Bu çalışma, Sağlık hibe, R01-HL70393 ve R01-NS060706 JCJC için Ulusal Sağlık Enstitüleri tarafından desteklenen Biyomedikal Mühendisliği profesörü Spencer T. Olin Endowment.

1. Oosit içine mRNA enjeksiyon <ol><li> Enjekte 0.05 - 50 ng mesajcı RNA Nanoject II Auto-Nanoliter Enjektör (Drummond Bilimsel Şirket, modeli 3-000-204) kullanarak Xenopus laevis oosit (Evre V-VI), in vitro transkripsiyonu da yapılmıştır . Her mRNA için bir düzine oosit enjeksiyon tekrarlayın. </li><li> Iki kez kullanarak enjekte oosit durulayın ND-96 solüsyonu (96 mM sodyum klorür (NaCl, Bay 58,44 g / mol), 2 mM potasyum klorür (KCl, Bay 74,56 g / mol), 1.8 mM kalsiyum klorür (CaCl 2 • 2H 2 O , Bay 147,02), 1 mM magnezyum klorür (MgCl 2 • 6H 2 O, Bay 203,31 g / mol), 5 mM 4 - (2-hidroksietil)-1-piperazineethanesulfonic asit (HEPES, Bay 238,31 g / mol), 2.5 mM sodyum piruvat (Bay 110,04 g / mol), 1x Penisilin-Streptomisin pH 7.6), daha sonra 18 yaşında bir kültür plaka ve inkübe koyun ° C 2 + gün. </li></ol> 2. Perfüzyon Sistemi hazırlanması <img src="/files/ftp_upload/2269/2269fig1.jpg" alt="Şekil 1" /> İllüstrasyon otomatikleştirin ValveLink 16 perfüzyon sistemi. Perfüzyon sistemi perfüzyon borulama ile perfüzyon çözümler, çözüm rezervuarların dışarı itmek için basınçlı azot kullanır ve perfüzyon kalem ve ucu. Perfüzyon çözüm her akarsuyun akış biri rezervuar valfi ve bir elektronik vana ile kontrol edilir. Bu protokol, rezervuarların bir atık toplayıcı yanı sıra bir basınç serbest bırakma mekanizması gibi basınçlı ve işlevleri değildir. (Resim üreticisinin web sitesine http://www.autom8.com adapte) <ol><li> Borulama perfüzyon kalem bağlayın. Elektronik supap kontrol açın ve elektronik vanalarının açık. </li><li> Deiyonize su ile dolu bir şırınga kullanarak her bir tüp yoluyla deiyonize (DI) su itme, borulama ve perfüzyon kalem yapışmasına neden olabilir ve hava kabarcıkları serbest olduğu sağlamak için. </li><li> Çözüm rezervuarları borulama bağlayın ve basınçlı azot uygulamak için silindir gaz valfi açık. </li><li> Rezervuar çözüm tüp doldurmak için rezervuar vanasını açın. Gerekirse çözüm kabarcıkları rezervuar valf Flick. Hortumun çözümü ile doldurulduktan sonra vanasını kapatın. </li><li> Perfüzyon ucunu su ile doldurun ve perfüzyon kalem üzerine vida. Ucu bağlarken herhangi bir kabarcıklar yakalamak için dikkatli olun. </li><li> Modelleme kil ile banyo aşamasına perfüzyon kalem sabitleyin. Perfüzyon ucu banyo alanı içinde olduğundan emin olun. Perfüzyon sistemi artık ayarlandı. </li><li> Banyo DI su ekleyin. Perfüzyon ucu suya batmış olduğundan emin olun. </li><li> Her perfüzyon çözüm perfüzyon ucu doğru geldiğini sınayın. Atık kolektör boru bağlı elektronik vanasını kapatın ve bir seferde bir perfüzyon çözeltisi (140 mM potasyum hidroksit (KOH, valfi açık M r 56.1 g / mol), 20 mM 4 - (2-hidroksi) -1 - piperazineethanesulfonic asit (Hepes, M r 238,31 g / mol), 2 mM potasyum klorür (KCl, M r 74,56 g / mol), 1 mM Etilen glikol-bis (2-aminoethylether)-N, N, N &#39;, N&#39;- tetraasetik asit (EGTA, M r 380,35 g / mol), pH metansülfonik asit (MESO 3, M r 96.1 g / mol), Ca 2 7,1-7,2 &#39;e ayarlanır + veya Mg2 + gerektiğinde) istenilen konsantrasyona eklenir . Mikroskop altında, perfüzyon ucu çıkan perfüzyon sıvısına jet gözlemliyoruz. Perfüzyon çözümü için vanayı kapatın ve atık toplayıcı valfi açık. Perfüzyon jet hemen hemen kaybolur. Perfüzyon tüm çözümler için aynı test tekrarlayın. </li><li> Doğru tüm perfüzyon çözümler akış kontrol ettikten sonra, banyo solüsyonu ile banyo (140 mM potasyum hidroksit (KOH, DI su yerine M r 56.1 g / mol), 20 mM 4 - (2-hidroksietil)-1-piperazineethanesulfonic asit (Hepes, M r 238,31 g / mol), 2 mM potasyum klorür (KCl, M r 74,56 g / mol), pH 7,1-7,2 metansülfonik asit (MESO 3, M r 96.1 g / mol)) tarafından ayarlanır. </li></ol> 3. Patch Sıkma <ol><li> İkinci kat uygulama, AgCl Ag kayıt elektrot her yama oturumda sıkma önce. Birincisi, pipet tutucu ve batığın en az 15 dakika süreyle taze çamaşır suyu içeren bir şişe elektrot teli ucu yarım elektrot teli çıkarın. Bu mevduat tel AgCl bir katman. </li><li> Elektrot teli deiyonize su ile çalkalayın ve kuru kurulayın. Sonra pipet tutucu Ag / AgCl elektrot geri yükleyin. </li><li> Headstage, banyo (referans) elektrodu bağlayın ve banyo yerleştirin. Bu kayıt için elektrotlar hazırlar. </li><li> Şimdi veri toplama için hazırlar. Bilgisayarınızı açın ve donanım anahtarı bilgisayarınızın yazıcı bağlantı noktasına takın. Heka darbe veri toplama yazılımı kullanmak için donanım anahtarı takılı