This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).

1. 지질 준비 <ol><li> 투명 유리 튜브로 원하는 양의 지질 나누어지는. 사용 E. 콜라이 극성 지질 추출물하지만 대부분 지질 조성물을 사용할 수있다. 지질이 분말 형태 인 경우, 20 ㎎ / ㎖의 농도로 이들을 클로로포름에 재현 탁. 모든 용매가 증발 할 때까지 N 2 가스에서 RT에서 지질을 건조. </li><li> 펜탄의 지질을 재현 탁 클로로포름의 남은 흔적을 제거하기 위해 다시 가스 N 2를 일정하게 건조시킨다. 드라이 모든 용매를 증발 할 때까지. 건조 동안 지질이 아래쪽 형성 치밀한 질량보다는 튜브의 아래쪽을 덮는 제 균일 한 막 분포되도록 부드럽게 회전 튜브. </li><li> 재구성 버퍼 함유 세제를 추가 (300 mM의 KCl을 50 mM의 시트르산, 25 mM의 K 2 HPO 4의 pH 4.5, 35 mM의 CHAPS)은 지질을 용해. 단백질이 CHAPS에 가난하게 허용하는 경우이 단계에서 다른 중성 세제를 사용하십시오. </li><li> Resuspen물 목욕 초음파기를 사용하여 선명도 지질 거라고. 짧은 (30 초 - 1 분)와 짧은 (30 초 - 1 분) 펄스를 최대 전력에서 물 초음파기 목욕의 중심에 튜브를 담가 일시 중지합니다. 이 솔루션은 명확하게해야한다. 주 : 최종 명확성 정도 달성​​ 사용될 특정 지질 조성에 의존한다. 심지어 동일한 명목상 지질이 단계 중 일부 뱃치 간 변이는 것도 가능하다. 잔류 헤이즈를 원심 분리에 의해 제거 될 수 현탁액 중의 큰 입자에 의한 광의 산란에 기인한다. </li><li> 20 ~ 60 분 동안 실온에서 재현 탁 지질을 품어. </li></ol> 2. Proteoliposome 형성 주 : 몇 가지 전략은 리포좀으로 세제 용해 된 단백질을 삽​​입하기 위해 사용될 수있다. CLC-EC1은 투석은 잘 작동하기 때문에 선택 6,9,10의 방법이다. <ol><li> 로 정제 된 단백질을 추가 원하는 단백질로 지질 μg의 O를 표현 (P / L) 비율 (<html> F 단백질 / mg의 지질). P / L 비의 선택은 실험의 목적에 의존한다. <ol><li> 목표는 관심있는 단백질의 활성을 평가 높은 P에 재구성하는 경우 각 리포좀 단백질의 여러 복사본을 포함합니다 / L&#39;들, 신호를 최대화한다. 각 소포 평균 한 단백질에 포함되도록 활성 및 / 또는 단백질의 단일 전송 레이트를 수치화하기 위해, 낮은 P / L&#39; s의 포아송 희석 정권 재구성한다. 각 체제를 사용하는 경우보다 깊이 분석을위한 7 단계 및 토론을 참조하십시오. </li></ol></li><li> 다양한 섭생에 대한 최적의 P / L 값을 결정하려면, 8,11-13 단백질 농도의 함수로서 활성의 완전한 적정을 수행한다. 그러나, 활성 단위의 분자량 (MW가 kDa의 표시)하는 모든 단백질을 다음 P / ℓ를 초기 값을 절반으로 사용될 수있는 공지되어있다 : PG &quot;/&gt; <img alt="식 (2)" fo:content-width="1.5in" src="/files/ftp_upload/52369/52369eq2.jpg" /></li><li> 미리 유체 접촉 투석 튜브 또는 카세트에 단백질과 지질 혼합물을로드합니다. 500-1,000x 지질 재구성 버퍼의 양을 함유하는 비이커에 투석 장치를 배치 (즉, 지질 부유 1 ㎖를 1 L 버퍼) 부드러운 교반하에. <ol><li> 변경 버퍼 간격&gt; 3 시간에 3-4 회. 모든 솔루션 변화에서, 카세트 / 튜브 및 증류수로 비커를 씻어 신선한 재구성 버퍼 비커를 입력합니다. 솔루션 변화 1-2 O / N 간격을 포함합니다. 용액 변경의 정확한 수와 기간이 사용될 정확한 지질 및 세제에 의존한다. </li></ol></li><li> 마지막 단계가 완료된 후, 투석 용기로부터 제거 리포좀 튜브들을 분취 플래시 -80 ° C에서 액상이 N 또는 동결 그들을 동결. </li></ol> 3. 