Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity. Berndt Sy J C Ee Aj H S

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Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity. 2015 Proceedings of the National Academy of Sciences of the United States of America 10.1073/pnas.1523341113 Berndt2015-structural-foundations-of-optogenetics-deter Berndt2015-structural-foundations-of-optogenetics-deter.txt, the project's staged copy da7722a672862e7f6baa427820e54b124da633f2 not-redistributable neuroscience

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0 spans located by searching the stored text for the quote's exact words, allowing any whitespace between them the structural determinants of ion selectivity in channelrhodopsin pores, and the inhibitory tools they make possible Quotation withheld because this record is not cleared for redistribution. whether an electrostatic model of the pore can be pushed further to build chloride channels usable for behavioural inhibition Quotation withheld because this record is not cleared for redistribution. builds 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 Quotation withheld because this record is not cleared for redistribution. method Quotation withheld because this record is not cleared for redistribution. channelrhodopsin ion selectivity Quotation withheld because this record is not cleared for redistribution. system-or-tool-building system-or-tool-building Quotation withheld because this record is not cleared for redistribution. mechanisms 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 Quotation withheld because this record is not cleared for redistribution. mechanisms 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 Quotation withheld because this record is not cleared for redistribution. mechanisms Inhibition works by shunting: opening the chloride conductance drops input resistance and shunts excitatory input, rather than by hyperpolarizing the cell Quotation withheld because this record is not cleared for redistribution. mechanisms Reduced input resistance lowers excitability in the same way endogenous GABA-A chloride channels do Quotation withheld because this record is not cleared for redistribution. mechanisms 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 Quotation withheld because this record is not cleared for redistribution. ions 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 Quotation withheld because this record is not cleared for redistribution. inhibition works by shunting excitatory input, as endogenous chloride channels do, rather than by hyperpolarising the cell Quotation withheld because this record is not cleared for redistribution. a 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 Quotation withheld because this record is not cleared for redistribution. the pH dependence of the first-generation channel was traced to protonatable pore residues and removed by further substitution Quotation withheld because this record is not cleared for redistribution. channels follow the cell's own chloride gradient while pumps move ions unidirectionally regardless of it Quotation withheld because this record is not cleared for redistribution. Spike inhibition by iC++ is not absolute: with intracellular chloride above the physiological range it can be partially overcome by very strong current injection. 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 Quotation withheld because this record is not cleared for redistribution. iC++ is not presented as universally superior; the choice between channel and pump depends on the preparation and hypothesis. 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 Quotation withheld because this record is not cleared for redistribution. because channels follow physiological gradients, which differ by cell type, subcellular region, developmental stage and pathology, the tool can even excite under some conditions 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 Quotation withheld because this record is not cleared for redistribution. pumps hyperpolarise far beyond physiological levels and destabilise membrane properties what this limits: the pump comparison: THE PUMPS CAN HYPERPOLARIZE CELLS FAR BEYOND PHYSIOLOGICAL LEVELS, CAUSING UNSTABLE MEMBRANE PROPERTIES Quotation withheld because this record is not cleared for redistribution. lowering pump light intensity fixes that but shrinks the inhibited tissue volume, with no clean solution 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 Quotation withheld because this record is not cleared for redistribution. the tool is explicitly not universally better - it must be chosen for the preparation and the hypothesis duplicate of: limitations[1] · what this limits: the same scope as limitations[1] - the tool is not claimed to be universally better Quotation withheld because this record is not cleared for redistribution. in both behavioural assays the three opsins performed equivalently, so the assays do not resolve the cellular differences the paper establishes 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 Quotation withheld because this record is not cleared for redistribution. a crystal structure and an electrostatic potential calculation, used as the design specification for a behavioural tool Quotation withheld because this record is not cleared for redistribution. shunting inhibition by GABA-A receptors, used as the model for how the engineered channel acts Quotation withheld because this record is not cleared for redistribution. matching iC++ protein expression against the parental iC1C2 construct, and comparing membrane properties of iC++-expressing cells against eYFP-only cells 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 Quotation withheld because this record is not cleared for redistribution. optogenetic 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 Quotation withheld because this record is not cleared for redistribution. inhibitory 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 Quotation withheld because this record is not cleared for redistribution. the 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 Quotation withheld because this record is not cleared for redistribution. iC++ is named for the count of charges added, so the construct name encodes the mechanistic claim Quotation withheld because this record is not cleared for redistribution. claim_type: mechanism 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. Quotation withheld because this record is not cleared for redistribution. method: structure-guided point mutation with whole-cell recording in cultured neurons, then viral expression and behavioural testing in freely moving mice 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. Quotation withheld because this record is not cleared for redistribution. phenomenon: ion selectivity in a light-gated channel pore 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. Quotation withheld because this record is not cleared for redistribution. extends → iC1C2, the first-generation structure-guided chloride-conducting channelrhodopsin Quotation withheld because this record is not cleared for redistribution. extends → the previously reported step-function variants SwiChR CA and SwiChR CT Quotation withheld because this record is not cleared for redistribution. builds-on → the naturally anion-conducting GtACR channelrhodopsins Quotation