olmalıdır. </li><li> Amplifikatör açın (Axon Instruments, AXOYAMA 200B) ve başlangıç ​​Heka Darbe. Stimulus Ölçek olarak, protokol dosya yükleyin ve yapılandırma ayarları. Yapılandırma ayarlarını ayarlamak için hedef test potansiyeli komut potansiyeli tam olarak eşit olduğunu güvence altına almak. Bu veri toplama ekipmanları hazırlar. </li><li> Oosit hazırlayın. Batmak sıyırma çözüm oosit (200 mM N-metil D-glucamine (NMG, M r 195,22 g / mol), 200 mM Aspartat (ve K +) (M r 133,1 g / mol), 2 mM potasyum klorür (KCl , M r 74,56 g / mol), 10 mM Etilen glikol-bis (2-aminoethylether)-N, N, N &#39;, N&#39;-tetraasetik asit (EGTA M r 380,35 g / mol), 1 mM magnezyum klorür (MgCl 2 • 6H 2 O, M r 203,31 g / mol), 10 mM 4 - (2-hidroksi)-1-piperazineethanesulfonic asit (Hepes, M r 238,31 g / mol), pH, sodyum hidroksit (NaOH, 7.4 &#39;e ayarlanır . 5 M r 40.0 g / mol) - 10 dakika vitellin membran şerit mümkün kılan plazma membran sıyırma çözüm vitellin zar ayırır. </li><li> Yavaşça, forseps, iki çift kullanarak oosit vitellin membran şerit. Devitellinized oosit az gerektiğinde son derece hassastır ve bu nedenle hareket ettirilmelidir. </li><li> Hava veya kabarcıklar devitellinized oosit dökülmesini önlemek için bakımı, banyo oosit transfer için yeterli bir çözüm ile dolu bir cam pipet kullanın. Bu kayıt için oosit hazırlar. </li><li> Yama pipetler hazırlayın. Sutter P-97 Flaming / Brown Mikropipet Çektirme içine bir cam kapiller tüp (VWR International, Kedi # 53.432-921) taktıktan sonra cam pipetler çekin. Ucu şeklini ve açılış çapı belirlemek için mikroskop altında pipetler dikkat edin. Açılış çapı 2-4 mikrometre olmalıdır. </li><li> Kapasitif akım kayıt sırasında azaltmak için düzgün bir ucu, mont, balmumu ile ucu sağlamak ve daha sonra yangın lehçe. </li><li> Pipet pipet ucu (2-hidroksi)-1-piperazineethanesulfonic asit (Hepes M r - pipet çözüm (140 mM potasyum hidroksit (KOH, M r 56.1 g / mol), 20 mM 4 içine koyarak doldurun 238,31 g / mol), 2 mM potasyum klorür (KCl, M r 74,56 g / mol), 2 mM magnezyum klorür (MgCl 2 • 6H 2 O, K r 203,31 g / mol), pH 7,1-7,2 için metansülfonik tarafından ayarlanır. asit (MESO 3, M r 96.1 g / mol)) ve piston çizimi. Bu ucu ıslatır. </li><li> Bir şırınga kullanarak pipet çözüm ile pipet tam 1 / 3 doldurun ve Ag / AgCl elektrot iç ve pipet çözüm ile temas pipetle tutucu pipet yerleştirmek. Bu yama pipet hazırlamaktadır. </li><li> Iç-dış yama hazırlanan oosit tüketim için, mikroskop altında yumurtanın açık kenarında bulabilirsiniz. Manipülatör kullanarak oosit yakın yama pipet hareket ettirin. Pipet seri direnç kaydedin. Ideal bir seri direnç yama pipet, pipet çözüm ile dolu ve banyo çözüm batmış M 1-1.5 arasında. </li><li> Direnci yaklaşık iki katına kadar oosit karşı pipet yavaşça itin. </li><li> Giga-ohm mühür elde edilinceye kadar pipet sahibine bağlı olan bir emme borusu yoluyla, ağız yoluyla membran nazik vakum uygulayın. Birkaç dakika için gerilim -30 mV tutarak yama sabitleyin. </li><li> Tersyüz olmuş bir yama, tüketim hızla oosit pipet içine daha fazla bastırıyor ve sonra yavaşça çekip yama elde etmek için. </li><li> Iç-dış yama şimdi sıkma için hazırdır. Iç-dış yama tutarak perfüzyon ucunun açılması uzaklıkta yaklaşık 100 mikron pipet hareket ettirin. </li><li> Elektronik atık toplamak için vanasını kapatın ve istenen perfüzyon çözümü için rezervuar valfi açık </li><li> Akımları karşısında yama kaydetmeye başlarsınız. Gerilim ve perfüzyon çözümleri istediğiniz gibi değiştirin. </li><li> Kayıt bittiğinde, takunya önlemek için deiyonize su ile iterek perfüzyon borulama, kalem ve bahşiş temizleyin. </li></ol> 4. Temsilcisi Sonuçlar Yama kelepçe için oosit hazırlanması sırasında, sıyırma çözüm 5-10 dakikalık tedavi vitellin vitellin membran sıyırma mümkün kılan plazma membranı, membran ayırma. Ideal bir seri direnç yama pipet, pipet çözüm ile dolu ve banyo çözüm batmış M 1-1.5 arasında. Aşağıda bir oosit membran yama yabani tip BK kanalları bir temsilci kayıt. 50 mV 50 mV artış ile 200 mV gerilim artar, ikinci adımda sol üst kare dalgalar, yama uygulanan gerilim gösterir. Aşağıda perfüzyon çözüm, nominal 0 Ca 2 + (ücretsiz [Ca 2 +] yaklaşık 0.5 nM) ilgili güncel izleri. IçindeBK kanalları açık olasılık geçerli genliği kırışıklık gerilimi ile arttığını gösterir. Sağ tarafta, aynı yama 1.0 mcM Ca 2 ile perfüze + aynı gerilim protokolü ile uygulanır. Geçerli genliği açık olasılık [Ca 2 +] ile arttığını belirterek, aynı gerilim altında daha fazla artar. <img src="/files/ftp_upload/2269/2269fig2.jpg" alt="Şekil 2" />

