Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

Berndt, Sy, J, C, Ee, Aj, H, S · Proceedings of the National Academy of Sciences of the United States of America · 2015 · neuroscience · 10.1073/pnas.1523341113 · record Berndt2015-structural-foundations-of-optogenetics-deter
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Where this record's text came from. faceted 2026-08 by reading the locally held text (html); every evidence string verified as an exact substring of that text before merging. The paper is a hybrid: protein engineering plus mechanism testing (it confirms the electrostatic pore-selectivity hypothesis) plus in vivo behavioural validation; claim type 'method' captures the primary contribution (the new tools) and 'system-or-tool-building' the method, but the electrophysiological characterisation (whole-cell patch clamp in cultured neurons, HEK cells and acute slices, in vivo extracellular recording) and the mouse behavioural assays are not visible in a single method slot. Self-stated limitations are sparse in this paper — the two recorded are the only explicit caveats about the authors' own tools; several other 'however' statements concern the competing pumps, not this work. GtACR relation is tagged builds-on rather than replicates because the convergence is evolutionary, not an experimental repetition. 2026-08-13 and 2026-08-15 schema-0.3 passes: every quote verified as an exact substring of the staged text and anchored at merge time; concept terms verified present; every edge endpoint an existing node.
11 quotations on this page contain a reference number where the paper printed a superscript. This journal style prints citations as raised numerals — parameters12,13 — and the extraction flattened them into the line, so the sentence reads parameters 12 , 13 . The digits are citation markers and not part of what the author wrote. The quotes keep them for the same reason they keep every other extraction fault: a quote is reproduced exactly or it proves nothing.
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Tags on this paper

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channelrhodopsin optogenetics chloride shunting hyperpolarization reversal potential photocurrent input resistance electrostatic pumps ion gradients place-aversion engram Ions are selected by electrostatic interaction with the charged residues lining the pore, so replacing glutamates with neutral/basic residues flips selectivity from cations to chloride Protonation of basic pore residues under acidic conditions creates favourable electrostatics for chloride, which is why the earlier iC1C2 conducted chloride better at low pH Inhibition works by shunting: opening the chloride conductance drops input resistance and shunts excitatory input, rather than by hyperpolarizing the cell Reduced input resistance lowers excitability in the same way endogenous GABA-A chloride channels do The longer arginine side chain substituted at position 134 sterically blocks channel closure, producing the dark leak current and low input resistance of the E83X mutants pore crystal structure mutation arginine glutamate amygdala CREB freezing bistable channelrhodopsins step-function opsin bistability shunting inhibition point mutation intracellular chloride concentration external pH V rev neuronal input resistance internal chloride concentration control cells photocurrent amplitude avoidance of the light-paired compartment ANOVA (two-way repeated measures) post hoc multiple comparison test (Holm-Sidak) Holm–Sidak post hoc tests interaction effect in factorial ANOVA light (on, off) × virus ANOVA yielded a significant interaction standard error of the mean two-way repeated-measures ANOVA with Holm-Sidak post hoc light by virus interaction ANOVA system-or-tool-building

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relation to other workclaim_relationshow this paper stands to another: agrees, contradicts, extends, qualifies
claim typeclaim_typethe shape of the claim: existence, mechanism, method, critique, prediction, null
concept edgeconcept_edgesan asserted relation between two concepts, with the sentence asserting it
content tagcontent_tagsa finding, definition, quantity, statistic or variable named in the paper
contributioncontributionwhat the paper claims to add
controlcontrolsan experiment run to eliminate a rival explanation
limitationlimitationswhat the paper says its evidence cannot support
mechanismmechanismshow the reported effect is said to come about
methodmethodhow the paper produced its evidence
object of studyobject_of_studywhat the paper is about — the thing it investigates
phenomenonphenomenonthe effect or regularity under study
research questionresearch_questionthe question the paper sets itself
sensesensesa distinct use of a term, quoted where the paper fixes it
transfertransfersa method or concept carried in from another field

The paper

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Every tag on this page

alternatives 3 · claim_relations 8 · claim_type 1 · concept_edges 2 · content_tags 51 · contribution 1 · controls 1 · limitations 7 · mechanisms 10 · method 1 · object_of_study 1 · phenomenon 1 · research_question 1 · senses 4 · transfers 2