녹화 환경 &lt;P 클래스 = &quot;jove_content&quot;&gt; 참고 : 녹화 설정 (그림 2A)이 두 개의 챔버 (평면 바닥 실린더, ~ 3-4 ml의 부피), CL로 구성 - 전극, 아날로그와 pH 측정기 (아래 참조) 디지털 전기 출력, 디지타이저 및 적절한 수집 소프트웨어와 컴퓨터 나. <ol><li> pH 미터 전극을, 컴퓨터에 연결되어 디지타이저 pH 미터의 출력 - CL을 연결한다. 하나의 챔버에서 기준 전극과 다른 (그림 2B)에서 레코딩 와이어를 놓습니다. 주 : 칼 ΔV가 양수 (단계 6.4 및 7.2 참조)의 경우에는 와이어 극성이 맞는지. </li><li> 기록 챔버 (그림 1B)에서 교반 막대와 볶음 접시에 챔버를 놓습니다. 교반 막대를 전극에 접촉하지 않도록하십시오. </li><li> 한천 다리 (그림 2B) (100 mM의 KCl을 2 % 아가로 오스)를 사용하여 챔버를 연결합니다. 100 mM의 KCl을의 한천 다리를 저장합니다. </리&gt; </ol> CL 4. 준비 - 전극 <ol><li> 과거와 -possibly- 작동하지 않는 pH 전극을 가져 가라. 완전히 실버 와이어를 공개 유리막 브레이크. </li><li> 조심스럽게 메스 블레이드를 사용하여 실버 와이어에서 코팅을 제거합니다. H 2 O와의 EtOH 풍부한 양을 사용하여 와이어를 청소합니다. </li><li> 전선까지 50ml을 (또는 표백제)의 포화 용액에 장소의 AgCl의 균일 한 어두운 코트 덮여있다. </li></ol> 단일 층 소포 5. 준비 <ol><li> 실험 전에 밤, 외부 버퍼 15 ㎖ (1 ㎜의 KCl, SO 4, 25 mM의 구연산, 산도 4.5 150 mM의 K 2) 흔들리는 부드러운와가 (저어하지 않음)에 세파 덱스 G-50 구슬의 1g 팽창 사용하기 전에 실온에서 최소 3 시간. 각 실험은 부어 구슬 ~ 3 ㎖가 필요합니다. 기껏해야 몇 일 동안 4 ° C에서 부어 구슬을 유지합니다. </li><li> 리포좀을 실온에서 해동하는 동안, 각 열 ~ 3m 부어부어 G-50 구슬의 리터. RT 중력 흐름에 의해 그들을 말리면; 이것은 일반적으로 1 ~ 2 분 걸립니다. </li><li> 0.4 m 테플론 차단을 사용하여 미니 압출기를 통해 테오에게 11 번을 돌출시켜 단일 층 소포를 준비합니다. </li><li> 플라스틱 둥근 바닥 튜브에 열을 놓습니다. 여분의 용액을 제거하기 위해 열을 스핀. 임상 원심 분리기에서 1,400 XG에 20 ~ 30 초 동안 원심 분리기. </li><li> 13 X 100mm 유리 튜브의 흐름을 통해, 장소 열을 버리고 컬럼에 돌출 된 소포의 100 μl를 추가합니다. 임상 원심 분리기 500 XG에서 1 분 동안 열을 스핀. </li><li> 단계 6.5의 기록 챔버에 첨가한다 관류 ~ 200 μL의 수집. </li></ol> 6. 유출 측정 <ol><li> 레퍼런스 (접지) 100mm의 챔버의 KCl 2㎖ 및 외부 버퍼의 1.8 ml의 배치 (1 mM의 KCl을을 150 mM의 K 2 SO 4, 25 mM의 시트르산, pH를 4.5) 기록 챔버. 내부 사이의 약간의 삼투압의 불균형외부 솔루션에 영향을주지 않습니다 </li><li> 취득 프로그램을 시작합니다. 이 몇 분 정도 걸릴 수 있습니다, 기준이 평형을 보자. </li><li> 신호를 안정적으로 기준선 (리포좀 준비하고 건조 컬럼)에 도달하면 (도 3a)을 촬영을 시작. </li><li> 시스템 (그림 2A)를 교정 된 10 mM의 KCl 용액 15 μl를 추가합니다. </li><li> 리포좀을 추가합니다. 테오 (그림 3A)에서 - 작은 점프 인해 외부 CL의 불완전한 제거로 볼 수 있습니다. 