<ol>
	<li>Methfessel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+measurements+on+Xenopus+laevis+oocytes:+currents+through+endogenous+channels+and+implanted+acetylcholine+receptor+and+sodium+channels.">Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels.</a> Pfl&#252;gers Arch. 407, 577-588 (1986).</li><li>Toro, L., Wallner, M., Meera, P., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maxi-K+(Ca),+Unique+Member+of+the+Voltage-Gated+K+Channel+Superfamily.">Maxi-K (Ca), Unique Member of the Voltage-Gated K Channel Superfamily.</a> News Physiol. Sci. 13, 112-117 (1998).</li><li>Fettiplace, R., Fuchs, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+hair+cell+tuning.">Mechanisms of hair cell tuning.</a> Annu. Rev. Physiol. 61, 809-834 (1999).</li><li>Du, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-sensitive+potassium+channelopathy+in+human+epilepsy+and+paroxysmal+movement+disorder.">Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder.</a> Nat. Genet. 37, 733-738 (2005).</li><li>Ledoux, J., Werner, M. E., Brayden, J. E., Nelson, M. T., T, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-activated+potassium+channels+and+the+regulation+of+vascular+tone.">Calcium-activated potassium channels and the regulation of vascular tone.</a> Physiology (Bethesda). 21, 69-78 (2006).</li><li>Salkoff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-conductance+potassium+channels+of+the+SLO+family.">High-conductance potassium channels of the SLO family.</a> Nat. Rev. Neurosci. 7, 921-931 (2006).</li><li>Shi, J., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Mg2++enhances+the+function+of+BK-type+Ca2+-activated+K++channels.">Intracellular Mg2+ enhances the function of BK-type Ca2+-activated K+ channels.</a> J. Gen. Physiol. 118, 589-606 (2001).</li><li>Magleby, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+mechanism+of+BK+(Slo1)+channels:+so+near,+yet+so+far.">Gating mechanism of BK (Slo1) channels: so near, yet so far.</a> J. Gen. Physiol. 121, 81-96 (2003).</li><li>Latorre, R., Brauchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+conductance+Ca2+-activated+K++(BK)+channel:+activation+by+Ca2++and+voltage.">Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage.</a> Biol. Res. 39, 385-401 (2006).</li><li>Cox, D. H., Cui, J., Aldrich, R. W., W, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+gating+of+a+large+conductance+Ca-activated+K++channel.">Allosteric gating of a large conductance Ca-activated K+ channel.</a> J. Gen. Physiol. 110, 257-281 (1997).</li><li>Cui, J., Aldrich, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+linkage+between+voltage+and+Ca2+-dependent+activation+of+BK-type+mslo1.">Allosteric linkage between voltage and Ca2+-dependent activation of BK-type mslo1.</a> K+ channels. Biochemistry. 39, 15612-15619 (2000).</li><li>Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Slo1+BK+channels+by+Mg2++coordinated+between+the+voltage+sensor+and+RCK1.">Activation of Slo1 BK channels by Mg2+ coordinated between the voltage sensor and RCK1.</a> 15, 1152-1159 (2008).</li><li>Lee, U. S., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term={beta}+subunit-specific+modulations+of+BK+channel+function+by+a+mutation+associated+with+epilepsy+and+dyskinesia.">{beta} subunit-specific modulations of BK channel function by a mutation associated with epilepsy and dyskinesia.</a> J. Physiol. , 587-1481 (2009).</li></ol>