Relations on this page carry a quotation for the claim. Where a relation was separately evidenced — and so tested against the vocabulary for its type — the row says so; none of 2 relations here carry that mark. The rest are the tagger’s reading of the sentence quoted beside them.

facettagfieldconf.whose words
object_of_studythe structural determinants of ion selectivity in channelrhodopsin pores, and the inhibitory tools they make possible
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object_of_studythe paper's words
research_questionwhether an electrostatic model of the pore can be pushed further to build chloride channels usable for behavioural inhibition
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research_questionthe paper's words
contributionbuilds enhanced chloride-conducting channelrhodopsins with 15-fold larger photocurrents, demonstrates the first behavioural control with them, and shows inhibition works by shunting rather than hyperpolarisation
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contributionthe paper's words
claim_typemethod
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claim_typethe paper's words
phenomenonchannelrhodopsin ion selectivity
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phenomenonthe paper's words
methodsystem-or-tool-building
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methodthe paper's words
mechanismsmechanisms
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mechanisms[0]the tagger's words
mechanismsmechanisms
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mechanisms[1]the tagger's words
mechanismsmechanisms
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mechanisms[2]the tagger's words
mechanismsmechanisms
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mechanisms[3]the tagger's words
mechanismsmechanisms
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mechanisms[4]the tagger's words
mechanismsions are selected by electrostatic interaction with the pore, so replacing negatively charged residues with neutral or positive ones flips the channel from cation- to anion-conducting
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mechanisms[5]1.0the tagger's words
mechanismsinhibition works by shunting excitatory input, as endogenous chloride channels do, rather than by hyperpolarising the cell
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mechanisms[6]1.0the tagger's words
mechanismsa structural argument diagnosed the leak: a longer arginine side chain where wild type has histidine could block channel closure, and reverting it restored input resistance
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mechanisms[7]1.0the tagger's words
mechanismsthe pH dependence of the first-generation channel was traced to protonatable pore residues and removed by further substitution
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mechanisms[8]1.0the tagger's words
mechanismschannels follow the cell's own chloride gradient while pumps move ions unidirectionally regardless of it
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mechanisms[9]1.0the tagger's words
limitationsSpike inhibition by iC++ is not absolute: with intracellular chloride above the physiological range it can be partially overcome by very strong current injection.
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what this limits: the completeness of the inhibition: nearly complete spike inhibition held even with elevated chloride, ALTHOUGH THE INHIBITION IN SUCH CASES COULD BE PARTIALLY OVERCOME WITH EXTREMELY STRONG (0.8-nA) CURRENT INJECTIONS
limitations[0]the tagger's words
limitationsiC++ is not presented as universally superior; the choice between channel and pump depends on the preparation and hypothesis.
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what this limits: any reading of the paper as naming a best inhibitory tool: iC++ AND RELATED CHANNELS ... LIKE ANY REAGENT IN BIOLOGY, SHOULD BE SELECTED BASED ON SUITABILITY FOR PREPARATION AND HYPOTHESIS
limitations[1]the tagger's words
limitationsbecause channels follow physiological gradients, which differ by cell type, subcellular region, developmental stage and pathology, the tool can even excite under some conditions
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what this limits: the SIGN of the effect, not just its size: gradients CAN DIFFER ACROSS CELL TYPES, SUBCELLULAR REGIONS, DEVELOPMENTAL STAGES, AND PATHOLOGICAL STATES ... EVEN GIVING RISE TO EXCITATION UNDER SOME CONDITIONS
limitations[2]1.0the tagger's words
limitationspumps hyperpolarise far beyond physiological levels and destabilise membrane properties
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what this limits: the pump comparison: THE PUMPS CAN HYPERPOLARIZE CELLS FAR BEYOND PHYSIOLOGICAL LEVELS, CAUSING UNSTABLE MEMBRANE PROPERTIES
limitations[3]1.0the tagger's words
limitationslowering pump light intensity fixes that but shrinks the inhibited tissue volume, with no clean solution
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what this limits: the remedy for item 3: lowering light intensity ALSO SEVERELY REDUCES THE VOLUME OF TISSUE INHIBITED BY THE PUMPS, so the trade-off has no clean side
limitations[4]1.0the tagger's words
limitationsthe tool is explicitly not universally better - it must be chosen for the preparation and the hypothesis
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duplicate of: limitations[1] · what this limits: the same scope as limitations[1] - the tool is not claimed to be universally better
