜片鉗技術(shù)是用玻璃微電極吸管把只含1-3個(gè)離子通道、面積為幾個(gè)平方微米的細(xì)胞膜通過(guò)負(fù)壓吸引封接起來(lái)，由于電極*與細(xì)胞膜的高阻封接，在電極*籠罩下的那片膜事實(shí)上與膜的其他部分從電學(xué)上隔離，因此，此片膜內(nèi)開(kāi)放所產(chǎn)生的電流流進(jìn)玻璃吸管，用一個(gè)極為敏感的電流監(jiān)視器（膜片鉗放大器）測(cè)量此電流強(qiáng)度，就代表單一離子通道電流。膜片鉗技術(shù)發(fā)展至今，已經(jīng)成為現(xiàn)代細(xì)胞電生理的常規(guī)方法。但是隨著研究的深入，目前發(fā)現(xiàn)膜片鉗主要有以下缺點(diǎn)：
? 測(cè)量非常耗時(shí)
? 需要高度熟練的操作者來(lái)進(jìn)行實(shí)驗(yàn)
? 需要建立千兆歐姆的封阻，但是千兆歐姆的封阻在測(cè)試時(shí)不穩(wěn)定
? 并不是所有離子通道都可以被測(cè)試，一般是配體或者在一側(cè)可以交換緩沖液的才可以被測(cè)量
? 膜片鉗無(wú)法對(duì)細(xì)胞器進(jìn)行分析
? 在測(cè)量過(guò)程中的細(xì)胞往往形成穿孔
? 結(jié)果在穩(wěn)定背景下經(jīng)過(guò)多次測(cè)量形成平均值，測(cè)量值離散度大
表1目前膜片鉗能夠與不能夠進(jìn)行檢測(cè)的疾?。?xì)胞）
可以被膜片鉗檢測(cè)的疾?。?xì)胞） 無(wú)法被膜片鉗檢測(cè)的疾病（細(xì)胞）
心律失常 骨質(zhì)疏松
焦慮 哮喘
癲癇 過(guò)敏
偏頭痛 癌癥
耳鳴 自身免疫性
失眠 慢性阻塞性肺病
失禁 囊性纖維化
精神分裂癥 腎結(jié)石
抑郁癥 糖尿病
高血壓
記憶障礙
肌強(qiáng)直
Ionovation Compact比膜片鉗的優(yōu)勢(shì)主要在于：
? 不需要建立千兆歐姆的封阻
? 是*一種采用雙層封阻的測(cè)量方式
? 可用于檢測(cè)各種細(xì)胞膜上的離子通道、囊泡、配體
? 容易實(shí)現(xiàn)對(duì)單個(gè)離子通道進(jìn)行分析檢測(cè)
? 可以在膜的兩側(cè)改變條件，形成雙信道進(jìn)行分析
系統(tǒng)應(yīng)用原理
Ionovation Compact采用了人工生物脂質(zhì)膜來(lái)構(gòu)建離子通道的測(cè)量方法。該系統(tǒng)使用兩個(gè)獨(dú)立的電生理測(cè)量室進(jìn)行測(cè)量。該雙層室由一個(gè)25μm厚、直徑120微米的聚四氟乙烯隔膜隔開(kāi)（a）。兩個(gè)聚碳酸酯小室的體積分別為1.2 ml。聚碳酸酯小室頂部有孔，方便接入的Ag / AgCl電極和注入緩沖液（a）。
當(dāng)兩個(gè)腔室中填充有合適的緩沖液時(shí)，溶解在正癸烷的脂質(zhì)被分散到直徑120微米的特氟隆隔膜的微孔（ b）中。緩沖液的液面經(jīng)過(guò)連續(xù)地降低和升高過(guò)程，直到特氟隆隔膜上的過(guò)量脂質(zhì)被除去，特氟隆隔膜上形成脂雙層自組織形成的人工生物脂質(zhì)膜（C）。
圖1 聚碳酸酯小室及聚四氟乙烯隔膜
圖2 正癸烷緩沖液以及形成的形成的人工生物脂質(zhì)膜
圖 3傳統(tǒng)膜片鉗與Ionovation Compact的測(cè)試原理對(duì)比
然后根據(jù)具體的目的蛋白或者細(xì)胞器等進(jìn)行操作。比如類(lèi)似于α-溶血素的毒素，可以直接加入聚碳酸酯小室中進(jìn)行測(cè)試。而需要重組的離子通道則需要將其與脂肪酸結(jié)合為重組脂蛋白，然后將這些脂蛋白或者從生物膜上“剪下”囊泡與人工生物脂質(zhì)膜通過(guò)離子梯度或融合肽方法進(jìn)行融合。
