Name: whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins
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Description: Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

優れた技術支援をしてくれたMaila Reh、Tharsana Tharmalingam、特にAltina Kleinに感謝します。この研究は、ドイツ研究財団（DFG）（SFB1078 B2、FOR1279 SPP1665〜PH）およびカタリシス、UniCat、BIG-NSE（JV）およびE4（PH）における優良概念クラスタのクラスターによって支持された。

<img alt="図1" src="/files/ftp_upload/55497/55497fig1.jpg" /> 図1：パッチクランプのセットアップ （1）光源、（2）光ファイバ、（3）プログラマブルシャッタ、（4）デジタイザ、（5）シャッタドライバ、（6）増幅器、（7）灌流システム、 （10）ファラデーケージ、（11）顕微鏡ステージ、（12）灌流入口、（13）灌流出口、（14）記録チャンバー、（15）液面セン?...

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	<li>Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algal+sensory+photoreceptors.">Algal sensory photoreceptors.</a> Annu. Rev. Plant Biol. 59, 167-189 (2008).</li><li>Nagel, G., 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=Channelrhodopsin-1:+a+light-gated+proton+channel+in+green+algae.">Channelrhodopsin-1: a light-gated proton channel in green algae.</a> Science. 296 (5577), 2395-2398 (2002).</li><li>Bruegmann, T., 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=Optogenetic+control+of+contractile+function+in+skeletal+muscle.">Optogenetic control of contractile function in skeletal muscle.</a> Nat. Commun. 6 (7153), 1-8 (2015).</li><li>Bruegmann, T., 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=Optogenetic+control+of+heart+muscle+in+vitro+and+in+vivo.">Optogenetic control of heart muscle in vitro and in vivo.</a> Nat. Methods. 7 (11), 897-900 (2010).</li><li>Zhang, F., 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=The+microbial+opsin+family+of+optogenetic+tools.">The microbial opsin family of optogenetic tools.</a> Cell. 147 (7), 1446-1457 (2011).</li><li>Nagel, G., 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=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Curr. Biol. 15 (24), 2279-2284 (2005).</li><li>Li, X., 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=Fast+noninvasive+activation+and+inhibition+of+neural+and+network+activity+by+vertebrate+rhodopsin+and+green+algae+channelrhodopsin.">Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin.</a> Proc. Natl. Acad. Sci. U. S. A. 102 (49), 17816-17821 (2005).</li><li>Ishizuka, T., Kakuda, M., Araki, R., Yawo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+evaluation+of+photosensitivity+in+genetically+engineered+neurons+expressing+green+algae+light-gated+channels.">Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels.</a> Neurosci. Res. 54 (2), 85-94 (2006).</li><li>Wietek, J., 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=Conversion+of+channelrhodopsin+into+a+light-gated+chloride+channel.">Conversion of channelrhodopsin into a light-gated chloride channel.</a> Science. 344 (6182), 409-412 (2014).</li><li>Berndt, A., 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=Structural+foundations+of+optogenetics:+Determinants+of+channelrhodopsin+ion+selectivity.">Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.</a> Proc. Natl. Acad. Sci. U. S. A. 113 (4), 822-829 (2016).</li><li>Tsunoda, S. P., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glu+87+of+Channelrhodopsin-1+Causes+pH-dependent+Color+Tuning+and+Fast+Photocurrent+Inactivation.">Glu 87 of Channelrhodopsin-1 Causes pH-dependent Color Tuning and Fast Photocurrent Inactivation.</a> Photochem. Photobiol. 85 (2), 564-569 (2009).</li><li>Schneider, F., Gradmann, D., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+selectivity+and+competition+in+channelrhodopsins.">Ion selectivity and competition in channelrhodopsins.</a> Biophys. J. 105 (1), 91-100 (2013).</li><li>Govorunova, E. G., Spudich, E. N., Lane, C. E., Sineshchekov, O. A., Spudich, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Channelrhodopsin+with+a+Red-Shifted+Spectrum+and+Rapid+Kinetics+from+Mesostigma+viride.">New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from Mesostigma viride.</a> MBio. 2 (3), 1-9 (2011).</li><li>Govorunova, E. G., Sineshchekov, O. A., Li, H., Janz, R., Spudich, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+highly+efficient+blue-shifted+channelrhodopsin+from+the+marine+alga+Platymonas+subcordiformis.">Characterization of a highly efficient blue-shifted channelrhodopsin from the marine alga Platymonas subcordiformis.