limitations[5]1.0the tagger's words
limitationsin both behavioural assays the three opsins performed equivalently, so the assays do not resolve the cellular differences the paper establishes
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what this limits: what the behavioural assays can resolve: EACH OPSIN WAS EQUALLY EFFECTIVE IN DRIVING REAL-TIME PLACE AVERSION, so the cellular differences do not appear at this readout
limitations[6]1.0the tagger's words
transfersa crystal structure and an electrostatic potential calculation, used as the design specification for a behavioural tool
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transfers[0]1.0the tagger's words
transfersshunting inhibition by GABA-A receptors, used as the model for how the engineered channel acts
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transfers[1]1.0the tagger's words
controlsmatching iC++ protein expression against the parental iC1C2 construct, and comparing membrane properties of iC++-expressing cells against eYFP-only cells
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eliminates: that the 16-fold larger photocurrent comes from higher expression rather than from the channel itself, and that expressing the opsin alters the cell's membrane properties · outcome: held
controls[0]0.9the tagger's words
sensesoptogenetic inhibition is not one thing: a chloride CHANNEL shunts along the cell's own gradient, a chloride PUMP moves ions against it, and the two differ in efficiency, stability and dependence on the preparation
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senses[0]1.0the tagger's words
sensesinhibitory is a property of the tool IN A PREPARATION, not of the tool - the same channel can excite where the chloride gradient runs the other way
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senses[1]1.0the tagger's words
sensesthe electrostatic pore model is treated as a predictive hypothesis, and each round of engineering is described as a test of it rather than as an application
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senses[2]1.0the tagger's words
sensesiC++ is named for the count of charges added, so the construct name encodes the mechanistic claim
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senses[3]0.85the tagger's words
alternativesclaim_type: mechanism
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a second reading DIFFERING from the recorded value. Recorded: method — note: a second reading, verbatim-verified, disagreeing with the value on the record. Not applied - a person decides.
alternatives.claim_typethe tagger's words
alternativesmethod: structure-guided point mutation with whole-cell recording in cultured neurons, then viral expression and behavioural testing in freely moving mice
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a second reading DIFFERING from the recorded value. Recorded: system-or-tool-building — note: a second reading, verbatim-verified, disagreeing with the value on the record. Not applied - a person decides.
alternatives.methodthe tagger's words
alternativesphenomenon: ion selectivity in a light-gated channel pore
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a second reading DIFFERING from the recorded value. Recorded: channelrhodopsin ion selectivity — note: a second reading, verbatim-verified, disagreeing with the value on the record. Not applied - a person decides.
alternatives.phenomenonthe tagger's words
claim_relationsextends → iC1C2, the first-generation structure-guided chloride-conducting channelrhodopsin
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claim_relations[0]the tagger's words
claim_relationsextends → the previously reported step-function variants SwiChR CA and SwiChR CT
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claim_relations[1]the tagger's words
claim_relationsbuilds-on → the naturally anion-conducting GtACR channelrhodopsins
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claim_relations[2]the tagger's words
claim_relationsbuilds-on → the structure-derived electrostatic model of channelrhodopsin pore selectivity
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claim_relations[3]the tagger's words
claim_relationsconfirms → the crystal structure-derived electrostatic model of channelrhodopsin pore selectivity
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claim_relations[4]1.0the tagger's words
claim_relationssupports → the naturally occurring GtACR chloride channels, whose pore residues match the engineered ones
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claim_relations[5]1.0the tagger's words
claim_relationsextends → the first-generation iC1C2 channel, which had small photocurrents and was never tested on behaviour
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status: the paper's own words: were not tested
claim_relations[6]1.0the tagger's words
claim_relationsqualifies → the use of light-activated chloride pumps for optogenetic inhibition
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claim_relations[7]1.0the tagger's words
concept_edgeschannelrhodopsin —distinct-from→ pumps