準(zhǔn)備好樣品后，開(kāi)始進(jìn)行電生理學(xué)測(cè)試。通過(guò)施加不同的電壓，來(lái)監(jiān)測(cè)細(xì)胞單離子通道的電流，其中在5-10 kHz帶寬的信號(hào)會(huì)存儲(chǔ)在系統(tǒng)的硬盤(pán)上，測(cè)試通常采用的模式是電壓鉗模式（voltage clamp mode），這種模式下應(yīng)用不同的電壓來(lái)得到目的信號(hào)。
圖4 Ionovation Compact測(cè)試系統(tǒng)工作狀態(tài)
圖5 Ionovation Compact測(cè)試系統(tǒng)得到的離子通道電流信號(hào)
圖6Ionovation Compact測(cè)試系統(tǒng)測(cè)量界面
系統(tǒng)功能亮點(diǎn)
（1） 實(shí)驗(yàn)成功率高
自動(dòng)膜生成技術(shù)以及結(jié)合運(yùn)用預(yù)制室保證了每個(gè)雙脂質(zhì)層試驗(yàn)成功。
（2） 實(shí)驗(yàn)平臺(tái)通用性好
系統(tǒng)設(shè)計(jì)時(shí)兼顧考慮到專(zhuān)家和初學(xué)者的使用水平，為專(zhuān)家和初學(xué)者均提供簡(jiǎn)單可靠的zui有效實(shí)驗(yàn)應(yīng)用工具。
（3） 實(shí)驗(yàn)流程簡(jiǎn)單、易操作
所有實(shí)驗(yàn)?zāi)康木堑玫嚼硐氲纳飳W(xué)分析結(jié)果，而實(shí)現(xiàn)生物測(cè)量中的膜準(zhǔn)備工作任務(wù)交由本系統(tǒng)直接自動(dòng)處理。
（4）系統(tǒng)運(yùn)用創(chuàng)造性的雙層電生理技術(shù)進(jìn)行
實(shí)驗(yàn)創(chuàng)新性好，解決了傳統(tǒng)膜片鉗的測(cè)量耗時(shí)、需要建立千兆歐姆的封阻等一系列缺點(diǎn)，相比于傳統(tǒng)方法，Ionovation Compact測(cè)量方法高度靈活，滿(mǎn)足不同的Idea。
（5）高度靈活的桌面系統(tǒng)
高度靈活的桌面系統(tǒng)整合了大量通道、根孔、轉(zhuǎn)運(yùn)體或膜囊膜的制備經(jīng)驗(yàn)。有很多高水平文章可做參考。
圖7 Ionovation Compact具有測(cè)量方法高度靈活的設(shè)計(jì)
2. 系統(tǒng)主要應(yīng)用領(lǐng)域
根據(jù)現(xiàn)有的已出版科研文獻(xiàn)報(bào)道，本系統(tǒng)主要科研應(yīng)用領(lǐng)域包括神經(jīng)科學(xué)，腦科學(xué)，心肌細(xì)胞，心血管，藥物學(xué)，藥理學(xué)，生理學(xué)，細(xì)胞生物學(xué)，生殖生理，病理生理，中藥學(xué)，植物細(xì)胞生理學(xué)等領(lǐng)域的研究，并可作為細(xì)胞生物學(xué)與分子生物學(xué)研究的橋梁。
• 植物通道與根孔
• 細(xì)菌通道與根孔
• 動(dòng)物通道與根孔
• 毒劑 比如肉毒毒素
• 膜活性劑比如突觸核蛋白
• 以上所有離子通道的結(jié)構(gòu)功能分析
• 脂質(zhì)觸發(fā)器
• 細(xì)胞膜轉(zhuǎn)運(yùn)處理
• 尋找活性藥物物質(zhì)
• 藥物安全分析
• 信號(hào)轉(zhuǎn)導(dǎo)模式分析
• 與帕金森和老年癡呆癥疾病等疾病相關(guān)的膜透性分析
• 該技術(shù)因其具有的自動(dòng)化、應(yīng)用方便特性，在藥物研發(fā)、藥物篩選中顯示了強(qiáng)勁的生命力
圖8 Ionovation Compact測(cè)試系統(tǒng)可用于多種離子通道的分析
系統(tǒng)主要性能參數(shù)