Oosit ifade sistemin endojen kanalları nispeten düşük arka nedeniyle voltaj bağımlı iyon kanallarının elektrofizyolojik karakterizasyonu için idealdir. Ayrıca, geçici bir ifade sistem olduğundan, bu kanalların mutagenez çalışma yerine etkili bir yöntem sağlar. Ancak, oosit ifade sistemi memeli hücrelerinde farklı olduğunu belirtmek gerekir, böylece farklı alt birimden post-translasyonel modifikasyonu ve dernek farklılıklar olabilir. Ayrıca, lipid konsantrasyonu kanalın fonksiyonel özellikler...

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<td>Nanoject II Auto-Nanoliter Injector</td>
<td>&nbsp;</td>
<td>Drummond Scientific Company</td>
<td>3-000-204</td>
<td>&nbsp;</td>
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<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Perfusion System and Electronic Controller</td>
<td>&nbsp;</td>
<td>AutoMate Scientific, Inc.</td>
<td>ValveLink 16</td>
<td>Inner diameter of perfusion tip: 100 microns</td>
</tr>
<tr>
<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>CKX31</td>
<td>&nbsp;</td>
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<td>Glass Pipettes</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>53432-921</td>
<td>&nbsp;</td>
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<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co.</td>
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<td>Amplifier</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>AXOPATCH 200B</td>
<td>&nbsp;</td>
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<td>Computer Interface</td>
<td>&nbsp;</td>
<td>INSTRUTECH Corporation</td>
<td>ITC-18</td>
<td>&nbsp;</td>
<tr>
<td>Headstage</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>CV 203BU</td>
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patch clamp and perfusion techniques for studying ion channels expressed in xenopus oocytes