베이스 라인이 안정 될 때까지 기다립니다. </li><li> 발리 노마 이신 (Valinomycin) 추가 (1 mg의 EtOH 중 / ㎖에서 1 μL)은 유출 (그림 3A)를 시작합니다. </li><li> 유출은 시간 물론 고원까지 (그림 3A)를 실행하자. </li><li> 모든 리포좀 (그림 3A)을 용해 할 수있는 외부 버퍼에 준비 1.5 M β-옥틸 글루코 시드 (β-OG)의 40 μl를 추가합니다. 촬영을 완료합니다. </li></ol> 7. 데이터nalysis <ol><li> ASCII 또는 텍스트 형식에서 추적 파일을 내보내고 선택의 분석 프로그램으로 가져옵니다. </li><li> 다음 사항 (도 3A)에서 전압을 측정한다 : 기록의 시작시 (0 V); 교정 펄스 (CAL V)을 첨가 한 후; 리포좀 (리포의 V)을 첨가 한 후; 유출 (V 지느러미)의 끝에; 세제 (V 어린 아이)을 첨가 한 후, 기준 전압 그래서 V 0 = 0. </li><li> 실험의 값을 결정 네른스트 - 플랑크 식을 사용하여 <img alt="식 (3)" fo:content-width=".3in" src="/files/ftp_upload/52369/52369eq3.jpg" /> 측정하여 <img alt="수학 식 4" fo:content-width="3in" src="/files/ftp_upload/52369/52369eq4.jpg" /> 여기서 권 칼 μL의 교정 펄스의 부피로 표현 된 실험의 시작에서 챔버 체적은 1800이다1 - 교정 펄스와 mm 단위로 외부 버퍼의 농도 ΔV CAL은 교정 펄스 후에 측정 MV에서 점프입니다 난, [CL] CAL /에서하면 CL 있습니다. 참고 : α의이 경험적인 결정은 세 가지 목적을 수행합니다 : 그것은 시스템이 제대로 응답하는지 보장, 실험 사이에 악기의 응답의 일관성의 측정을 제공뿐만 아니라 실수는 솔루션 결정에 만든되지 않았 음을 확인합니다. </li><li> 다음 각 단계 후에 CL 농도를 계산한다 : <img alt="식 (5)" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq5.jpg" /> <img alt="식 (6)" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq6.jpg" /> <img alt="식 (7)" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq7.jpg" /> 400분의 3 인자는 2000 μL의 최종 부피로 15 μL 교정 펄스의 희석에서 온다. </li><li> Calcul각 단계, 지느러미 ΔCl의 사러가 / / TOT에서 [CL]의 변화를 먹었다. </li><li> 와 CL (t)에 V (t)를 변환 <img alt="식 (8)" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq8.jpg" /> 직접 중요한 지점에서 [CL] 값을 결정하고 내부 통제로 계산 된 값으로 비교합니다. </li><li> 추적을 정상화 CL 확인해의 리포 = 0, CL 확인해 무비 = 1 그래서 <img alt="식 (9)" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq9.jpg" /></li><li> 하기 식 유출 경시 맞는 : <img alt="식 (10)" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq10.jpg" /> CL 실험적으로 측정함으로써 결정된다 누설 - - L는 Cl의 비율이다 τ와 동일한 조건에서 제조 된 무 단백질 리포좀으로부터 유출이 공정의 시간 상수이다. </li><li> V, 초기 속도를 계산한다 : 여기서 ΔCl 핀은 밀리몰의 표현 및 V는 μL의 챔버의 체적이다. </li><li> 전송 속도 / 전도도를 계산 <img alt="식 (12)" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq12.jpg" /> P는 P / L이고, MW는 활성 착물 ​​kDa의 분자량으로 표현 </li></ol>