測(cè)量對(duì)象：可實(shí)現(xiàn)對(duì)上萬(wàn)種離子通道、轉(zhuǎn)運(yùn)體和孔隙活動(dòng)的精確測(cè)量；
軟件：電生理數(shù)據(jù)采集分析軟件Patchmaster，采用用戶(hù)界面友好的軟件實(shí)現(xiàn)測(cè)量全部控制；具備預(yù)定義協(xié)議式的用戶(hù)定義實(shí)驗(yàn)工作流程；
自動(dòng)操作：全自動(dòng)設(shè)備操作（雙層生產(chǎn)和驗(yàn)證、雙層完整性的電容控制、膜雙側(cè)灌注系統(tǒng)）；•雙層分子可視化操作；
電流測(cè)量：穩(wěn)定低噪聲Ag/AgCl電極，采用鹽橋記錄電流范圍從pA到幾個(gè)nA；
耗材：一次性雙分子層室可簡(jiǎn)單、快速置換；
參考文獻(xiàn)
1. Weiß K, Neef A, Van Q, Kramer S, Gregor I, Enderlein J.Quantifying the diffusion of membrane proteins and peptides in black lipid membranes with 2-focus fluorescence correlation spectroscopy.Biophys J. 2013 Jul 16;105(2):455-62. doi: 10.1016/j.bpj.2013.06.004.
2. Weingarth M, Prokofyev A, van der Cruijsen EA, Nand D, Bonvin AM, Pongs O, Baldus M.Structural determinants of specific lipid binding to potassium channels.J Am Chem Soc. 2013 Mar 13;135(10):3983-8. doi: 10.1021/ja3119114. Epub 2013 Mar 4.
3. Theis T, Mishra B, von der Ohe M, Loers G, Prondzynski M, Pless O, Blackshear PJ, Schachner M, Kleene R.Functional role of the interaction between polysialic acid and myristoylated alanine-rich C kinase substrate at the plasma membrane.J Biol Chem. 2013 Mar 1;288(9):6726-42. doi: 10.1074/jbc.M112.444034. Epub 2013 Jan 17.
4. Köstler K, Werz E, Malecki E, Montilla-Martinez M, Rosemeyer H. Nucleoterpenes of thymidine and 2'-deoxyinosine: synthons for a biomimetic lipophilization of oligonucleotides Chem Biodivers. 2013 Jan;10(1):39-61. doi: 10.1002/cbdv.201100338.
5. Schmidt F, Levin J, Kamp F, Kretzschmar H, Giese A, Bötzel K. Single-channel electrophysiology reveals a distinct and uniform pore complex formed by α-synuclein oligomers in lipid membranes. PLoS One. 2012;7(8):e42545. doi: 10.1371/journal.pone.0042545. Epub 2012 Aug 3.
