We thank Susanne Fenz for discussing the possibility of reconstituting proteins by agarose swelling, Feng Ching Tsai for current measurements, and present and former members of the Bassereau group for support and assistance. The project was funded by the Agence Nationale de la Recherche (grant BLAN-0057-01), by the European Commission (NoE SoftComp), by the Universit&#233; Pierre et Marie Curie (grant from the FED21, Dynamique des Syst&#232;mes Complexes). M.G. was supported by an Institut Curie International PhD Fellowship, S.A. by a fellowship from the Fondation pour la Recherche M&#233;dicale, G.E.S.T. by a Marie Curie Incoming International Fellowship from the European Commission and a grant from the Universit&#233; Pierre et Marie Curie. The publication fees were covered by the Labex &#8216;CelTisPhyBio&#8217; (ANR-11-LABX0038).

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The authors have nothing to disclose.

نظم نموذج المحاكاة البيولوجية هي أداة هامة لدراسة خصائص وتفاعلات البروتينات والأغشية. مقارنة بأنظمة أخرى مثل إعادة تشكيل BLMs أو الأغشية الدهنية المعتمدة، GUV النظم القائمة الحالية عدة فرص بما في ذلك سيطرة كبيرة من تكوين الغشاء والتوتر والهندسة، فضلا عن كونه حقا خالية...