</a> J. Biol. Chem. 288 (41), 29911-29922 (2013).</li><li>Hou, S. -. Y., 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=Diversity+of+Chlamydomonas+Channelrhodopsins.">Diversity of Chlamydomonas Channelrhodopsins.</a> Photochem. Photobiol. 88 (1), 119-128 (2012).</li><li>Klapoetke, N. C., 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nat. Methods. 11 (3), 338-346 (2014).</li><li>Lin, J. Y., Lin, M. Z., Steinbach, P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+engineered+channelrhodopsin+variants+with+improved+properties+and+kinetics.">Characterization of engineered channelrhodopsin variants with improved properties and kinetics.</a> Biophys. J. 96 (5), 1803-1814 (2009).</li><li>Yizhar, O., 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=Neocortical+excitation/inhibition+balance+in+information+processing+and+social+dysfunction.">Neocortical excitation/inhibition balance in information processing and social dysfunction.</a> Nature. 477 (7363), 171-178 (2011).</li><li>Berndt, A., Yizhar, O., Gunaydin, L. a., Hegemann, P., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bi-stable+neural+state+switches.">Bi-stable neural state switches.</a> Nat. Neurosci. 12 (2), 229-234 (2009).</li><li>Berndt, A., Lee, S. Y., Ramakrishnan, C., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-Guided+Transformation+of+Channelrhodopsin+into+a+Light-Activated+Chloride+Channel.">Structure-Guided Transformation of Channelrhodopsin into a Light-Activated Chloride Channel.</a> Science. 344 (6182), 420-424 (2014).</li><li>Govorunova, E. G., Sineshchekov, O. A., Spudich, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomonas+sulcata+ACR1:+A+Fast+Anion+Channelrhodopsin.">Proteomonas sulcata ACR1: A Fast Anion Channelrhodopsin.</a> Photochem. Photobiol. 92 (2), 257-263 (2016).</li><li>Wietek, J., Broser, M., Krause, B. S., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+Natural+Green+Light+Absorbing+Chloride+Conducting+Channelrhodopsin+from+Proteomonas+sulcata.">Identification of a Natural Green Light Absorbing Chloride Conducting Channelrhodopsin from Proteomonas sulcata.</a> J. Biol. Chem. 291 (8), 4121-4127 (2016).</li><li>Govorunova, E. G., Sineshchekov, O. A., Janz, R., Liu, X., Spudich, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+light-gated+anion+channels:+A+family+of+microbial+rhodopsins+for+advanced+optogenetics.">Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics.</a> Science. 349 (6248), 647-650 (2015).</li><li>Neher, E., Sakmann, B., Steinbach, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+patch+clamp:+A+method+for+resolving+currents+through+individual+open+channels+in+biological+membranes.">The extracellular patch clamp: A method for resolving currents through individual open channels in biological membranes.</a> Pfl&#252;gers Arch. Eur. J. Physiol. 375 (2), 219-228 (1978).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pfl&#252;gers Arch. Eur. J. Physiol. Arch. Eur. J. Physiol. 391 (2), 85-100 (1981).</li><li>Barry, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JPCalc,+a+software+package+for+calculating+liquid+junction+potential+corrections+in+patch-clamp,+intracellular,+epithelial+and+bilayer+measurements+and+for+correcting+junction+potential+measurements.">JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements.</a> J. Neurosci. Methods. 51 (1), 107-116 (1994).</li><li>Amtmann, A., Sanders, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unified+procedure+for+the+correction+of+liquid+junction+potentials+in+patch+clamp+experiments+on+endo-+and+plasma+membranes.">A unified procedure for the correction of liquid junction potentials in patch clamp experiments on endo- and plasma membranes.</a> J. Exp. Bot. 48, 361-364 (1997).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat. Protoc. 1 (5), 2406-2415 (2006).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques:+plating+methods.">Aseptic laboratory techniques: plating methods.