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differs in: THE AXIS IS LIGHT SENSITIVITY, AND A SECOND ONE IS NAMED BESIDE IT. 'the pumps, Quotation withheld.' Two dimensions in one clause - how much light each needs, and whether that requirement can be engineered away. The second is the one that decides which tool a laboratory can actually use. · strength of the relation: 1 · note: differ in light sensitivity, bistability, membrane stability and whether they respect physiological gradients Audit 2026-09-28: DOUBTFUL - read against its span and kept, but the span contrasts pumps with channels on light sensitivity; distinct-from is loose.
concept_edges[3]1.0the tagger's words
concept_edgesshunting —contrasted-with→ hyperpolarization
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strength of the relation: 1 · note: RE-GROUNDS another row of this record: the old span named ONE endpoint.
concept_edges[5]1.0the tagger's words
content_tagsANOVA (two-way repeated measures)
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content_tags.statistics[0]the tagger's words
content_tagspost hoc multiple comparison test (Holm-Sidak)
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content_tags.statistics[1]the tagger's words
content_tagsinteraction effect in factorial ANOVA
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content_tags.statistics[2]the tagger's words
content_tagsstandard error of the mean
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content_tags.statistics[3]the tagger's words
content_tagstwo-way repeated-measures ANOVA with Holm-Sidak post hoc
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content_tags.statistics[4]0.9the tagger's words
content_tagslight by virus interaction ANOVA
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content_tags.statistics[5]0.9the tagger's words
content_tagsThe paper tests the hypothesis that channelrhodopsin selectivity is set by the pore's surface potential: a negatively charged pore conducts cations, and a pore with neutral and positive residues conducts chloride.
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content_tags.findings[0]the tagger's words
content_tagsStructure-guided redesign produced iC++ and the bistable SwiChR++, with more than 15-fold larger photocurrents and a reversal potential about 15 mV more negative than the previous generation.
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content_tags.findings[1]the tagger's words
content_tagsUnlike its predecessor iC1C2, iC++ shows a reversal potential independent of the proton gradient, i.e. it does not conduct protons.
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content_tags.findings[2]the tagger's words
content_tagsiC++ passes other halides (iodide, bromide) but excludes physiological small organic anions.
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content_tags.findings[3]the tagger's words
content_tagsThe purely model-driven engineered pore converged on the same residue solution that evolution produced in the natural anion channel GtACR2.
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content_tags.findings[4]the tagger's words
content_tagsThe chloride channel inhibited neurons far more efficiently than the chloride pump eNpHR3.0, blocking any tested input in over 90% of expressing cells.
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content_tags.findings[5]the tagger's words
content_tagsSwiChR++ can be switched off immediately by red light, giving temporally bounded bistable control of chloride conductance and neuronal inhibition.
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content_tags.findings[6]the tagger's words
content_tagsUnder these conditions iC++, iC1C2 and eNpHR3.0 were equally effective at driving real-time place aversion.
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content_tags.findings[7]the tagger's words
content_tagsOptogenetically silencing CREB-overexpressing amygdala neurons reduced tone-evoked freezing, while silencing an equal number of non-allocated neurons did not.
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content_tags.findings[8]the tagger's words
content_tagsnet photocurrents increased more than 15-fold under physiological conditions
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content_tags.findings[9]1.0the tagger's words
content_tagseffective photocurrent was more than 16 times larger than the first generation at comparable expression
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content_tags.findings[10]1.0the tagger's words
content_tagsthe advantage over the first generation is largest for short 5 ms pulses needing over 500 pA to drive spiking
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content_tags.findings[11]1.0the tagger's words
content_tagsthe engineered pore residues match those of a naturally occurring chloride channel discovered afterwards
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content_tags.findings[12]1.0the tagger's words
content_tagsReverting one arginine to the wild-type histidine (R134H) increased input resistance and photocurrent size.
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content_tags.findings[13]1.0the tagger's words
content_tagsthe channel inhibits spikes more efficiently than the chloride pump, through stronger shunting
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content_tags.findings[14]1.0the tagger's words
content_tagsall three opsins produced equivalent real-time place aversion
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content_tags.findings[15]1.0the tagger's words
content_tagssilencing CREB-overexpressing amygdala neurons reduced fear freezing, while silencing the same number of non-CREB neurons did not
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content_tags.findings[16]1.0the tagger's words