عند دراسة المبادئ الفيزيائية التي تحكم الأنظمة الحية، ونهج من أسفل إلى أعلى تسمح للالتجريبي للسيطرة على تكوين النظام وغيرها من المعالم التي لم يتم التلاعب بها بسهولة في النظم القائمة على خلية 1. للعمليات التي تعتمد على الأغشية، العملاق Unilamellar الحويصلات (GUVs، قطر ~ 1-100 ميكرون) وقد ثبت أن نظام بيوميمتيك مفيدة جدا 2-7 كما هي مناسبة تماما للدراسات الفحص المجهري والمجهرية 8-10. في حين أن هناك العديد من البروتوكولات المختلفة لإنتاج GUVs، ومعظم تقع في فئتين - مستحلب أساس النهج 11،12 والتقنيات التي تعتمد على إعادة إماهة فيلم الدهون 13-16. في الأساليب المستندة إلى مستحلب، يتم تجميع النشرات الداخلية والخارجية من الأغشية GUV بالتتابع من الطبقات الوحيدة الدهون في واجهات المياه / النفط. هذا النهج هو المثالي لتغليف البروتينات القابلة للذوبان معفي GUVs، وتشكيل GUVs مع غير المتماثلة تكوين النشرة الدهون. ومع ذلك، يمكن GUVs تشكلت من المستحلبات تحتفظ آثار مذيب أن تغيير خصائص الغشاء والميكانيكية 17، والنهج ليس وخاصة مناسبة تماما لالعابرة للغشاء البروتين إعادة. طرق معالجة الجفاف الفيلم تعتمد على حقيقة أن تجفيف (الجفاف) يسبب العديد من خليط الدهن لتشكيل كومة متعددة رقائقي من الأغشية. إذا ثم يتم وضع هذا المكدس في اتصال مع وجود مخزن مؤقت مائي، والأغشية في المكدس تتحرك التدفقات بصرف النظر المذيبات فيما بينها وعلى سطح المكدس، يمكن الأغشية الفردية فصل لتشكيل GUVs 13،18 (وكذلك حديقة حيوانات حقيقية ل الأجسام شحمي أخرى). ومع ذلك، حتى بالنسبة المثلى العازلة والدهون التراكيب، وهذا &quot;تورم عفوية&quot; الكلاسيكية الأسلوب لديه العائد المنخفض نسبيا من GUVs خالية من العيوب. واحد الطريقة المستخدمة على نطاق واسع لزيادة العائد من GUVs خالية من العيوب هو &quot;electroformation221 ؛، التي يتم تطبيق (AC) الحقل التيار المتردد خلال فيلم الإماهة. في حين لا تزال الآلية غير مفهومة، &quot;electroformation&quot; يمكن أن تعطي عوائد GUV مذهلة (&gt; 90٪ في الظروف مواتية) لمخازن تركيز الملح منخفضة (&lt;5 ملم) 14،19، ويمكن أن تعمل حتى في مخازن الفسيولوجية (~ 100 ملم) باستخدام تردد أعلى (500 هرتز مقابل 10 هرتز) AC المجال وأقطاب البلاتين 15. نهج بديل لزيادة العائد من GUVs خالية من العيوب هو &quot;بمساعدة هلام تورم&quot;، الذي وتودع الحل الدهون على هلام الركيزة البوليمرية بدلا من السلبي (على سبيل المثال، والزجاج، PTFE) ركائز المستخدمة في الكلاسيكية &quot;تورم العفوي &quot;. عندما يتم ممهى الناتجة الفيلم الدهون / هلام، يمكن GUVs تشكل بسرعة حتى لمخازن الفسيولوجية 16،20. ويمكن لجميع هذه الأساليب تنتج GUVs-الدهون الوحيد الذي يمكن استخدامه لدراسة غشاء المرتبطة الظواهر مثلالتفاعل بين البروتينات القابلة للذوبان والأغشية. ومع ذلك، لإدراج البروتين العابر للغشاء في GUVs، هناك حاجة إلى تعديلات كبيرة لضمان أن البروتين لا يزال في حالة وظيفية في جميع أنحاء الإجراء إعادة. في حين حلول الدهون في المذيبات العضوية (على سبيل المثال، والكلوروفورم، الهكسان الحلقي) مثالية لإنتاج الأفلام الدهون والبروتينات عبر الغشاء وعادة ما تكون مستقرة فقط عندما يتم تضمين مسعور المجال الخاص بها عبر الغشاء في طبقة ثنائية الدهون، أو محاطة مذيلة المنظفات ( على سبيل المثال، خلال تنقية البروتين). وهكذا، فإن المواد بدءا من أجل إعادة هي عادة الأغشية الأم، البروتين النقي في حل المنظفات، أو الحويصلات التي تحتوي على البروتين unilamellar صغيرة (PROTEO-سيارات الدفع الرباعي) و / أو حويصلات متعددة رقائقي (PROTEO-MLVs) التي شكلتها إزالة المنظفات في وجود الدهون. معظم طرق لدمج هذه البروتينات الغشاء إلى GUVs تقع في ثلاث فئات. مغرز مباشرةن: يتم خلط عبر غشاء البروتين معلقة في المنظفات مع، الدهون فقط، GUVs تذوب المنظفات أقل ما يقال شكلت مسبقا، والمنظفات ثم إزالة باستخدام biobeads 21. في حين بسيطة من الناحية المفاهيمية، يتطلب هذا الأسلوب مراقبة دقيقة من تركيز المنظفات، وتركيز عالية جدا المنظفات يمكن أن يحل GUVs في حين منخفضة جدا تركيز يمكن أن يسبب البروتين لتتكشف أو مجموع المباراتين. يتم الجمع بين البروتين في