</a> J. Vis. Exp. (63), e1-e18 (2012).</li><li>Felgner, P. L., 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=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proc. Natl. Acad. Sci. U. S. A. 84 (21), 7413-7417 (1987).</li><li>Halliwell, J., Whitaker, M., Ogden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+microelectrodes.">Using microelectrodes.</a> Microelectrode Tech. Plymouth Work. Handb. 3, 1-15 (1949).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+patch-pipettes+and+sharp+electrodes+with+a+programmable+puller.">Making patch-pipettes and sharp electrodes with a programmable puller.</a> J. Vis. Exp. (20), e1-e2 (2008).</li><li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> J. Pharmacol. Toxicol. Methods. 51 (3), 187-200 (2005).</li><li>Pusch, M., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rates+of+diffusional+exchange+between+small+cells+and+a+measuring+patch+pipette.">Rates of diffusional exchange between small cells and a measuring patch pipette.</a> Pfl&#252;gers Arch. Eur. J. Physiol. 411 (2), 204-211 (1988).</li><li>Prigge, 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=Color-tuned+channelrhodopsins+for+multiwavelength+optogenetics.">Color-tuned channelrhodopsins for multiwavelength optogenetics.</a> J. Biol. Chem. 287 (38), 31804-31812 (2012).</li><li>Gradmann, D., Berndt, A., Schneider, F., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rectification+of+the+channelrhodopsin+early+conductance.">Rectification of the channelrhodopsin early conductance.</a> Biophys. J. 101 (5), 1057-1068 (2011).</li><li>Beppu, K., 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=Optogenetic+countering+of+glial+acidosis+suppresses+glial+glutamate+release+and+ischemic+brain+damage.">Optogenetic countering of glial acidosis suppresses glial glutamate release and ischemic brain damage.</a> Neuron. 81 (2), 314-320 (2014).</li><li>Wietek, J., Prigge, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+Channelrhodopsins:+An+Overview.">Enhancing Channelrhodopsins: An Overview.</a> Methods Mol. Biol. 1408, 141-165 (2016).</li><li>Ferenczi, E. A., 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=Optogenetic+approaches+addressing+extracellular+modulation+of+neural+excitability.">Optogenetic approaches addressing extracellular modulation of neural excitability.</a> Sci. Rep. 6, 1-20 (2016).</li><li>Zhang, F., 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=Red-shifted+optogenetic+excitation:+a+tool+for+fast+neural+control+derived+from+Volvox+carteri.">Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri.</a> Nat. Neurosci. 11 (6), 631-633 (2008).</li><li>Sugiyama, Y., 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=Photocurrent+attenuation+by+a+single+polar-to-nonpolar+point+mutation+of+channelrhodopsin-2.">Photocurrent attenuation by a single polar-to-nonpolar point mutation of channelrhodopsin-2.</a> Photochem. Photobiol. Sci. 8 (3), 328-336 (2009).</li><li>Kleinlogel, S., 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=Ultra+light-sensitive+and+fast+neuronal+activation+with+the+Ca2+-permeable+channelrhodopsin+CatCh.">Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh.</a> Nat. Neurosci. 14 (4), 513-518 (2011).</li><li>Wang, H., 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=Molecular+determinants+differentiating+photocurrent+properties+of+two+channelrhodopsins+from+chlamydomonas.">Molecular determinants differentiating photocurrent properties of two channelrhodopsins from chlamydomonas.</a> J. Biol. Chem. 284 (9), 5685-5696 (2009).</li><li>Gradinaru, V., Thompson, K. R., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=eNpHR:+a+Natronomonas+halorhodopsin+enhanced+for+optogenetic+applications.">eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications.</a> Brain Cell Biol. 36 (1-4), 129-139 (2008).</li><li>Berglund, K., 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=Luminopsins+integrate+opto-+and+chemogenetics+by+using+physical+and+biological+light+sources+for+opsin+activation.">Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation.</a> Proc. Natl. Acad. Sci. 113 (3), 358-367 (2016).</li></ol>

