Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity
The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed e...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2016-01, Vol.113 (4), p.822-829 |
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| creator | Berndt, Andre Lee, Soo Yeun Wietek, Jonas Ramakrishnan, Charu Steinberg, Elizabeth E. Rashid, Asim J. Kim, Hoseok Park, Sungmo Santoro, Adam Frankland, Paul W. Iyer, Shrivats M. Pak, Sally Ährlund-Richter, Sofie Delp, Scott L. Malenka, Robert C. Josselyn, Sheena A. Carlén, Marie Hegemann, Peter Deisseroth, Karl |
| description | The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor–based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure–function relationships of the light-gated pore. |
| doi_str_mv | 10.1073/pnas.1523341113 |
| format | Article |
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The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor–based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure–function relationships of the light-gated pore.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.1523341113</identifier><identifier>PMID: 26699459</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Action Potentials ; Amino Acid Sequence ; Amino acids ; Animals ; Arginine - chemistry ; Avoidance Learning - physiology ; Avoidance Learning - radiation effects ; Basolateral Nuclear Complex - physiology ; Basolateral Nuclear Complex - radiation effects ; Binding sites ; Biological Sciences ; Cells, Cultured ; Cellular biology ; Chlorides - metabolism ; Dependovirus - genetics ; Electroshock ; Electrostatics ; Fear ; Fiber Optic Technology ; Genetic Vectors - administration & dosage ; Genetic Vectors - genetics ; HEK293 Cells ; Hippocampus - cytology ; Histidine - chemistry ; Humans ; Hydrogen-Ion Concentration ; INAUGURAL ARTICLES ; Ion Channel Gating - physiology ; Ion Channel Gating - radiation effects ; Ions ; Male ; Memory - physiology ; Memory - radiation effects ; Mice ; Mice, Inbred C57BL ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Neurons - physiology ; Optogenetics ; Protein Conformation ; Proteins ; Rats ; Rats, Sprague-Dawley ; Rhodopsin - chemistry ; Rhodopsin - metabolism ; Rhodopsin - radiation effects ; Sequence Alignment ; Ventral Tegmental Area - physiology</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2016-01, Vol.113 (4), p.822-829</ispartof><rights>Volumes 1–89 and 106–113, copyright as a collective work only; author(s) retains copyright to individual articles</rights><rights>Copyright National Academy of Sciences Jan 26, 2016</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c569t-2c263bc5a7578a213c7f45bd6ac1ec59547f4fb4cc5ce132daeae78cb808de1e3</citedby><cites>FETCH-LOGICAL-c569t-2c263bc5a7578a213c7f45bd6ac1ec59547f4fb4cc5ce132daeae78cb808de1e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/113/4.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/26467496$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/26467496$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,550,723,776,780,799,881,27901,27902,53766,53768,57992,58225</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26699459$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttp://kipublications.ki.se/Default.aspx?queryparsed=id:132833112$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><creatorcontrib>Berndt, Andre</creatorcontrib><creatorcontrib>Lee, Soo Yeun</creatorcontrib><creatorcontrib>Wietek, Jonas</creatorcontrib><creatorcontrib>Ramakrishnan, Charu</creatorcontrib><creatorcontrib>Steinberg, Elizabeth E.</creatorcontrib><creatorcontrib>Rashid, Asim J.</creatorcontrib><creatorcontrib>Kim, Hoseok</creatorcontrib><creatorcontrib>Park, Sungmo</creatorcontrib><creatorcontrib>Santoro, Adam</creatorcontrib><creatorcontrib>Frankland, Paul W.</creatorcontrib><creatorcontrib>Iyer, Shrivats M.</creatorcontrib><creatorcontrib>Pak, Sally</creatorcontrib><creatorcontrib>Ährlund-Richter, Sofie</creatorcontrib><creatorcontrib>Delp, Scott L.</creatorcontrib><creatorcontrib>Malenka, Robert C.</creatorcontrib><creatorcontrib>Josselyn, Sheena A.</creatorcontrib><creatorcontrib>Carlén, Marie</creatorcontrib><creatorcontrib>Hegemann, Peter</creatorcontrib><creatorcontrib>Deisseroth, Karl</creatorcontrib><title>Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor–based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure–function relationships of the light-gated pore.