6. Betaneli V, Petrov EP, Schwille P. The role of lipids in VDAC oligomerization. Biophys J. 2012 Feb 8;102(3):523-31. doi: 10.1016/j.bpj.2011.12.049. Epub 2012 Feb
7. Weiß K., Enderlein J. Lipid Diffusion within Black Lipid Membranes Measured with Dual-Focus Fluorescence Correlation Spectroscopy. Chemphyschem. 2012 Mar;13(4):990-1000.
8. Werz E, Korneev S, Montilla-Martinez M, Wagner R, Hemmler R, Walter C, Eisfeld J, Gall K, Rosemeyer H. Specific DNA Duplex Formation at an Artificial Lipid Bilayer: towards a New DNA Biosensor Technology. Chem Biodivers. 2012; Feb;9(2):272-81.
9. Schmidt F, Levin J, Kamp F, Kretzschmar H, Giese A, Bötzel K. Single-channel electrophysiology reveals a distinct and uniform pore complex formed by α-synuclein oligomers in lipid membranes. PLoS One. 2012;7(8):e42545. doi: 10.1371/journal.pone.0042545. Epub 2012 Aug 3.
10. Betaneli V, Petrov EP, Schwille P. The role of lipids in VDAC oligomerization Biophys J. 2012 Feb 8;102(3):523-31. doi: 10.1016/j.bpj.2011.12.049. Epub 2012 Feb 7.
11. Weiß K., Enderlein J. Lipid Diffusion within Black Lipid Membranes Measured with Dual-Focus Fluorescence Correlation Spectroscopy. Chemphyschem. 2012 Mar;13(4):990-1000.
12. Werz E, Korneev S, Montilla-Martinez M, Wagner R, Hemmler R, Walter C, Eisfeld J, Gall K, Rosemeyer H. Specific DNA Duplex Formation at an Artificial Lipid Bilayer: towards a New DNA Biosensor Technology. Chem Biodivers. 2012; Feb;9(2):272-81
13. Erika Kovács-Bogdán, J Philipp Benz, Jürgen Soll, Bettina Bölter Tic20 forms a channel independent of Tic110 in chloroplasts BMC Plant Biol. 2011; 11: 133.
14. Honigmann A, Walter C, Erdmann F, Eggeling C, Wagner R. Characterization of horizontal lipid bilayers as a model system to study lipid phase separation. Biophys J. 2010 Jun 16;98(12):2886-94.
15. Schneider R, Etzkorn M, Giller K, Daebel V, Eisfeld J, Zweckstetter M, Griesinger C, Becker S, Lange AThe native conformation of the human VDAC1 N terminus. Angew Chem Int Ed Engl. 2010 Mar 1;49(10):1882-5.
16. Kostka M, H?gen T, Danzer KM, Levin J, Habeck M, Wirth A, Wagner R, Glabe CG, Finger S, Heinzelmann U, Garidel P, Duan W, Ross CA, Kretzschmar H, Giese A. Single-particle characterization of iron-induced pore-forming alpha -synuclein oligomers. J Biol Chem. 2008 Feb 7.
17. van der Laan M, Meinecke M, Dudek J, Hutu DP, Lind M, Perschil I, Guiard B, Wagner R, Pfanner N, Rehling P. Motor-free mitochondrial presequence translocase drives membrane integration of preproteins. Nat Cell Biol. 2007; 9(10):1152-9.
18. Pagliuca C, Goetze TA, Wagner R, Thiel G, Moroni A, Parcej D. Molecular properties of Kcv, a virus encoded K+ channel. Biochemistry. 2007; 46(4):1079-90.
19. Goetze TA, Philippar K, Ilkavets I, Soll J, Wagner R. OEP37 is a new member of the chloroplast outer membrane ion channels J Biol Chem. 2006; 281(26):17989-98. Epub 2006 Apr 19
20. Kovermann P, Truscott KN, Guiard B, Rehling P, Sepuri NB, Muller H, Jensen RE, Wagner R, Pfanner N. Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel Mol Cell. 2002; 9(2):363-73.
21. Meuser D, Splitt H, Wagner R, Schrempf H. Mutations stabilizing an open conformation within the external region of the permeation pathway of the potassium channel KcsA. Eur Biophys J. 2001; 30(5):385-91.
