Name: whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins
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Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

אנו מודים Maila Reh, Tharsana Tharmalingam ובמיוחד Altina Klein לעזרה טכנית מעולה. עבודה זו נתמכה על ידי קרן המחקר הגרמנית (DFG) (SFB1078 B2, FOR1279 SPP1665 ל- PH) ואשכול המצוינות המאחד קונספטים בקטליזה, UniCat, BIG-NSE (JV) ו- E4 (PH).

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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>

The authors have nothing to disclose.

קביעת הפוטנציאלים היפוך בתנאים יוניים וחשמליים מוגדר מספק מידע על מינים יון מועבר לאחר הפעלה קלה של CHR. אם באופן בלעדי יון אחד הוא מגוון במדיום פיזיולוגי מורכב ואת השינויים הפוטנציאליים היפוך המתקבל על פי הפוטנציאל Nernst תיאורטית, זה מין יון הוא היחיד מועבר אחד. <p class...

Channelrhodopsins (CHR) הם ערוצי יון אור מגודלים המתרחשים בנקודת העין של אצות ירוקות נעות, ומשמשים כמחזירים ראשוניים עבור פוטוטקסיס ותגובות פוביות 1 . מאז התיאור הראשון שלהם בשנת 2002, ChRs סללו את הדרך עבור השדה המתעוררים של optogenetics ויכול להיות מיושם במגוון של תאים נרגשים למשל בתוך שרירי השלד, הלב, או את המוח 3 , 4 , 5 . ביטוי של CHRs בתאי היעד גורם חדירות אור יון לשליטה של ​​התא בהתאמה. בהקשר נוירוני, זה מאפשר הפעלה 6 , 7 , 8 או עיכוב 9 , 10 של פעולה פוטנציאליים (AP) ירי - בהתאם יון שנערך - עם הזמן המרחבידיוק של אור המדגיש כיצד סלקטיביות יון של גרסה ChR קובעת יישום optogenetic שלה. גידולי CHR הראשונים שהתגלו מ - Chlamydomonas reinhardtii ו- Volvox carteri חדירים לפרוטונים, אך גם לקטיונים מונוולנטיים כמו נתרן, אשלגן, ובמידה פחותה גם לקטיונים דו-קלים כגון סידן ומגנזיום 11 , 12 , 13 . כיום, יותר מ -70 קטיונים טבעיים (cCR) 14 , 15 , 16 , 17 וריאנטים מהונדסים שונים 18 , 19 , 20 עם מאפיינים שונים כגון גודל פוטו, רגישות ספקטרלית, קינטיקה, סלקטיביות קטיון זמינים. בעוד בתחום מדעי המוח, CCRs אששימשו להפעלת תאים ומפעילי APPS, משאבות מונעות על ידי אור, היו היריבים הזמינים רק עבור נוירונים משתיקים במשך שנים. בשנת 2014, שתי קבוצות הראו בו זמנית כי CCRs ניתן להמיר anion-conductrhopsops ניצח אניון (ACRs) על ידי שינוי של הקוטביות לאורך יון putative ניצוח באמצעות הנדסה מולקולרית 9 , 21 . לאחר מכן, ACRs טבעיים זוהו בכמה קריפטופיטים 22 , 23 , 24 . והכי חשוב, הפעלה קלה של ACRs מתווכת זרמי כלוריד נוירונים מבוגרים המאפשר עיכוב של הפעילות העצבית בעוצמות האור הרבה יותר נמוך מאשר משאבות מיקרוביאליות כי רק הובלה יחיד חיובי לכל פוטון נספג. פעילות ChR ניתן לטפל ישירות על ידי אלקטרו קליפ הקלטות אלקטרומגנטית של זרמים האור המושרה בתאים HEK293. מהדק התיקוןהטכניקה פותחה במקור בסוף שנות ה -70 25 ושיפר עוד יותר על ידי Hamill et al. , המאפשר הקלטה של ​​ישות של זרמים מתא קטן (מצב תא שלם) עם רזולוציה גבוהה הנוכחי שליטה ישירה של מתח קרום 26 . יישומי בתרבית תאים, טכניקה זו מספקת שליטה מדויקת של תנאי ההקלטה יונית כמו גם חשמל, ומאפשר לימוד סלקטיביות יון יחד עם התרומה היחסית של יונים לסך הנוכחי. כאן אנו מדגימים את בדיקת סלקטיביות יון עבור anion מנצח transrhodopsin של Proteomonas sulcata (פס ACR1) 22 , 23 באמצעות הקלטה של ​​יחסי מתח הנוכחי תחת ריכוזי כלוריד תאיים שונים כדי להוכיח מוליכות כלוריד גבוהה.