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A., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+purified+human+Hv1+H++channels.">Functional reconstitution of purified human Hv1 H+ channels.</a> Journal of Molecular Biology. 387, 1055-1060 (2009).</li><li>Terashima, H., Picollo, A., Accardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+TMEM16A+is+sufficient+to+form+Ca2++activated+Cl-+channels.">Purified TMEM16A is sufficient to form Ca2+ activated Cl- channels.</a> Proc Natl Acad Sci U S A. 110, 19354-19359 (2013).</li><li>Malvezzi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-dependent+phospholipid+scrambling+by+a+reconstituted+TMEM16+ion+channel.">Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel.</a> Nature Communications. 4, 2367 (2013).</li><li>Picollo, A., Malvezzi, M., Houtman, J. C., Accardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basis+of+substrate+binding+and+conservation+of+selectivity+in+the+CLC+family+of+channels+and+transporters.">Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters.</a> Nat Struct Mol Biol. 16, 1294-1301 (2009).</li><li>Eckford, P. D., Li, C., Ramjeesingh, M., Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+transmembrane+conductance+regulator+(CFTR)+potentiator+VX-770+(ivacaftor)+opens+the+defective+channel+gate+of+mutant+CFTR+in+a+phosphorylation-dependent+but+ATP-independent+manner.">Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner.</a> J Biol Chem. 287, 36639-36649 (2012).</li><li>Stockbridge, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoride+resistance+and+transport+by+riboswitch-controlled+CLC+antiporters.">Fluoride resistance and transport by riboswitch-controlled CLC antiporters.</a> Proc Natl Acad Sci U S A. 109, 15289-15294 (2012).</li><li>Menon, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opsin+is+a+phospholipid+flippase.">Opsin is a phospholipid flippase.</a> Curr Biol. 21, 149-153 (2011).</li><li>Nimigean, C. M., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na++block+and+permeation+in+a+K++channel+of+known+structure.">Na+ block and permeation in a K+ channel of known structure.</a> J Gen Physiol. 120, 323-335 (2002).</li><li>Picollo, A., Xu, Y., Johner, N., Bern&#232;che, S., Accardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+substrate+binding+determines+the+stoichiometry+of+transport+of+a+prokaryotic+H(+)/Cl(-)+exchanger.">Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H(+)/Cl(-) exchanger.</a> Nat Struct Mol Biol. 19, 525-531 (2012).</li><li>Tsai, M. F., Fang, Y., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sided+functions+of+an+arginine-agmatine+antiporter+oriented+in+liposomes.">Sided functions of an arginine-agmatine antiporter oriented in liposomes.</a> Biochemistry. 51, 1577-1585 (2012).</li><li>Heginbotham, L., Kolmakova-Partensky, L., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+a+prokaryotic+K++channel.">Functional reconstitution of a prokaryotic K+ channel.</a> J Gen Physiol. 111, 741-749 (1998).</li><li>Lundbaek, J. A., Collingwood, S. A., Ing&#243;lfsson, H. I., Kapoor, R., Andersen, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+bilayer+regulation+of+membrane+protein+function:+gramicidin+channels+as+molecular+force+probes.">Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes.</a> J R Soc Interface. 7, 373-395 (2010).</li><li>Lim, H. H., Stockbridge, R. B., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoride-dependent+interruption+of+the+transport+cycle+of+a+CLC+Cl(-)/H(+)+antiporter.">Fluoride-dependent interruption of the transport cycle of a CLC Cl(-)/H(+) antiporter.</a> Nat Chem Biol. 9, 712-715 (2013).</li><li>Stockbridge, R. B., Robertson, J. L., Kolmakova-Partensky, L., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+fluoride-specific+ion+channels+with+dual-topology+architecture.">A family of fluoride-specific ion channels with dual-topology architecture.</a> Elife. 2, e01084 (2013).</li><li>Nimigean, C., Shane, T., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cyclic+nucleotide+modulated+prokaryotic+K++channel.">A cyclic nucleotide modulated prokaryotic K+ channel.</a> J Gen Physiol. 124, 7 (2004).</li><li>Brohawn, S. G., del M&#225;rmol, ., J, R., MacKinnon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structure+of+the+Human+K2P+TRAAK,+a+Lipid-+and+Mechano-Sensitive+K++Ion+Channel.">Crystal Structure of the Human K2P TRAAK, a Lipid- and Mechano-Sensitive K+ Ion Channel.</a> Science. 335, 436-441 (2012).</li><li>Jayaram, H., Robertson, J. L., Wu, F., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+slow+CLC+Cl-/H++antiporter+from+a+cyanobacterium.">Structure of a slow CLC Cl-/H+ antiporter from a cyanobacterium.</a> Biochemistry. 50, 788-794 (2011).</li></ol>