PROTEO-سيارات الدفع الرباعي مع شكلت مسبقا، الدهون فقط GUVs ويتم تسهيل الاندماج مع الببتيدات fusogenic الخاصة 22 أو المنظفات 21: GUV / PROTEO-SUV فيوجن. وعادة ما مدى الانصهار ومحدودة يؤدي إلى GUVs مع انخفاض كثافة البروتين. الجفاف / الإماهة: يتم تشكيل لفيلم الدهون التي تحتوي على البروتين بسبب الجفاف الجزئي لPROTEO-SUV (أو PROTEO-MLV) حل وGUVs يتم بعد ذلك نمت كما لفيلم الدهن النقي. التحدي الواضح هو حماية البروتين خلال dehydrati جزئيعلى الخطوة 23، ولكن الأسلوب وقد استخدم بنجاح لإعادة البروتينات العابرة للغشاء مثل رودوبسين جرثومي، الكالسيوم أتباز، إنتغرين وVDAC إلى GUVs 7،23 - 25. توضح هذه المقالة بروتوكولات الجفاف / الإماهة لجعل GUVs تحتوي على قناة الجهد بوابات البوتاسيوم، KvAP، من فرط حرارة الأركيا، Aeropyrum pernix. KvAP لديه درجة عالية من التماثل إلى الجهد حقيقية النواة قنوات البوتاسيوم تعتمد 26 والتركيب البلوري المعروف 27 ، مما يجعلها نموذجا جيدا لدراسة آلية الجهد النابضة. وقد وصفت إنتاج PROTEO-سيارات الدفع الرباعي بالتفصيل سابقا وليس جزءا من هذا البرنامج التعليمي 26،28،29. الأهم من ذلك، KvAP PROTEO-سيارات الدفع الرباعي لا يجب أن يتم إنتاجها لكل إعداد GUV، لأنها يمكن خزنها في صغيرة (على سبيل المثال، 10 ميكرولتر) aliquots في -80 ° C لفترات طويلة من الزمن (&gt; 1 سنة). Electroformationأو بمساعدة هلام تورم ويمكن بعد ذلك أن تستخدم لزراعة GUVs من KvAP PROTEO-سيارات الدفع الرباعي (أو PROTEO-MLVs). ويوضح الخطوات الرئيسية لبروتوكول electroformation في الشكل 1. تترسب قطرات من محلول سيارات الدفع الرباعي التي تحتوي على البروتين على أسلاك البلاتين (كما هو موضح في الشكل 2). الجفاف الجزئي للتعليق SUV يؤدي إلى تشكيل لفيلم البروتين الدهني من خلال مزيج من سيارات الدفع الرباعي. أثناء الإماهة، يتم تطبيق حقل AC إلى الأقطاب لمساعدة طبقات الدهون لdelaminate وتشكيل GUVs. حقل 10 هرتز يعمل بشكل جيد عند استخدام &quot;قليل الملح&quot; (&lt;5 مم) الإماهة عازلة 28 و GUVs يستغرق عدة ساعات لتنمو. في المقابل، مخازن الفسيولوجية (التي تحتوي على ~ 100 ملم الملح) تعمل بشكل جيد مع الجهد أقل، 500 هرتز AC المجال ولكن تحتاج إلى فترات طويلة (~ 12 ساعة) مدة 15 التورم. ويستند هذا الأسلوب على بروتوكول سابق باستخدام الشرائح ITO 24، ولكن يستخدم تابع غرفة مخصصةaining اثنين من الأسلاك البلاتين كما هو مبين في الشكل 2 (انظر مناقشة للحصول على تفاصيل تصميم واقتراحات لبساطة وبدائية غرف). ويوضح الشكل (3) طريقة تورم بمساعدة هلام. بروتوكول يعمل بشكل جيد مع مخازن مع تركيز الملح الفسيولوجية، سريع، وينتج GUVs مع توزيع البروتين أكثر تجانسا. ومع ذلك، فإن العائد من معزولة، GUVs المجانية عيب، على ما يبدو (أي الغشاء GUV موحد في موازين طول البصرية ولا أرفق أي كائنات) هو أقل من ذلك، على الرغم من أنها توفر عدد كاف لالتصحيح، المشبك والتجارب الصغيرة التلاعب . واستند هذا الأسلوب على البروتوكول باستخدام هلام الاغاروز لإنتاج الدهون الوحيد GUVs 16 ويتطلب معدات متخصصة أقل من الطريقة electroformation. يوصف توصيف GUVs مع مضان المجهري، وكذلك الإجراءات باستخدام معيار التصحيح، المشبك مجموعة المتابعة لقياس النشاط KvAP في &quot;الداخل إلى الخارج&quot; رفعه بقع الغشاء. يمكن المتزايدة GUVs التي تحتوي على البروتين أن يكون أكثر صعوبة من GUVs-الدهون الوحيد. على وجه الخصوص، يمكن أن العائد GUV النهائي يعتمد على بحساسية بالضبط كيف وتودع الحل SUV والمجففة لتشكيل كومة الغشاء. لشخص دون أي خبرة سابقة مع GUVs، قد يكون من المفيد أن ينمو أولا GUVs-الدهون الوحيد التالية بروتوكول التقليدي 15،16 في الذي يتكون الفيلم الغشاء عن طريق إيداع الدهون من المذيبات العضوية. وبمجرد أن البروتوكول التقليدي يعمل بشكل جيد، SUV ترسب والجفاف الجزئي ويمكن بعد ذلك أن يتقن استخدام سيارات الدفع الرباعي-الدهون فقط، التي هي أيضا مفيدة جدا عند ضبط بروتوكول لتكوين الدهون الجديد. عندما تنمو GUVs موثوق من سيارات الدفع الرباعي-الدهون فقط، وعندها فقط خطوة صغيرة لإنتاج GUVs التي تحتوي على البروتين من PROTEO-سيارات الدفع الرباعي.