Burada sunulan protokol büyük iletkenlik, voltaj ve Ca 2 + ile aktive olan K + (BK) kanalları aktivasyon çalışma için tasarlanmıştır. Protokol, aynı zamanda, diğer iyon kanalları ve nörotransmitter reseptörlerinin 1 için yapı-işlev ilişkisi çalışma için kullanılan olabilir. BK kanalları farklı dokularda yaygın olarak ifade edilir ve düz kas kasılması düzenlenmesi, iç saç hücrelerinin frekans ayarı ve nörotransmitter sürüm 2-6 düzenlenmesi de dahil olmak üzere birçok önemli fizyolojik fonksiyonları, suçlanmıştır . BK kanalları membran depolarizasyon ve hücre içi Ca 2 + ve Mg 2 + 6-9 tarafından aktive edilir. Bu nedenle, protokol, membran voltaj ve hücre içi bir çözüm kontrol etmek üzere tasarlanmıştır. Bu protokol, BK kanalları mesajcı RNA 2-5 gün inkübasyon 18 ° C 10-13 takip Xenopus laevis oosit (Evre V-VI) enjekte edilir. Tek veya birden fazla BK kanalları içeren Membran yamalar yama klemp teknikleri 10-13, iç-dış yapılandırma ile eksize. Yama intraselüler tarafı, farklı koşullar altında kanal aktivasyon incelenir, böylece kayıt sırasında istenen çözümleri ile perfüze. Özetlemek gerekirse, BK kanalları mRNA oosit zarı kanal proteinleri ifade Xenopus laevis oosit içine enjekte edilir; yama klemp teknikleri, kontrollü gerilim ve hücre içi çözümleri altında kanallar aracılığıyla akıntılar kaydetmek için kullanılır .

Watch this Scientific Journal Video about Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes at JoVE.com

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

BK kanalları İyonik akım yama klemp teknikleri kullanılarak kaydedildi. BK kanalları ifade edilmiştir.<em&gt; Xenopus oositleri</em&gt; Mesajcı RNA enjekte edilerek. Yama kelepçe kayıtları sırasında hücre içi bir çözüm perfüzyon sistemi tarafından kontrol edilir.

İyon Kanalları incelenmesi için yama Kelepçe ve Perfüzyon Teknikleri cinsinden ifade edilir Xenopus Oosit

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

patch-clamp-perfusion-techniques-for-studying-ion-channels-expressed

Research

JoVE Journal

Biology

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

20.9K Görüntüleme.  Washington University in St. Louis. BK kanalları İyonik akım yama klemp teknikleri kullanılarak kaydedildi. BK kanalları ifade edilmiştir. Xenopus oositleri Mesajcı RNA enjekte edilerek. Yama kelepçe kayıtları sırasında hücre içi bir çözüm perfüzyon sistemi tarafından kontrol edilir.

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

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.

Kaliks Patch Kelepçe Kapasitans Kayıtlar Yöntemleri

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

Biyoloji

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.

Xenopus Oosit ifade İyon Kanalları Patch Kelepçe Kaydı

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.

Geliştirilmiş Patch-kelepçe kayıt için Basınç-parlatma Pipetler

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

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Xenopus laevis Oosit, Mikroenjeksiyon

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Mitoz incelenmesi için Mikroenjeksiyon Teknikleri Drosophila melanogaster Sinsityal Embriyo

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Görselleştirme RNA Yerelleştirme Xenopus Oosit

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

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.

İyon Kanalları ve Fonksiyonel Analiz Mutagenez Heterologously Memeli Hücreleri cinsinden ifade edilir

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 ...

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.

Görüntüleme ve Patch-Kelepçe Elektrofizyoloji için dev Liposome Hazırlık

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

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

<em&gt; Xenopus</emFluorometri ile&gt; Yumurta Kes-açık Vazelin Gap Gerilim-kelepçe Tekniği

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Tek kanallı bir hücre-bağlı Patch-clamp kayıt

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...