定義されたイオン状態および電気状態での反転電位の決定は、ChRの光活性化後に輸送されるイオン種に関する情報を提供する。排他的に1つのイオン種が複雑な生理学的媒体中で変化し、得られた逆電位が理論上のネルンスト電位に従ってシフトするならば、このイオン種は唯一の輸送種である。 しかし、ChRの場合、反転電位シフトは通常、異なるイオン種への浸透性お?...

チャンネルロドプシン（ChR）は、運動性の緑藻の眼球のスポットに存在する光依存性イオンチャネルであり、光走性および恐怖反応の主要な光センサーとして機能する1 。 2002年2月の最初の記述以来、ChRは新興オプトジェネティクス分野への道を開き、様々な興奮性細胞、例えば骨格筋、心臓、または脳内に適用することができる3,4,5。標的細胞におけるChRの発現は、それぞれの細胞の光制御可能なイオン透過性をもたらす。これは、ニューロンの状況において、活動電位（AP）の発火（伝導イオンに依存する）の活性化6,7,8または阻害9,10を空間的および時間的ChR変異体のイオン選択性がどのようにその光発生適用を決定するかを強調する光の精度。 Chlamydomonas reinhardtiiおよびVolvox carteri由来の最初に発見されたChRは、プロトンに対して透過性であるが、ナトリウム、カリウム、およびカルシウムおよびマグネシウムなどの二価陽イオンにはあまり関わらない一価カチオンにも透過性である11,12,13 。今日では、70種類以上の天然カチオン伝導性チャネルロドプシン（CCR）14,15,16,17およびいくつかの改変された改変体18,19,20が 、 光電流サイズ、分光感度、カイネティックス、およびカチオン選択性が利用可能である。神経科学では、CCRはa細胞を活性化させ、APを誘発するために再使用される場合、光駆動の微生物ポンプは、ニューロンをサイレンシングするために唯一利用可能なアンタゴニストであった。 2014年に2つのグループが同時に、CCRが分子工学を介して推定イオン伝導ポアに沿って極性を変化させることにより、陰イオン伝導チャネルロドプシン（ACR）に変換できることを示した9,21。続いて、いくつかのクリプトフィト藻類において天然ACRが同定された22,23,24。最も重要なことに、ACRの軽い活性化は、成人ニューロンにおける塩化物電流を媒介し、吸収された光子につき単一の電荷のみを輸送する微生物ポンプよりもはるかに低い光強度でニューロン活性の阻害を可能にする。 ChR活性は、HEK293細胞における光誘起電流の電気生理学的パッチクランプ記録によって直接対処することができる。パッチクランプ技術はもともと1970年代後半に開発されたものであり、Hamill et al。高い電流分解能および膜電圧26の直接制御を伴う小さなセル（全細胞モード）からの電流実体の記録を可能にする。細胞培養に適用されるこの技術は、イオンおよび電気記録条件の正確な制御を提供し、イオン選択性を総電流に対するイオンの相対的な寄与と共に研究することを可能にする。ここでは、様々な細胞外クロライド濃度下で電流 - 電圧関係を記録して、高塩化物コンダクタンスを証明することにより、Proteomonas sulcata（ Ps ACR1） 22,23の陰イオン伝導性チャネルロドプシンのイオン選択性の試験を例示する。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >HEK293 cells</td>
			<td >Sigma Aldrich</td>
			<td >85120602</td>
			<td >Human embryonic kidney cells</td>
		</tr>
		<tr >
			<td >Retinal</td>
			<td >Sigma Aldrich</td>
			<td>R2500</td>
			<td>all-trans retinal</td>
		</tr>
		<tr >
			<td >FuGENE HD</td>
			<td >Promega</td>
			<td>E2312</td>
			<td>Transfection reagent</td>
		</tr>
		<tr >
			<td >DMEM</td>
			<td >Biochrome</td>
			<td>FG 0445</td>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
		</tr>
		<tr >
			<td >Agarose</td>
			<td >Roth</td>
			<td>3810</td>
			<td>Agar bridges</td>
		</tr>
		<tr >
			<td >CaCl2</td>
			<td >Roth</td>
			<td>5239</td>
			<td>CaCl2 2H2O</td>
		</tr>
		<tr >
			<td >CsCl</td>
			<td >Biomol</td>
			<td>2452</td>
			<td></td>
		</tr>
		<tr >
			<td >EGTA</td>
			<td >Roth</td>
			<td>3054</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS</td>
			<td >Biochrome</td>
			<td>S0615</td>
			<td>Cell culture</td>
		</tr>
		<tr >
			<td >Glucose</td>
			<td >Roth</td>
			<td>HN06</td>
			<td>D(+)-Glucose</td>
		</tr>
		<tr >
			<td >KCl</td>
			<td >Roth</td>
			<td>6781</td>
			<td></td>
		</tr>
		<tr >
			<td >MgCl2</td>
			<td >Roth</td>
			<td>2189</td>
			<td>MgCl2 6H2O</td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >Roth</td>
			<td>3957</td>
			<td></td>
		</tr>
		<tr >
			<td >NMG</td>
			<td >Sigma Aldrich</td>
			<td>M2004</td>
			<td>N-Methyl-D-glucamine</td>
		</tr>
		<tr >
			<td >Na-Aspartate</td>
			<td >Sigma Aldrich</td>
			<td>A6683</td>
			<td>L-Aspartic acid sodium salt monohydrate</td>
		</tr>
		<tr >
			<td >Citric acid</td>
			<td >Roth</td>