<table border="0" cellpadding="0" cellspacing="0" width="599">
	<tbody>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Name of the 
			Material/Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td style="width:149px;">Comments/ Description</td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine)</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>850356P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Egg PC L-&#945;-phosphatidylcholine (Egg, Chicken)&#160;</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>840051P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Egg PA L-&#945;-phosphatidic acid (Egg, Chicken)</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>840101P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">18:1 (&#916;9-Cis) PC (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>850375P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">cholesterol (ovine wool, &#62;98%)&#160;</td>
			<td style="width:132px;">Avanti Polar Lipids&#160;</td>
			<td>700000P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">TRed-DHPE</td>
			<td style="width:132px;">Invirtogen</td>
			<td>T-1395MP</td>
			<td style="width:149px;">labeled lipid</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">BPTR-Ceramide</td>
			<td style="width:132px;">Invirtogen</td>
			<td>D-7540&#160;</td>
			<td style="width:149px;">labeled lipid</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Choloroform</td>
			<td style="width:132px;">VWR</td>
			<td>22711.290</td>
			<td style="width:149px;">AnalaR Normapur</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Acetone</td>
			<td style="width:132px;">VWR</td>
			<td>20066.296</td>
			<td style="width:149px;">AnalaR Normapur</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Ethanol</td>
			<td style="width:132px;">VWR</td>
			<td>20821.330</td>
			<td style="width:149px;">AnalaR Normapur</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Kimwipe</td>
			<td style="width:132px;">Kimtech</td>
			<td>7552</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Hamilton syringes&#160;</td>
			<td style="width:132px;">Hamilton Bonaduz AG</td>
			<td></td>
			<td style="width:149px;">diverse</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Amber vials and teflon cups</td>
			<td style="width:132px;">Sigma</td>
			<td>SU860083 and SU860076</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Parafilm</td>
			<td style="width:132px;">VWR</td>
			<td>291-1214</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">microcentrifuge tube</td>
			<td style="width:132px;">eppendorf</td>
			<td></td>
			<td style="width:149px;">diverse</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Agarose</td>
			<td style="width:132px;">Euromex</td>
			<td style="width:155px;">LM3 (1670-B, Tg 25.7C, Tm 64C)</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Sucrose</td>
			<td style="width:132px;">Sigma</td>
			<td>84097-1kg</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Glucose&#160;</td>
			<td style="width:132px;">Sigma</td>
			<td>G8270-1kg</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Trehalose</td>
			<td style="width:132px;">Sigma</td>
			<td>T9531-25G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">KCl</td>
			<td style="width:132px;">Sigma</td>
			<td>P9333-1kg</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Hepes</td>
			<td style="width:132px;">Sigma</td>
			<td>H3375-100G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">TRIS</td>
			<td style="width:132px;">Euromex</td>
			<td>EU0011-A</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Osmometer</td>
			<td style="width:132px;">Wescor</td>
			<td>Vapro</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">pH meter</td>
			<td style="width:132px;">Schott instruments</td>
			<td>Lab850</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Sonicator</td>
			<td style="width:132px;">Elmasonic</td>
			<td>S 180 H</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Syringe filter 0.2&#181;m</td>
			<td style="width:132px;">Sartorius steim</td>
			<td>Minisart 16532</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Function generator</td>
			<td style="width:132px;">TTi</td>
			<td>TG315</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Platinum wires</td>
			<td style="width:132px;">Goodfellow</td>
			<td>LS413074</td>
			<td style="width:149px;">99.99+%, d=0.5mm</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Polytetrafluoroethylene</td>
			<td style="width:132px;">Goodfellow</td>
			<td></td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Dow Corning &#39;high vacuum grease&#39;</td>
			<td style="width:132px;">VWR</td>
			<td>1597418</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">sealing paste</td>
			<td style="width:132px;">Vitrex medical A/S, Denmark</td>
			<td>REF 140014</td>
			<td style="width:149px;">Sigillum Wax</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Cover slides 22x40 mm No1,5</td>
			<td style="width:132px;">VWR&#160;</td>
			<td>631-0136</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Cover slides 22x22 mm No1,5</td>
			<td style="width:132px;">VWR&#160;</td>
			<td>631-0125</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">plasma cleaner</td>
			<td style="width:132px;">Harrick</td>
			<td>PDC-32G</td>
			<td style="width:149px;">airplasma, setting &#39;high&#39;</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">petri dishes</td>
			<td style="width:132px;">Falcon BD</td>
			<td>REF 353001</td>
			<td style="width:149px;">3.5 cmx1cm</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">patch clamp amplifier</td>
			<td style="width:132px;">Axon instruments</td>
			<td>Multiclamp700B</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">DAQ Card</td>
			<td style="width:132px;">National Instruments</td>
			<td>PCI-6221</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Labview</td>
			<td style="width:132px;">National Instruments</td>
			<td>version 8.6</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Glass pipettes boro silicate OD 1mm ID 0.58mm</td>
			<td style="width:132px;">Harvard Apparatus</td>
			<td>GC100-15</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Electrode holder</td>
			<td style="width:132px;">Warner Instruments&#160;</td>
			<td>Q45W-T10P</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Micromanipulator</td>
			<td style="width:132px;">Sutter&#160;</td>
			<td>MP-285</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">pipette puller</td>
			<td style="width:132px;">Sutter&#160;</td>
			<td>P-2000&#160;</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Camera</td>
			<td style="width:132px;">ProSilica&#160;</td>
			<td>GC1380</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Zeiss microscope</td>
			<td style="width:132px;">Zeiss</td>
			<td>Axiovert 135</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">Objective</td>
			<td style="width:132px;">Zeiss</td>
			<td></td>
			<td style="width:149px;">40x long working distance, Phase contrast</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Objective&#160;</td>
			<td style="width:132px;">Zeiss</td>
			<td></td>
			<td style="width:149px;">100x Plan-Apochromat NA 1.3</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Filterset GFP (for Alexa-488)</td>
			<td style="width:132px;">Horiba</td>
			<td>XF100-3</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">Filterset Cy3 (for TexasRed)</td>
			<td style="width:132px;">Horiba</td>
			<td>XF101-2</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">beta-casein</td>
			<td style="width:132px;">Sigma&#160;</td>
			<td>C6905-1G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:163px;">confocal microscope</td>
			<td style="width:132px;">Nikon Eclipse TE 2000-E</td>
			<td></td>
			<td style="width:149px;">D-Eclipse C1 confocal head</td>
		</tr>
		<tr height="20">
			<td dir="LTR" height="20" style="height:20px;width:163px;">Objective</td>
			<td style="width:132px;">Nikon</td>
			<td></td>
			<td style="width:149px;">Plan Fluor 100&#215; NA1.3</td>
		</tr>
		<tr height="60">
			<td dir="LTR" height="60" style="height:60px;width:163px;">matlab</td>
			<td style="width:132px;">Mathworks</td>
			<td></td>
			<td style="width:149px;">for image processing and analysis of the current traces</td>
		</tr>
	</tbody>
</table>

