이 작품은 건강 보조금 R01 - HL70393 및 JCJC에 R01 - NS060706 국립 연구소에 의해 지원되었다 스펜서 T. 올린 엔다우먼트에 의생명 공학 교수이다.

1. Oocytes에 mRNA의 분사 <ol><li> 0.05을 주사 - Nanoject II 자동 Nanoliter 주입기 (드루먼드 과학 회사, 모델 3-000-204)를 사용하여 Xenopus laevis의 oocytes (무대 V - VI)로 체외에서 베꼈는데했습니다 50 NG 전령 RNA합니다. 각각의 mRNA에 대한 열 oocytes에서 주사를 반복합니다. </li><li> 두 번 사용하여 주입 oocytes 씻어 ND를 - 96 솔루션 (96 MM 나트륨 염화물 (NaCl, 미스터 58.44 g / 몰), 2 MM의 칼륨 염화물 (KCl, 미스터 74.56 g / 몰), 1.8 MM의 염화칼슘 (CaCl 2 • 2 시간 2 O 씨 147.02), 1 MM 마그네슘 염화물 (MgCl 2 • 6H 2 O 씨, 203.31 g / 몰), 5 MM 4 - (2 - 히드록시 에틸) - 1 - piperazineethanesulfonic 산 (HEPES, 미스터 238.31 g / 몰), 2.5 MM 나트륨 pyruvate (씨 110.04 g / 몰) 및 1X 페니실린 - 스트렙토 마이신. 산도 7.6)은 다음 18 문화 판과 부화에 배치 ° 2 +의 일 C. </li></ol> 2. 재관류 시스템 준비 <img src="/files/ftp_upload/2269/2269fig1.jpg" alt="그림 1" /> 자동화 ValveLink 16 재관류 시스템의 일러스트. 재관류 시스템은 재관류의 tubings 통해 솔루션 저수지의 재관류 솔루션을 밖으로 밀어 가압 질소를 사용하고, 재관류의 연필과 팁합니다. 재관류 솔루션의 각 스트림의 흐름을 한 저수지 밸브 하나의 전자 밸브에 의해 제어됩니다. 이 프로토콜에서는, 저수지 중 하나는 폐기물 수집뿐 아니라 압력 릴리스 메커니즘으로 가압 및 기능하지 않습니다. (사진은 공급 업체의 웹 사이트 http://www.autom8.com에서 적응) <ol><li> 재관류의 연필에 tubings를 연결합니다. 전자 밸브 컨트롤러의 전원을 켜고 전자 밸브를 엽니다. </li><li> 탈이온수 가득한 주사기를 사용하여 각 튜브를 통해 deionized (DI) 물을 밀어 tubings 및 재관류의 연필이 나막신과 공기 거품없는 것을 확신합니다. </li><li> 솔루션 저수지에 tubings를 연결하고 가압 질소를 적용하는 실린더 가스의 밸브를 엽니다. </li><li> 저수지 솔루션 튜빙을 채우기 위해 저수지 밸브를 엽니다. 필요한 경우 솔루션에 거품을 제거하기 위해 저수지 밸브를 버려. 튜빙이 솔루션으로 가득 후 밸브를 닫습니다. </li><li> 물로 세례 팁을 입력하고 재관류의 연필에 그것을 나사. 팁을 연결할 때 모든 거품을 함정하지 않도록주의하십시오. </li><li> 모델링 점토를 사용하여 목욕 단계에 재관류의 연필을 수정. 재관류 팁는 목욕 공간에 있는지 확인하십시오. 재관류 시스템은 지금 설정됩니다. </li><li> 욕조에 물을 DI를 ​​추가합니다. 재관류 팁이 물 속에 빠져들 있는지 확인하십시오. </li><li> 각 재관류 솔루션이 제대로 재관류 팁 나오는 것 테스트합니다. 쓰레기 수집기의 튜브에 연결된 전자 밸브를 닫고 한 번에 하나의 재관류 솔루션 (140 MM의 칼륨 수산화 (코에 대한 밸브를 열어 M R 56.1 g / 몰), 20 MM 4 - (2 - 히드록시 에틸) -1 - piperazineethanesulfonic 산 (HEPES, M R 238.31 g / 몰), 2 MM의 칼륨 염화물 (KCl, M R 74.56 g / 몰), 1 MM 에틸렌 글리콜 - 비스 (2 - aminoethylether) - N, N, N &#39;, N&#39; - tetraacetic 산 (EGTA, M R 380.35 g / 몰)는 산도가 methanesulfonic 산성 (MeSO 3, M R 96.1 g / 몰), CA 2 7.1-7.2하도록 조정됩니다 +이나 Mg2 + 필요할 때) 원하는 농도에 추가됩니다.