			<td>6490</td>
			<td></td>
		</tr>
		<tr >
			<td >AgeI</td>
			<td >ThermoFischerScientific</td>
			<td>ER1462</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr >
			<td >XhoI</td>
			<td >ThermoFischerScientific</td>
			<td>ER0695</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr >
			<td >NheI</td>
			<td >ThermoFischerScientific</td>
			<td>ER0975</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr >
			<td >XL1Blue E.coli/</td>
			<td >Agilent Technologies</td>
			<td>200249</td>
			<td>Chemocompetent E.coli</td>
		</tr>
		<tr >
			<td >Kanamycin</td>
			<td >Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr >
			<td >Lysogeny broth medium</td>
			<td >Roth</td>
			<td>X964</td>
			<td></td>
		</tr>
		<tr >
			<td >Agar-Agar</td>
			<td >Roth</td>
			<td>6494</td>
			<td>Agar plates</td>
		</tr>
		<tr >
			<td >Plasmid purification kit</td>
			<td >Marchery-Nagel</td>
			<td>740727.25</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicilin/Streptomycin</td>
			<td >Biochrome</td>
			<td>A 2213</td>
			<td>Cell culture</td>
		</tr>
		<tr >
			<td >Poly-D-lysine hydrobromide</td>
			<td >Sigma Aldrich</td>
			<td>P6407-5MG</td>
			<td>Cover slip coating</td>
		</tr>
		<tr >
			<td >Microforge</td>
			<td >Custom made</td>
			<td></td>
			<td>Fire polishing</td>
		</tr>
		<tr >
			<td >Serological pipettes</td>
			<td >TPP</td>
			<td></td>
			<td>Different sizes</td>
		</tr>
		<tr >
			<td >Clean bench</td>
			<td >Kojair</td>
			<td>Biowizard SL130</td>
			<td></td>
		</tr>
		<tr >
			<td >Stirrer</td>
			<td >IKA</td>
			<td>RCT classic</td>
			<td></td>
		</tr>
		<tr >
			<td >Silver wire</td>
			<td >Science Products</td>
			<td>AG-T25; AG-T10</td>
			<td>Electrodes, 0.64 mm (bath); 0.25 mm (electrode)</td>
		</tr>
		<tr >
			<td >pH-meter</td>
			<td >Knick</td>
			<td>765 Calimetric</td>
			<td></td>
		</tr>
		<tr >
			<td >Osmometer</td>
			<td >Vogel</td>
			<td>OM 815</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td >Carl Zeiss</td>
			<td>ID03</td>
			<td>Fire polishing</td>
		</tr>
		<tr >
			<td >CO2 incubator</td>
			<td >Binder</td>
			<td>CB150</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell culture dishes</td>
			<td >TPP</td>
			<td>93040</td>
			<td>34 mm internal diameter</td>
		</tr>
		<tr >
			<td >Cover slips</td>
			<td >Roth</td>
			<td>P232</td>
			<td>15 mm diameter</td>
		</tr>
		<tr >
			<td >Thermometer</td>
			<td >R&#246;ssel Messtechnik</td>
			<td>MTM12</td>
			<td></td>
		</tr>
		<tr >
			<td >Beamsplitter</td>
			<td >Chroma</td>
			<td>21011</td>
			<td>90/10 transmission</td>
		</tr>
		<tr >
			<td >Pipette holder</td>
			<td>ALA Scientific Instruments</td>
			<td>PPH-1P-AXU-0-1.5</td>
			<td></td>
		</tr>
		<tr >
			<td >Headstage</td>
			<td >Molecular Devices</td>
			<td>CV203BU</td>
			<td></td>
		</tr>
		<tr >
			<td >Amplifier</td>
			<td >Molecular Devices</td>
			<td >AxoPatch200B</td>
			<td></td>
		</tr>
		<tr >
			<td >Digitizer</td>
			<td >Molecular Devices</td>
			<td >DigiData1400</td>
			<td>Digital analog converter</td>
		</tr>
		<tr >