The authors declare no competing financial interests.

정제 음이온 선택적 채널 또는 리포좀에 재구성 수송에 의해 매개 교통 - 우리는 상세한 CL을 측정 할 수있는 프로토콜을 설명했다. CLC-EC1을 기 - 사용 된 예는 원핵 H + / CL이었다. 고려중인 이온에 적합한 하나의 AgCl 전극 : 그러나, 방법론 용이 리간드 12,13,15, 11,12 전압 또는 AG로 대체하여 다른 음이온 선택성 15,16 스포츠 의해 게이트 된 채널을 연구하기에 ?...

마지막 두 과발현과 막 수송 단백질을 정제 할 수있는 능력이 크게 증가하고있다 수십 년 : 이온 채널, 일차 및 이차 전송기들은 보통 이종 발현 시스템뿐만 아니라, 천연 공급원으로부터 정제된다. 이 단백질의 안정성을, 표현을 모니터링 개선하고 추출을 용이하게하고 강화하는 새로운 접근 방식은 끊임없이 1-5 개발되고있다. 이러한 기술 혁신은 차례로, 그 기능의 구조적 기지에 대한 우리의 이해를 강화, 막 단백질에 원자 수준의 구조 정보의 폭발을 유발하는 데 큰 도움이되고있다. 반면에, 우리의 능력은 어떤 경우에는 고해상도 구조적 정보가 따라서 정량적 구조 기반 예측을 테스트 할 수있는 능력을 제한 질적 기능 데이터에 의해 수반되도록, 동일한 비율로 증가하지 않은 정제 된 단백질의 기능적 특성을 프로빙. 따라서, developmen는양적 일반화 기능 분석의 t는 막 단백질의 기능 기계론 토대 해명 향해 중요한 단계이다. 채널 및 전송기 - 준비 정량적 정제하고 재구성 CL의 주요 기능적 특성을 결정하기 위해 사용될 수 유출 분석을 기술한다. 분석을위한 원칙은 교통 시스템뿐만 아니라 다양한 비 이온 수송 단백질로 일반화 될 수있다. 채널 / CL 대형의 존재 수송차 - - 정제 된 리포솜 CL로 녹이고 구배 (도 1A, B). CL이 - 유출가 우리의 경우에는, 반대 이온 플럭스 있도록 이온 운반체의 첨가에 의해 개시되는 K + 이온 운반체 발리 노마 이신 (Valinomycin), CL에 의해 설정된 전압 션트되는 - 구배 및 K의 평형 전위로 초기 막 전위를 설정할 + 6,7. t없이이 횡단 전위의 발생에 의해 방지로 유출이 발생, - 그는 더 중요한 순 (CL)을 이온 운반체가 없습니다. - 유출 및 F 0, 활성 단백질을 함유하지 않은 리포좀 분획 τ, CL의 시상수 : 정량적 데이터는 두 개의 측정 파라미터 (도 1C)에 의해 설명된다. τ와 f 0 CL에서 단일 - 전송 속도, 활성 단백질의 활성 분획물 및 복합체의 분자량은 8 유도 될 수있다. 알려진 구조와 기능의 교환기 -이 기술은 CLC-EC1, 잘 특성화 된 CLC 형 H + / CL로 재구성 테오를 사용하여 여기에 예시되어있다. 상기 분석 용이 전압 활성 및 / 또는 리간드의 존재에 따라 상이한 또는 이온 선택성 채널들 또는 컨베이어로 일반화된다. 또한,이 분석은 소분자 직접 영향을 미치는 단백질 재구성 여부를 결정하는데 사용될 수있다,이들 화합물 및 방법에 막 조성물 또는 지질 - 변경 시약 재구성 채널들 및 수송의 기능에 영향을 미치는 영향을 정량.