이 현미경, 재관류 팁 나오는 재관류 유체의 제트를 관찰합니다. 재관류 솔루션을위한 밸브를 닫고 쓰레기 수집기를 위해 밸브를 엽니다. 재관류 제트는 거의 사라질 것입니다. 모든 재관류 솔루션에 대해 동일한 시험을 반복합니다. </li><li> 제대로 모든 재관류 솔루션 흐름을 확인했으면, 목욕 솔루션 욕조 (140 MM의 칼륨 수산화 (코에서 DI 워터를 교체 M R 56.1 g / 몰), 20 MM 4 - (2 - 히드록시 에틸) - 1 - piperazineethanesulfonic 산 (HEPES, M R 238.31 g / 몰), 2 MM의 칼륨 염화물 (KCl, M R 74.56 g / 몰)는 산도가 methanesulfonic 산성 (MeSO 3, M R 96.1 g / 몰))에 의해 7.1-7.2로 조정됩니다. </li></ol> 3. 패치 클램핑 <ol><li> Recoat AgCl과 자세 기록 전극은 각 패치는 세션을 클램핑하기 전에. 첫째, 피펫 홀더 및 잠수함 적어도 15 분 동안 신선한 표백제가 들​​어있는 유리병에 전극 와이어의 끝에 절반의 전극 와이어를 제거합니다. 와이어에 AgCl의이 예금은 레이어. </li><li> 탈이온수으로 전극 와이어를 씻어 건조 얼룩. 다음 피펫 홀더에 다시 자세 / AgCl 전극을 설치합니다. </li><li> headstage에 목욕 (참조) 전극을 연결하고 목욕에 놓으십시오. 이것은 기록을위한 전극을 준비합니다. </li><li> 이제 데이터 수집을위한 준비. 컴퓨터를 켜고 컴퓨터의 프린터 포트에 하드웨어 키를 꽂습니다. 당신이 HEKA 파동있는 데이터 수집 소프트웨어를 사용하는 하드웨어 키를 연결해야합니다. </li><li> (액슨 인 스트 루먼트, AXO 앰프에 스위치패치 200B) 및 시작 HEKA 펄스. 같은 자극 규모로, 프로토콜 파일을로드하고 구성 설정을 조정합니다. 구성 설정을 조정 목표는 테스트 가능성이 명령 잠재 정확히 같다는 것을 보증하는 것입니다. 이것은 데이터 수집 장비를 준비합니다. </li><li> oocytes를 준비합니다. 스트립 솔루션에서 잠수함 oocyte (200 MM N - 메틸 - D - glucamine (NMG, M R 195.22 g / 몰), 200 MM의 Aspartate (NO K +) (M R 133.1 g / 몰), 2 MM 염화칼륨 (KCl , M R 74.56 g / 몰), 10 MM 에틸렌 글리콜 - 비스 (2 - aminoethylether) - N, N, N &#39;, N&#39; - tetraacetic 산 (EGTA, M R 380.35 g / 몰), 1 MM 마그네슘 염화물 (MgCl 2 • 6H 2 O, M R 203.31 g / 몰), 10 MM 4 - (2 - 히드록시 에틸) - 1 - piperazineethanesulfonic 산 (HEPES, M R 238.31 g / 몰)는 산도가 (NaOH, 수산화 나트륨에 의해 7.4로 조정됩니다 5 M R 40.0 g / 몰) -. 10 분 스트립 솔루션은 가능한 vitelline 막 제거하기 위해서 만드는 플라즈마 막에서 vitelline 멤브레인를 분리합니다. </li><li> 부드럽게 포셉 두 쌍을 사용하여 oocyte에서 vitelline 막을 스트립. devitellinized oocyte 필요로 작은로 매우 연약하기 때문에 이동해야합니다. </li><li> 공기 또는 거품에 devitellinized oocyte를 노출되지 않도록 돌보는 것은 욕조에 oocyte를 전송하기에 충분한 해결책 가득한 유리 피펫을 사용합니다. 이 기록에 대한 oocyte를 준비합니다. </li><li> 패치 pipettes 준비. 서터 P - 97 플라밍 / 브라운 Micropipette 풀러에 유리 모세관 (VWR 인터내셔널, 고양이 # 53432-921)을 삽입 후 유리 pipettes을 당기세요. 