content_tagsthe reduction in freezing did not differ significantly between the three opsins
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content_tags.findings[17]1.0the tagger's words
content_tagschannelrhodopsins
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content_tags.definitions[0]the tagger's words
content_tagsoptogenetics
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also in: optogenetics
content_tags.definitions[1]the tagger's words
content_tagsstep-function opsin bistability
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content_tags.definitions[2]the tagger's words
content_tagsoptogenetics
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also in: optogenetics
content_tags.definitions[3]0.85the tagger's words
content_tagsshunting inhibition
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content_tags.definitions[4]1.0the tagger's words
content_tagspoint mutation
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content_tags.variables[0]the tagger's words
content_tagsintracellular chloride concentration
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content_tags.variables[1]the tagger's words
content_tagsexternal pH
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also in: external pH
content_tags.variables[2]the tagger's words
content_tagsV rev
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also in: V rev
content_tags.variables[3]the tagger's words
content_tagsneuronal input resistance
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content_tags.variables[4]the tagger's words
content_tagsinternal chloride concentration
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content_tags.variables[5]the tagger's words
content_tagscontrol cells
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also in: control cells
content_tags.variables[6]the tagger's words
content_tagsreversal potential
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content_tags.variables[7]1.0the tagger's words
content_tagsphotocurrent amplitude
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content_tags.variables[8]1.0the tagger's words
content_tagsavoidance of the light-paired compartment
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content_tags.variables[9]0.9the tagger's words
content_tagsmore than 16 times — increase in effective photocurrent size of iC++ over iC1C2 at comparable protein expression
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content_tags.quantities[0]the tagger's words
content_tags9,500-fold (τ off,dark = 115 s ± 9) — slowing of channel closure produced by the C128A mutation, creating the bistable SwiChR++
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content_tags.quantities[1]the tagger's words
content_tags488 nm — peak activation wavelength of iC++, indicating an intact retinal-binding pocket
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content_tags.quantities[2]the tagger's words
content_tagsnear −65 mV — reversal potential of the first-generation chloride-conducting channelrhodopsins
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content_tags.quantities[3]the tagger's words
content_tags9 and 10 residues — number of amino acid replacements in iC1C2 and iC++ relative to the parental cation channel C1C2
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content_tags.quantities[4]the tagger's words
content_tagsat least seven times — efficiency advantage of the iC++ channel over the eNpHR3.0 pump in inhibiting current-driven spiking
Quotation withheld — this record is not cleared for redistribution. The sentence is in the paper at the link above.
content_tags.quantities[5]the tagger's words
content_tagsmore than 90% — proportion of iC++-expressing cells in which light blocked every current input tested
Quotation withheld — this record is not cleared for redistribution. The sentence is in the paper at the link above.
content_tags.quantities[6]the tagger's words
content_tags∼15% — share of lateral amygdala pyramidal neurons transduced before fear conditioning in the memory-engram experiment
Quotation withheld — this record is not cleared for redistribution. The sentence is in the paper at the link above.
content_tags.quantities[7]the tagger's words
content_tagsmore than 15-fold, and more than 16 times at matched expression — photocurrent improvement over the first generation
Quotation withheld — this record is not cleared for redistribution. The sentence is in the paper at the link above.
content_tags.quantities[8]0.9the tagger's words
content_tagsreversing near -65 mV, decreased by a further about 15 mV — reversal potential of the first and second generation channels
Quotation withheld — this record is not cleared for redistribution. The sentence is in the paper at the link above.
content_tags.quantities[9]0.9the tagger's words
content_tags9 and 10 residues replaced — how heavily engineered the two constructs are
Quotation withheld — this record is not cleared for redistribution. The sentence is in the paper at the link above.
content_tags.quantities[10]1.0the tagger's words
content_tagsabout 15% of lateral amygdala pyramidal neurons transduced — the engram experiment's targeting
Quotation withheld — this record is not cleared for redistribution. The sentence is in the paper at the link above.
content_tags.quantities[11]0.8the tagger's words

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© 2026 Shir Sivroni · ORCID 0009-0000-2597-2824