<table border="0" cellpadding="0" cellspacing="0" width="995">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Name of Reagent/ Equipment</td>
			<td style="width:337px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liposomicator, Avanti Polar Lipids Inc.</td>
			<td style="width:337px;">&#160;Avanti Polar Lipids Inc.</td>
			<td>610200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IEC Centra CL2 Benchtop</td>
			<td style="width:337px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orion Research Model 701A Digital pH-mV meter</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>These can be found on Ebay.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Non-functional pH probe</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160; DI-710 Data Logger</td>
			<td style="width:337px;">DATAQ instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WinDAQ acquisition software</td>
			<td style="width:337px;">DATAQ instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Disposable Plastic Columns, Gravity-flow, 2ml</td>
			<td style="width:337px;">Pierce (Thermo Scientific)</td>
			<td>29922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KIMAX Culture Tubes, Disposable, Borosilicate Glass</td>
			<td style="width:337px;">Kimble Chase</td>
			<td>73500-13100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extruder Set With Holder/Heating Block</td>
			<td style="width:337px;">Avanti Polar Lipids Inc.</td>
			<td align="right" style="width:119px;">610000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a proteoliposome-based efflux assay to determine single-molecule properties of cl- channels and transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

교환기, 리포좀에 재구성 - 정제 CLC-EC1, 원핵 CLC 형 H + / CL에 의해 매개 교통 - 우리는 상세하고 강력한 CL을 측정 할 수있는 프로토콜을 설명합니다. 실험 장치의 개략적 인 대표도도 3에 도시되어 정제 된 CLC-EC1 및 높은 내부 CL 함유 재구성은 테오 -. 낮은 CL 함유 용액 욕에 침지 -. 이러한 조건 그물 CL에서 - 유출은 리포좀 (그림 3A)...

Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Proteoliposome 기반 유출 분석은 CL의 단일 분자의 속성을 확인하는<sup&gt; -</sup&gt; 채널 및 전송기

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

a-proteoliposome-based-efflux-assay-to-determine-single-molecule

Research

JoVE Journal

Biology

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

9.7K 조회수.  Weill Cornell Medical College. Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

비디오: A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

이중층 탄성 등록 정보 모니터링 변경에 대한 단일 분자 방법

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

연구

생물학

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

형광 현미경과 함께 머릿속으로 단일 분자의 DNA 복제

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

표면 고정화 Biomolecules의 단일 분자 형광 측정을위한 자동화된 시스템

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

효소 복제 DNA의 직접 관찰 어세을 스트레칭 단일 분자 DNA를 사용하여

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

GIRK 채널의 변조기를 식별을위한 형광 검사 분석

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

유전자 규정의 단일 분자 이미징<em&gt; 생체에</em&gt; 절단하여 Cotranslational 인증 (CoTrAC)를 사용

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

단백질-DNA 상호 작용의 연구에 대한 단일 분자 조작 및 이미지를 결합

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

단일 분자 FRET에 의해 DNA 루핑을 공부

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

세포 외 산 생산의 측정 및 분석은 당분 속도를 확인하는

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

전기 중립 전송기의 활동을 연구 전기 생리학 측정의 응용

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...