22. Hill K, Model K, Ryan MT, Dietmeier K, Martin F, Wagner R, Pfanner N. Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins [see comment] Nature. 1998; 395(6701):516-21.
案例1 Thomas Theis. Functional roles of transient receptor potential canonical channels and myristoylated alanine-rich protein kinase C substrate as novel interaction partners of the neural cell adhesion molecule NCAM and polysialic acid in Mus musculus，2013 博士論文
Figure 5.31: Schematic drawing of the setup to measure the capacitance of an artificially lipid bilayer. The Ionovation Compact V02 system was used to build up an artificially lipid bilayer and to measure the capacitance of the bilayer. The lipid bilayer separates adjacent chambers， which were filled with electrode buffer. The artificially lipid bilayer contains a mixture of 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) (1:1). In the circuit diagram the resistance circuit (red) and the capacitor circuit (cyan) is shown. The resistance of the red marked circuit is increased when the lipid bilayer is formed. As a consequence more current flows through the capacitor circuit and， thus， lead to an increase of capacitance. When colominic acid and a peptide derived from the ED of MARCKS (ED-Peptide) are added to the trans chamber and the cis chamber， respectively， and if they interact through the lipid bilayer， the lipid bilayer would be more instable from both sides and change its capacitance. Therefore the resistance would decrease and less current would flow through the capacitor circuit which would diminish the capacitance (A). Chondroitin sulfate (ChS) has similar chemical properties as colominic acid， but it is not known that neither it attaches to the membrane nor that it can interact with MARCKS. The control-peptide is also derived from the ED of MARCKS， but the phenylalanine residues were changed to alanine residues. Both were used as controls， which would not penetrate in the lipid bilayer and therefore the bilayer would be stable. The resistance in the red marked circuit would be higher in the controls and therefore more current would flow through the cyan marked circuit followed by an increase of the capacitance (B， C， D).
案例2 Citation: Schmidt F， Levin J， Kamp F， Kretzschmar H， Giese A， et al. (2012) Single-Channel Electrophysiology Reveals a Distinct and Uniform Pore Complex Formed by a-Synuclein Oligomers in Lipid Membranes. PLoS ONE 7(8): e42545. doi:10.1371/journal.pone.0042545
實(shí)驗(yàn)方法：
Influence of a-syn on electrophysiological properties of planar lipid bilayers Planar lipid bilayers were produced in the Ionovation Compact (Ionovation， Osnabruck， Germany) by the painting technique [20]. Two bath chambers separated by a Teflon-septum were filled with 250 mM KCl， 10 mM MOPS， pH= 7.2 (Merck). In the cis-chamber， 2 ml of a 100 mg/ml-solution of purified azolectin in n-Decane (Ionovation) was applied to a pinhole of 120 mmin diameter. After 30 min incubation at RT， lipid was thinned out by repetitive lowering and re-raising of the buffer-level until a bilayer was formed. Bilayer formation was monitored optically and by capacitance- and conductance-measurements. The resulting bilayers had a typical capacitance of 60–80pF and a resistance of .100GV. The monitoring of the bilayer was performed using Ag/AgCl-electrodes (Ionovation)， an EPC 10-amplifier and Patchmaster-software (HEKA， Lambrecht/Pfalz， Germany). The electrode in the cis-chamber was directly connected to the amplifier， so all potentials are referred to this compartment. The noise was ，0.4pA (r.m.s.) at 3 kHz bandwidth. After bilayer formation， we waited for 10 min to ensure application of the protein to a stable bilayer-system. Then， a-syn aggregation samples (total assay volume: 200 ml) were added in aliquots of 20 ml close to the membrane in the trans-chamber. The electrophysiological properties were monitored using +/220 mV- squarewave-voltage pulses. Pore formation resulted in an increase in the current flow over the membrane compared to an intact bilayer (Fig. 1A). Threshold for pore detection was set to a conductance of 70pS. If no increase in bilayer conductance beyond the threshold was detected for 5 min， the next aliquot of the sample was added. Pore detection rate was defined as the probability of pore detection per a-syn aggregation sample. In the event of an increase in bilayer conductance， a standardized recording-protocol was employed， consisting of a voltage-ramp reaching from 2100 to +100 mV over 10 sec and different squarewave-voltage pulses.