팁 모양과 개방 직경을 결정하는 현미경 pipettes을 준수. 여는 직경 2-4 micrometers해야합니다. </li><li> 녹화 중에 용량성 전류를 감소하고 부드러운 팁, 코팅 왁스로 끝부분을 보장하기 위해 다음을 해고 - 폴란드어. </li><li> (2 - 히드록시 에틸) - 1 - piperazineethanesulfonic 산 (HEPES, M R - 피펫 솔루션 (140 MM의 칼륨 수산화 (코, M R 56.1 g / 몰), 20 MM 네 안에있는 피펫의 팁을 배치하여 피펫 팁을 작성 238.31 g / 몰), 2 MM의 칼륨 염화물 (KCl, M R 74.56 g / 몰), 2 MM 마그네슘 염화물 (MgCl 2 • 6H 2 O, M R 203.31 g / 몰)는 산도가 methanesulfonic으로 7.1-7.2로 조정됩니다 산성 (MeSO 3, M R 96.1 g / 몰))와 플런저를 그리기. 이것은 끝에 오줌 싼다. </li><li> 주사기를 사용하여 피펫 솔루션 피펫 전체 1 / 3 입력하고 자세 / AgCl 전극 내부 피펫 솔루션 문의와 피펫 홀더에 피펫을 놓으십시오. 이것은 패치 피펫을 준비합니다. </li><li> 준비 oocyte에서 소비세 내부 아웃 패치를하려면, 현미경 oocyte의 명확한 가장자리를 찾으십시오. 조작하는 사람을 사용하여 oocyte 가까운 패치 피펫을 이동합니다. 피펫의 일련 저항을 기록합니다. 패치 피펫의 이상적인 직렬 저항은 피펫 솔루션 가득, 욕실 솔루션에 빠져들 때 MΩ 1-1.5 사이입니다. </li><li> 저항이 약 두 배로 때까지 천천히 oocyte에 대한 피펫 밀어. </li><li> 기가 - 옴 밀봉을 얻을 때까지 피펫 홀더에 연결된 흡입 튜브를 통하여 입안으로 멤브레인에 부드러운 흡입을 적용합니다. 몇 분 동안은 전압 -30 MV에서 개최하여 패치를 안정화. </li><li> 빨리 oocyte에 추가로 피펫을 밀고하고 부드럽게 그것을 철수하여 내부 아웃 패치, 소비세에게 패치를 구하십시오. </li><li> 내부 아웃 패치 클램핑 이제 준비가되었습니다. 재관류 팁의 개통 떨어져 약 100 미크론에 내부 아웃 패치를 들고 피펫 이동합니다. </li><li> 폐기물 수집에 대한 전자 밸브를 닫고 원하는 재관류 솔루션을위한 저수지의 밸브를 열어 </li><li> 패치를 통해 전류를 녹음 시작합니다. 원하는대로 전압과 재관류 솔루션을 변경합니다. </li><li> 당신은 녹음을 완료하면, 나막신을 피하기 위해 탈이온수을 통해 추진하여 재관류의 tubings, 연필 및 팁을 청소하십시오. </li></ol> 4. 대표 결과 패치 클램프에 대한 oocytes의 준비 기간 동안, 스트립 솔루션의 50-10 분 치료 vitelline 막가 벗겨진 가능하게 플라즈마 막에서 vitelline 멤브레인를 분리합니다. 패치 피펫의 이상적인 직렬 저항은 피펫 솔루션 가득, 욕실 솔루션에 빠져들 때 MΩ 1-1.5 사이입니다. 다음은 oocyte 막 패치에 야생 형 BK 채널의 대표적인 기록이다. 50 MV 50 MV의 증가 200 MV 전압 증가의 두 번째 단계 - 왼쪽 상단에서 광장 파도 패치에 적용된 전압을 나타냅니다. 아래 재관류 솔루션 공칭 0 칼슘 2 + (무료 [칼슘 2 +]는 약 0.5 nm의)이 해당 현재의 흔적입니다. 에현재 진폭의 포복 절도는 BK 채널의 개방 가능성이 전압 증가시킬 수있다는 것을 나타냅니다. 오른쪽에서 동일한 패치는 1.0 μm의 칼슘 2 perfused입니다 + 같은 전압 프로토콜 적용하면. 현재 진폭은 오픈 확률 [칼슘 2 +]로 증가시킬 수있다는 것을 나타내는 같은 전압에 따라 더 증가합니다. <img src="/files/ftp_upload/2269/2269fig2.jpg" alt="그림 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>