reconstitution of a transmembrane protein, the voltage-gated ion channel, kvap, into giant unilamellar vesicles for microscopy and patch clamp studies

عملاق Unilamellar الحويصلات (GUVs) هي نظام بيوميمتيك شعبية لدراسة الظواهر المرتبطة الغشاء. ومع ذلك، وتستخدم عادة بروتوكولات لتنمو يجب أن يتم تعديل GUVs من أجل تشكيل GUVs تحتوي على البروتينات عبر الغشاء وظيفية. توضح هذه المقالة طريقتين الجفاف-الإماهة - electroformation وبمساعدة هلام تورم - لتشكيل GUVs تحتوي على قناة البوتاسيوم الجهد بوابات، KvAP. في كلتا الطريقتين، وهو حل من الحويصلات التي تحتوي على البروتين unilamellar صغيرة هو المجففة جزئيا لتشكيل كومة من الأغشية، والتي يتم بعد ذلك يسمح للتضخم في منطقة عازلة الإماهة. للتعرف على طريقة electroformation، وتودع الفيلم على أقطاب البلاتين بحيث يمكن تطبيق حقل AC خلال فيلم الإماهة. في المقابل، يستخدم الأسلوب بمساعدة هلام تورم هلام الركيزة الاغاروز لتعزيز فيلم الإماهة. ويمكن لكل من طرق إنتاج GUVs في (على سبيل المثال، 100 ملم) تركيزات منخفضة (على سبيل المثال، 5 ملم) والفسيولوجية الملح. وتتميز GUVs الناتجة عن طريق المجهر مضان، وظيفة من القنوات أعيد قياسها باستخدام التكوين التصحيح، المشبك من الداخل إلى الخارج. في حين تورم في وجود حقل كهربائي متناوب (electroformation) يعطي عالية الغلة من GUVs خالية من العيوب، وتنتج طريقة بمساعدة هلام تورم توزيع البروتين أكثر تجانسا ويتطلب أي معدات خاصة.