<table><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&#039;s Modified Eagle Medium</td></tr><tr><td>Agarose</td><td>Roth</td><td>3810</td><td>Agar bridges</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>5239</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>2189</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; 6H&lt;sub&gt;2&lt;/sub&gt;O</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 &lt;em&gt;E. coli&lt;/em&gt;</td><td>Agilent Technologies</td><td>200249</td><td>Chemocompetent &lt;em&gt;E. coli&lt;/em&gt;</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>CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;ouml;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&amp;nbsp; 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&amp;auml;tebau</td><td>MPCU</td><td></td></tr><tr><td>Tripleband filterset</td><td>Chroma</td><td>69008</td><td>Fluorescence filter&amp;nbsp; ECFP/EYFP/mCherry&amp;nbsp;</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&amp;nbsp; 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>&lt;em&gt;Ps&lt;/em&gt;ACR1</td><td>GenBank or Addgene</td><td>KF992074.1 or Addgene plasmid #85465</td><td>Gene encoding for &lt;em&gt;Ps&lt;/em&gt;ACR1</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

במהלך העשור האחרון, channelrhodopsins הפך חיוני במחקר neuroscientific שבו הם משמשים כלים לא פולשני לתפעל תהליכים חשמליים בתאי היעד. בהקשר זה, סלקטיביות יון של channelrhodopsin הוא בעל חשיבות מיוחדת. מאמר זה מתאר את החקירה של סלקטיביות כלוריד עבור לאחרונה זיהו anion-channelrhodopsin ניטור של Proteomonas sulcata באמצעות הקלטות קליפ תיקון אלקטרו על תאים HEK293. ההליך הניסויי למדידת צילומי האור הנשלטים על ידי האור דורש תמסורת מהירה - מונוכרומטית - מקור אור צמוד למיקרוסקופ של תצורת טלאי קונבנציונאלי. ההליכים ההכנה לפני הניסוי מתוארים הכנת פתרונות שנאגרו, שיקולים על פוטנציאל צומת נוזלים, זריעה transfection של תאים, ו משיכת טפיחות תיקון. ההקלטה בפועל של יחסי המתח הנוכחיS כדי לקבוע את הפוטנציאל היפוך עבור ריכוזי כלוריד שונים מתרחש 24 שעות עד 48 שעות לאחר transfection. לבסוף, נתונים אלקטרופיזיולוגיים מנותחים ביחס לשיקולים תיאורטיים של הולכה כלוריד.

<html> איור 2 מציג תוצאות מייצגות המתקבלות ממדידות בעקבות הפרוטוקול המתואר. במהלך התאורה עם אור ירוק, Ps ACR1 תכונות זרם חולף מהיר אשר דועך במהירות לרמה הנוכחית נייח. לאחר כיבוי האור, התאים מתפרקים לאפס בתוך אלפיות השנייה ( איור 2 א ). ?...

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.

תא שלם תיקון מהדק הקלטות עבור קביעת electrophysiological של יון סלקטיביות ב Channelrhodopsins

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

whole-cell-patch-clamp-recordings-for-electrophysiological

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.0K Views.  Humboldt-Universität zu Berlin. 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.

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

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.

הצמד תיקון הקלטה של ​​תעלות יונים מתבטא ביציות Xenopus

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

Biology

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

כל תא הקלטות בו זמנית מקולטני אור ועל נוירונים מסדר שני בהכנה פורסים רשתית אמפיבי

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ...

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

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

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

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

הצמד תיקון וטכניקות זלוף עבור לימוד תעלות יונים מתבטא Xenopus ביציות

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

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

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

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

Transfection אסטרטגיה אופטימיזציה עבור הביטוי הקלטה של ​​אלקטרו Voltage-מגודרת ערוצי רקומביננטי יון ב HEK-293T תאים

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

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

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

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

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

שיטה אלקטרו-קולית לקולקציית מתח מלא<em&gt; תסיסנית</em&gt; פוטורספטורים

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

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.

הקלטת תיקון מהדק מצורף תא אחד ערוצים

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

Optogenetic Electrophysiology Recording of Cells with Light-Dependent Ion Channels

Source: Grimm, C., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55497">Whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins.</a> J. Vis. Exp. (2017)
In this video, embryonic kidney cell cultures expressing light-dependent ion channel proteins fused with a fluorescent reporter demonstrate the effect of light on ion movement. Whole-cell patch clamp is used to monitor the movement of ions.