			<td >Lightsource</td>
			<td >TILL Photonics</td>
			<td >Polychrome V</td>
			<td>Set to 540 nm full intensity</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td >Carl Zeiss</td>
			<td >Axiovert 100</td>
			<td></td>
		</tr>
		<tr >
			<td >Shutter</td>
			<td >Vincent Associates</td>
			<td >VS25</td>
			<td></td>
		</tr>
		<tr >
			<td >Shutter driver</td>
			<td >Vincent Associates</td>
			<td >VCM-D1</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass capilarries</td>
			<td >Warner Instruments</td>
			<td>G150F-3</td>
			<td >Boresilicate capillaries with fire polished ends OD 1.5 mm ID&#160; 0.86 mm</td>
		</tr>
		<tr >
			<td >Micropipette puller</td>
			<td >Sutter Instruments</td>
			<td >P1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Bath handler</td>
			<td >Lorenz Messger&#228;tebau</td>
			<td >MPCU</td>
			<td></td>
		</tr>
		<tr >
			<td >Tripleband filterset</td>
			<td >Chroma</td>
			<td>69008</td>
			<td>Fluorescence filter&#160; ECFP/EYFP/mCherry&#160;</td>
		</tr>
		<tr >
			<td >CCD camera</td>
			<td >Watec</td>
			<td >Wat-221SCCD</td>
			<td></td>
		</tr>
		<tr >
			<td >Optometer</td>
			<td >Gigahertz Optik</td>
			<td >P9710</td>
			<td>Measure light intensities</td>
		</tr>
		<tr >
			<td >Objective</td>
			<td >Carl Zeiss</td>
			<td>421462-9900-000</td>
			<td >W Plan-Apochromat 40X/1.0 DIC</td>
		</tr>
		<tr >
			<td >Micromanipulator</td>
			<td >Scientifica</td>
			<td>PatchStar</td>
			<td></td>
		</tr>
		<tr >
			<td >Recording chamber</td>
			<td >Custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Power supply</td>
			<td >Manson</td>
			<td>HCS-3202</td>
			<td>Avoids electrical noise from microscope built-in power supply</td>
		</tr>
		<tr >
			<td >Vibration isolated table</td>
			<td >Newport</td>
			<td>M-VW-3636-OPT-01</td>
			<td></td>
		</tr>
		<tr >
			<td >Faraday cage</td>
			<td >Custom made or any commercial matching table</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Hoses</td>
			<td >Any comercial; e.g. Roth</td>
			<td></td>
			<td>Different sizes and materials for bath handling and application of pipette pressure; agar bridges</td>
		</tr>
		<tr >
			<td >Linear shaker</td>
			<td >Sunlab Instruments</td>
			<td>SU 1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Liquid junction potential calculator</td>
			<td >Molecular Devices or directly from Peter H. Barry</td>
			<td></td>
			<td>Program is included in the Clampex aquisition software or can be obtained from&#160; p.barry@unsw.edu.au</td>
		</tr>
		<tr >
			<td >Data acquisition software</td>
			<td >Molecular Devices</td>
			<td>Clampex 10.X</td>
			<td></td>
		</tr>
		<tr >
			<td >Data evaluation software</td>
			<td >Molecular Devices</td>
			<td>Clampfit 10.X</td>
			<td></td>
		</tr>
		<tr >
			<td >PsACR1</td>
			<td>GenBank or Addgene</td>
			<td >KF992074.1 or Addgene plasmid #85465</td>
			<td>Gene encoding for PsACR1</td>
		</tr>
		<tr >
			<td >Amplifier guide</td>
			<td >Molecular Devices</td>
			<td>The Axon Guide</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins

過去10年間で、チャネルロドプシンは神経科学的研究において不可欠となり、標的細胞における電気的プロセスを非侵襲的に操作するツールとして使用されている。これに関連して、チャネルロドプシンのイオン選択性は特に重要である。この記事では、HEK293細胞の電気生理学的パッチクランプ記録を介して、Proteomonas sulcataの最近同定された陰イオン伝導性チャネルロドプシンの塩化物選択性の研究について説明する。光ゲート光電流を測定するための実験手順は、従来のパッチクランプセットアップの顕微鏡に結合された、高速で切り替え可能な、理想的には単色の光源を必要とする。実験前の準備手順は、緩衝液の調製、液絡部電位の検討、細胞の播種およびトランスフェクション、およびパッチピペットの引き上げを含む概略を述べる。電流 - 電圧関係の実際の記録トランスフェクションの24時間後から48時間後に異なる塩化物濃度の逆転電位を測定することができます。最後に、電気生理学的データを塩化物伝導の理論的考察に関して分析する。

<html> 図2は、記載されたプロトコルに従った測定から得られた代表的な結果を示す。緑色光を照射している間、 Ps ACR1は速い過渡電流を特徴とし、急速に定常電流レベルまで減衰します。光が消灯した後、光電流はミリ秒以内にゼロに減衰します（ 図2A ）。細胞外の塩化物濃度を交換すると、獲得した光電流トレースに直接見ることが?...

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

チャンネルロドプシンにおけるイオン選択性の電気生理学的測定のための全細胞パッチクランプ記録

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Research

JoVE Journal

Neuroscience

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

内耳有毛細胞におけるパッチクランプ記録は、単離されたゼブラフィッシュから

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

赤外線神経刺激のメカニズムを調査するための全細胞パッチクランプ

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

胚ゼブラフィッシュマウスナー細胞からパッチクランプ記録

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

急性脳スライスにおけるニューロンマイクロ回路の電気生理学的および形態学的特徴は、ペアパッチクランプ記録を使用して、

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

脳スライスにおける全細胞パッチクランプ記録

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

全細胞電圧クランプ記録のための電気生理学的方法<em&gt;ショウジョウバエ</em&gt;感光体

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

聴性脳幹反応と生後ラットの外有毛細胞細胞パッチ ・ クランプ記録

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

準備やアフリカツメガエルの視蓋ニューロンの全体セル パッチ ・ クランプ記録のためのプロトコル

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

膠様質ニューロンの細胞パッチク ランプ記録のため急性脊髄スライスの準備

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...