案例3 Citation: Smeazzetto S， Saponaro A， Young HS， Moncelli MR， Thiel G, (2013) .Structure-Function Relation of Phospholamban: Modulation of Channel Activity as a Potential Regulator of SERCA Activity. PLoS ONE 8(1): e52744. doi:10.1371/journal.pone.0052744
實(shí)驗(yàn)方法
Planar lipid bilayer and single channel measurements Experiments with planar lipid bilayers were carried out as described previously [23] using the folding method with a 10 mg/ ml solution of diphytanoylphosphatidylcholine (DPhPC) (Avanti- Polar， AL， USA) in pentane. The experimental chambers used to assemble the planar bilayer were either custom made or disposable chambers (Ionovation， Osnabruck， Germany). The measurements were performed in a buffer containing 500 mM KCl， 10 mM Mops/Tris pH 7. The Ag/AgCl electrode in the cis compartment was directly connected to the head stage of a current amplifier (EPC 7， List， Darmstadt， Germany); the trans chamber was grounded. Currents were recorded and stored by an analogue/digital-converter (LIH 1600， HEKA electronics， Lam- brecht， Germany) with a sampling rate of 3.571 kHz after low pass filtering at 1 kHz. Data were recorded by Patchmaster-Software (HEKA electronics， Lambrecht， Germany) and analyzed with the Fitmaster-Software (HEKA electronics， Lambrecht， Germany) and the KielPatch program (University of Kiel， www.zbm.uni-kiel. de/aghansen/software.html) and Origin (OriginLab. Northamp- ton， MA， USA). The apparent single channel current amplitudes (Iapp) were determined by visual inspection of the current traces using the KielPatch software. The open probability (Po) was calculated with the KielPatch software. In the case that more than one channel was present in a bilayer we estimated the number of channels from the maximal number of concomitant open events. The protein， which was dissolved in water， was added directly to the trans chamber at a final concentration of ca. 0.3 mM. Before addition of the protein the bilayer conductance was routinely recorded for approximay 1 hour in order to exclude artefacts from contaminations. Only bilayers without artefacts were used for reconstitution of PLN. To perform experiments of phosphorylated wt-PLN， the protein was added to the bilayer after 3 h incubation at 30uC under the following conditions: 1 mg/ml wt-PLN， 20 mM imidazole， 100 mM KCl， 1 mM DTT， 10 mM MgCl2， 0.5 mM EGTA， 1 mM ATP and (5 units/10 mg PLN) PKA (Sigma) diluted in storage buffer solution.
案例4：Erika Kovács-Bogdán, et al. Tic20 forms a channel independent of Tic110 in chloroplasts.BMC plant biology, 2011, 11:133—134
Results: We performed a comprehensive biochemical and electrophysiological study to characterize Tic20 in more detail and to gain a deeper insight into its potential role in protein import into chloroplasts. Firstly, we compared transcript and protein levels of Tic20 and Tic110 in both Pisum sativum and Arabidopsis thaliana. We found the Tic20 protein to be generally less abundant, which was particularly pronounced in Arabidopsis. Secondly, we demonstrated that Tic20 forms a complex larger than 700 kilodalton in the inner envelope membrane, which is clearly separate from Tic110, migrating as a dimer at about 250 kilodalton. Thirdly, we defined the topology of Tic20 in the inner envelope, and found its N- and C-termini to be oriented towards the stromal side. Finally, we successfully reconstituted overexpressed and purified full-length Tic20 into liposomes. Using these Tic20- proteoliposomes, we could demonstrate for the first time that Tic20 can independently form a cation selective channel in vitro.
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