</description><subject>Action Potentials</subject><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Animals</subject><subject>Arginine - chemistry</subject><subject>Avoidance Learning - physiology</subject><subject>Avoidance Learning - radiation effects</subject><subject>Basolateral Nuclear Complex - physiology</subject><subject>Basolateral Nuclear Complex - radiation effects</subject><subject>Binding sites</subject><subject>Biological Sciences</subject><subject>Cells, Cultured</subject><subject>Cellular biology</subject><subject>Chlorides - metabolism</subject><subject>Dependovirus - genetics</subject><subject>Electroshock</subject><subject>Electrostatics</subject><subject>Fear</subject><subject>Fiber Optic Technology</subject><subject>Genetic Vectors - administration & dosage</subject><subject>Genetic Vectors - genetics</subject><subject>HEK293 Cells</subject><subject>Hippocampus - cytology</subject><subject>Histidine - chemistry</subject><subject>Humans</subject><subject>Hydrogen-Ion Concentration</subject><subject>INAUGURAL ARTICLES</subject><subject>Ion Channel Gating - physiology</subject><subject>Ion Channel Gating - radiation effects</subject><subject>Ions</subject><subject>Male</subject><subject>Memory - physiology</subject><subject>Memory - radiation effects</subject><subject>Mice</subject><subject>Mice, Inbred C57BL</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Neurons - physiology</subject><subject>Optogenetics</subject><subject>Protein Conformation</subject><subject>Proteins</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Rhodopsin - chemistry</subject><subject>Rhodopsin - metabolism</subject><subject>Rhodopsin - 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PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>2016-01-26</date><risdate>2016</risdate><volume>113</volume><issue>4</issue><spage>822</spage><epage>829</epage><pages>822-829</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near −65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor–based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure–function relationships of the light-gated pore.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>26699459</pmid><doi>10.1073/pnas.1523341113</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Proceedings of the National Academy of Sciences - PNAS, 2016-01, Vol.113 (4), p.822-829 |
| issn | 0027-8424 1091-6490 |
| language | eng |
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| source | Jstor Complete Legacy; MEDLINE; PubMed Central; Alma/SFX Local Collection; SWEPUB Freely available online; Free Full-Text Journals in Chemistry |
| subjects | Action Potentials Amino Acid Sequence Amino acids Animals Arginine - chemistry Avoidance Learning - physiology Avoidance Learning - radiation effects Basolateral Nuclear Complex - physiology Basolateral Nuclear Complex - radiation effects Binding sites Biological Sciences Cells, Cultured Cellular biology Chlorides - metabolism Dependovirus - genetics Electroshock Electrostatics Fear Fiber Optic Technology Genetic Vectors - administration & dosage Genetic Vectors - genetics HEK293 Cells Hippocampus - cytology Histidine - chemistry Humans Hydrogen-Ion Concentration INAUGURAL ARTICLES Ion Channel Gating - physiology Ion Channel Gating - radiation effects Ions Male Memory - physiology Memory - radiation effects Mice Mice, Inbred C57BL Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Neurons - physiology Optogenetics Protein Conformation Proteins Rats Rats, Sprague-Dawley Rhodopsin - chemistry Rhodopsin - metabolism Rhodopsin - radiation effects Sequence Alignment Ventral Tegmental Area - physiology |
| title | Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-06T23%3A08%3A43IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-jstor_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Structural%20foundations%20of%20optogenetics:%20Determinants%20of%20channelrhodopsin%20ion%20selectivity&rft.jtitle=Proceedings%20of%20the%20National%20Academy%20of%20Sciences%20-%20PNAS&rft.au=Berndt,%20Andre&rft.date=2016-01-26&rft.volume=113&rft.issue=4&rft.spage=822&rft.epage=829&rft.pages=822-829&rft.issn=0027-8424&rft.eissn=1091-6490&rft_id=info:doi/10.1073/pnas.1523341113&rft_dat=%3Cjstor_cross%3E26467496%3C/jstor_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1765603552&rft_id=info:pmid/26699459&rft_jstor_id=26467496&rfr_iscdi=true |