רישום אלקטרופיזיולוגיה אופטוגנטית של תאים עם תעלות יונים תלויות אור

1. Setup Prior to Recordings

	Prepare the recording solutions with ion concentrations as indicated in Table 1.&#8203;
	
		Prepare 15 mL of 2 M stock ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

Source:&#160; Katz, B., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55627">Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors.</a> J. Vis. Exp. (2017)
The video demonstrates recording electrical activity in the photoreceptor neurons of Drosophila&#39;s ommatidium by establishing a whole-cell configuration. It highlights the activation of transient receptor potential channels by light pulses, resulting in measurable quantum bumps.

הקלטות מהדק מתח של תא שלם מפוטורצפטורים של דרוזופילה

NOTE: During all of the following steps, use only dim red light illumination and ensure that the ommatidia&#39;s exposure to light is minimal (i.e., work ...

Electrophysiology Techniques

Patch Clamp

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively and negatively charged ions across the cell membrane. One competent method to record current flowing across the whole cell or single ion channel is the patch-clamp technique.
In this method, a glass micropipette containing electrolyte solution is tightly sealed against a small portion of the cell membrane. As a result, a patch of the cell membrane is electrically isolated, ensuring that the ions moving across the channels flow only into the micropipette. Further, this tight sealing of the cell membrane prevents ions from escaping into the bath solution in which the cell is suspended.
Different Patch-clamp Methods:
Depending upon the research interest, several variations of the patch-clamp method can be used for measuring the cell&#39;s biophysical properties. For example, researchers can clamp or control the membrane voltage and measure the current passing through it. Alternatively, they can clamp the current and measure any variation in the membrane voltage.
In cell-attached mode, the membrane patch containing a single or a few ion channels remains intact. As a result, the current flowing through the membrane patch alone can be measured. In contrast, the whole-cell method involves disruption of the membrane patch by briefly applying strong suction. Consequently, the interior of the pipette becomes continuous with the cytoplasm. This mode enables the measurement of electrical current and voltage from the entire cell.
Another patch-clamp method requires gentle retraction of the attached pipette. As a result, the membrane patch is excised without affecting the tight seal. In this inside-out configuration, the intracellular portion of the membrane is exposed to the bath solution, allowing the study of intracellular factors affecting the channel functions.

מהדק טלאי

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively ...

תא שלם תיקון מהדק הקלטות עבור קביעת electrophysiological של יון סלקטיביות ב Channelrhodopsins

Summary

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הצמד תיקון הקלטה של תעלות יונים מתבטא ביציות Xenopus

כל תא הקלטות בו זמנית מקולטני אור ועל נוירונים מסדר שני בהכנה פורסים רשתית אמפיבי

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

הצמד תיקון וטכניקות זלוף עבור לימוד תעלות יונים מתבטא Xenopus ביציות

Transfection אסטרטגיה אופטימיזציה עבור הביטוי הקלטה של אלקטרו Voltage-מגודרת ערוצי רקומביננטי יון ב HEK-293T תאים

שיטה אלקטרו-קולית לקולקציית מתח מלא

הקלטת תיקון מהדק מצורף תא אחד ערוצים

רישום אלקטרופיזיולוגיה אופטוגנטית של תאים עם תעלות יונים תלויות אור

הקלטות מהדק מתח של תא שלם מפוטורצפטורים של דרוזופילה

מהדק טלאי

תא שלם תיקון מהדק הקלטות עבור קביעת electrophysiological של יון סלקטיביות ב Channelrhodopsins

Summary

Explore More Videos

Chapters in this video

הצמד תיקון הקלטה של ​​תעלות יונים מתבטא ביציות Xenopus

כל תא הקלטות בו זמנית מקולטני אור ועל נוירונים מסדר שני בהכנה פורסים רשתית אמפיבי

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

הצמד תיקון וטכניקות זלוף עבור לימוד תעלות יונים מתבטא Xenopus ביציות

Transfection אסטרטגיה אופטימיזציה עבור הביטוי הקלטה של ​​אלקטרו Voltage-מגודרת ערוצי רקומביננטי יון ב HEK-293T תאים

שיטה אלקטרו-קולית לקולקציית מתח מלא

הקלטת תיקון מהדק מצורף תא אחד ערוצים

רישום אלקטרופיזיולוגיה אופטוגנטית של תאים עם תעלות יונים תלויות אור

הקלטות מהדק מתח של תא שלם מפוטורצפטורים של דרוזופילה

מהדק טלאי

הצמד תיקון הקלטה של תעלות יונים מתבטא ביציות Xenopus

Transfection אסטרטגיה אופטימיזציה עבור הביטוי הקלטה של אלקטרו Voltage-מגודרת ערוצי רקומביננטי יון ב HEK-293T תאים