نمو GUVs يمكن تقييمها بسرعة عن طريق فحص غرفة النمو تحت المجهر. لelectroformation، وGUVs تميل إلى النمو في عناقيد على طول الأسلاك البلاتين، كما هو مبين في الشكل (4). وخلال بمساعدة هلام تورم، وتظهر GUVs عن الهياكل الكروية التي تنمو وتلتحم مع بعضها (الشكل 5) بسرعة. <p c...

Watch this Scientific Journal Video about Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies at JoVE

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

ويتجلى إعادة تشكيل البروتين الغشاء، KvAP، إلى الحويصلات unilamellar العملاقة (GUVs) للطريقتين الجفاف-الإماهة - electroformation، وبمساعدة هلام التورم. في كلتا الطريقتين، وتنصهر الحويصلات unilamellar صغيرة تحتوي على البروتين معا لتشكيل GUVs التي يمكن بعد ذلك دراستها من قبل المجهري مضان والتصحيح، المشبك الكهربية.

إعادة تشكيل البروتين عبر الغشاء، وايون القناة بوابات الجهد، KvAP، إلى العملاق Unilamellar الحويصلات للفحص المجهري وتصحيح الدراسات المشبك

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

reconstitution-transmembrane-protein-voltage-gated-ion-channel-kvap

Research

JoVE Journal

Biology

18.9K Views.  Université Pierre et Marie Curie.  ويتجلى إعادة تشكيل البروتين الغشاء، KvAP، إلى الحويصلات unilamellar العملاقة (GUVs) للطريقتين الجفاف-الإماهة - electroformation، وبمساعدة هلام التورم. في كلتا الطريقتين، وتنصهر الحويصلات unilamellar صغيرة تحتوي على البروتين معا لتشكيل GUVs التي يمكن بعد ذلك دراس?...

Video: Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

أساليب لتصحيح تسجيلات السعة المشبك من كاليكس

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

التصحيح تسجيل المشبك من القنوات ايون معبرا عنه البويضات القيطم

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

الضغط تلميع الماصات لتسجيل باتش تحسين المشبك

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

أعربت المشبك التصحيح وتقنيات الإرواء لدراسة في قنوات ايون القيطم البويضات

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

الاستراتيجية الأمثل للتعبير وترنسفكأيشن تسجيل الكهربية المؤتلف من القنوات ايون الجهد بوابات - 293T كلوة الجنين البشري في خلايا

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

قياس إزفاء الببتيد في الحويصلات الكبيرة Unilamellar

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

استكشاف الشرياني العضلات الملساء قناة البوتاسيوم Kv7 الدالة باستخدام المشبك التصحيح الكهربية وتخطيط العضل الضغط

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

إعداد الحويصلية عملاق التصوير والتصحيح، المشبك الكهربية

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

إعادة تشكيل قناة كيلو فولت في الأغشية الدهنية للدراسات الهيكلية والوظيفية

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...