관심 없음 충돌 선언하지 않습니다.

oocyte 표현 시스템은 내생 채널 상대적으로 낮은 배경에 의한 전압 - 의존 이온 채널의 electrophysiological 특성에 이상적입니다. 또한, 그것이 일시적 표현 시스템이기 때문에, 그것은 이러한 채널의 돌연변이 유발 연구를 수행하는 효율적인 방법을 제공합니다. 그러나, 그것이 oocyte 표현 시스템은 포유 동물 세포에서 다른 것을 지적해야하므로 다른 subunits와 포스트 translational 수정 및 결사의 차이가?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Nanoject II Auto-Nanoliter Injector</td>
<td>&nbsp;</td>
<td>Drummond Scientific Company</td>
<td>3-000-204</td>
<td>&nbsp;</td>
<tr>
<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>Glass Pipettes</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>53432-921</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co.</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Amplifier</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>AXOPATCH 200B</td>
<td>&nbsp;</td>
<tr>
<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

patch clamp and perfusion techniques for studying ion channels expressed in xenopus oocytes

여기에 제시 프로토콜은 대형 전도, 전압 및 칼슘 2 + - 활성 K + (BK)이 채널의 활성화를 연구하기 위해 설계되었습니다. 프로토콜은 다른 이온 채널과 신경 전달 물질 수용체 1 구조 - 기능 관계를 연구하는 데 사용할 수 있습니다. BK 채널 널리 여러 조직에서 표현되고 평활근의 수축 규제, 내부 세포의 주파수 튜닝 및 신경 전달 물질 릴리스 2-6 규제 등 많은 생리 기능에 연루되어있다. BK 채널 막에 의해 탈분극 및 세포내 칼슘 2 +와 MG 2 + 6-9로 활성화됩니다. 따라서 프로토콜은 막 전압과 세포내 솔루션을 모두 제어할 수 있도록 설계되었습니다. 이 프로토콜에서는 BK 채널 메신저 RNA는 18 ° C 10-13에서 부화 후 2-5 일 뒤에 Xenopus laevis의 oocytes (무대 V - VI)에 주입합니다. 하나 또는 여러 개의 BK 채널을 포함하는 막 패치는 내부 아웃 구성을 패치 클램프 기법 10-13 사용에 excised됩니다. 패치의 세포 측면은 서로 다른 조건에서 채널 정품 인증 심사 수 있도록 녹음 중 원하는 솔루션을 perfused 있습니다. 요약하면, BK 채널의 mRNA는 oocyte 막에 채널 단백질을 표현하는 Xenopus laevis의 oocytes로 주입되며 패치 클램프 기법 제어 ​​전압과 세포내 솔루션에서 채널을 통해 흐르는 전류를 기록하는 데 사용됩니다.

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 채널 이온 전류는 패치 클램프 기법을 사용하여 기록됩니다. BK 채널로 표현됩니다<em&gt; Xenopus의 oocytes</em&gt; 메신저 RNA를 주입하여. 패치 클램프 레코딩하는 동안 세포 솔루션은 재관류 시스템에 의해 제어됩니다.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 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 ...

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 조회수.  Washington University in St. Louis. BK 채널 이온 전류는 패치 클램프 기법을 사용하여 기록됩니다. BK 채널로 표현됩니다 Xenopus의 oocytes 메신저 RNA를 주입하여. 패치 클램프 레코딩하는 동안 세포 솔루션은 재관류 시스템에 의해 제어됩니다.

비디오: 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.

칼리에서 패치 클램프 커패시턴스 레코딩을위한 방법

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

연구

생물학

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 Oocytes의 표현 이온 채널 패치 클램프 녹음

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.

향상된 패치 - 클램프 녹음 압력 연마 Pipettes

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 Laevis의 Oocytes의 Microinjection

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.

의 유사 분열 유학을위한 Microinjection 기법 Drosophila melanogaster Syncytial 엠브료

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.

머릿속 RNA 현지화에서 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 ...

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.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

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.

이미징 및 패치 클램프 전기 생리학에 대한 거대한 리포좀 준비

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; Xenopus의</em형광 측정으로&gt; 난자 절개 바셀린 갭 전압 클램프 기술

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.

1 채널의 세포 부착 된 패치 클램프 녹음

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