withheld because this record is not cleared for redistribution. builds-on → the structure-derived electrostatic model of channelrhodopsin pore selectivity Quotation withheld because this record is not cleared for redistribution. confirms → the crystal structure-derived electrostatic model of channelrhodopsin pore selectivity Quotation withheld because this record is not cleared for redistribution. supports → the naturally occurring GtACR chloride channels, whose pore residues match the engineered ones Quotation withheld because this record is not cleared for redistribution. extends → the first-generation iC1C2 channel, which had small photocurrents and was never tested on behaviour status: the paper's own words: were not tested Quotation withheld because this record is not cleared for redistribution. qualifies → the use of light-activated chloride pumps for optogenetic inhibition Quotation withheld because this record is not cleared for redistribution. channelrhodopsin —distinct-from→ pumps channelrhodopsin pumps 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. Quotation withheld because this record is not cleared for redistribution. shunting —contrasted-with→ hyperpolarization shunting hyperpolarization strength of the relation: 1 · note: RE-GROUNDS another row of this record: the old span named ONE endpoint. Quotation withheld because this record is not cleared for redistribution. ANOVA (two-way repeated measures) ANOVA (two-way repeated measures) Quotation withheld because this record is not cleared for redistribution. post hoc multiple comparison test (Holm-Sidak) post hoc multiple comparison test (Holm-Sidak) Quotation withheld because this record is not cleared for redistribution. interaction effect in factorial ANOVA interaction effect in factorial ANOVA Quotation withheld because this record is not cleared for redistribution. standard error of the mean standard error of the mean Quotation withheld because this record is not cleared for redistribution. two-way repeated-measures ANOVA with Holm-Sidak post hoc two-way repeated-measures ANOVA with Holm-Sidak post hoc Quotation withheld because this record is not cleared for redistribution. light by virus interaction ANOVA light by virus interaction ANOVA Quotation withheld because this record is not cleared for redistribution. The 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. Quotation withheld because this record is not cleared for redistribution. Structure-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. Quotation withheld because this record is not cleared for redistribution. Unlike its predecessor iC1C2, iC++ shows a reversal potential independent of the proton gradient, i.e. it does not conduct protons. Quotation withheld because this record is not cleared for redistribution. iC++ passes other halides (iodide, bromide) but excludes physiological small organic anions. Quotation withheld because this record is not cleared for redistribution. The purely model-driven engineered pore converged on the same residue solution that evolution produced in the natural anion channel GtACR2. Quotation withheld because this record is not cleared for redistribution. The chloride channel inhibited neurons far more efficiently than the chloride pump eNpHR3.0, blocking any tested input in over 90% of expressing cells. Quotation withheld because this record is not cleared for redistribution. SwiChR++ can be switched off immediately by red light, giving temporally bounded bistable control of chloride conductance and neuronal inhibition. Quotation withheld because this record is not cleared for redistribution. Under these conditions iC++, iC1C2 and eNpHR3.0 were equally effective at driving real-time place aversion. Quotation withheld because this record is not cleared for redistribution. Optogenetically silencing CREB-overexpressing amygdala neurons reduced tone-evoked freezing, while silencing an equal number of non-allocated neurons did not. Quotation withheld because this record is not cleared for redistribution. net photocurrents increased more than 15-fold under physiological conditions Quotation withheld because this record is not cleared for redistribution. effective photocurrent was more than 16 times larger than the first generation at comparable expression Quotation withheld because this record is not cleared for redistribution. the advantage over the first generation is largest for short 5 ms pulses needing over 500 pA to drive spiking Quotation withheld because this record is not cleared for redistribution. the engineered pore residues match those of a naturally occurring chloride channel discovered afterwards Quotation withheld because this record is not cleared for redistribution. Reverting one arginine to the wild-type histidine (R134H) increased input resistance and photocurrent size. Quotation withheld because this record is not cleared for redistribution. the channel inhibits spikes more efficiently than the chloride pump, through stronger shunting Quotation withheld because this record is not cleared for redistribution. all three opsins produced equivalent real-time place aversion Quotation withheld because this record is not cleared for redistribution. silencing CREB-overexpressing amygdala neurons reduced fear freezing, while silencing the same number of non-CREB neurons did not Quotation withheld because this record is not cleared for redistribution. the reduction in freezing did not differ significantly between the three opsins Quotation withheld because this record is not cleared for redistribution. channelrhodopsins channelrhodopsins Quotation withheld because this record is not cleared for redistribution. optogenetics optogenetics Quotation withheld because this record is not cleared for redistribution. step-function opsin bistability step-function opsin bistability Quotation withheld because this record is not cleared for redistribution. optogenetics optogenetics Quotation withheld because this record is not cleared for redistribution. shunting inhibition shunting inhibition Quotation withheld because this record is not cleared for redistribution. point mutation point mutation Quotation withheld because this record is not cleared for redistribution. intracellular chloride concentration intracellular chloride concentration Quotation withheld because this record is not cleared for redistribution. external pH external pH Quotation withheld because this record is not cleared for redistribution. V rev V rev Quotation withheld because this record is not cleared for redistribution. neuronal input resistance neuronal input resistance Quotation withheld because this record is not cleared for redistribution. internal chloride concentration internal chloride concentration Quotation withheld because this record is not cleared for redistribution. control cells control cells Quotation withheld because this record is not cleared for redistribution. reversal potential reversal potential Quotation withheld because this record is not cleared for redistribution. photocurrent amplitude photocurrent amplitude Quotation withheld because this record is not cleared for redistribution. avoidance of the light-paired compartment avoidance of the light-paired compartment Quotation withheld because this record is not cleared for redistribution. more than 16 times — increase in effective photocurrent size of iC++ over iC1C2 at comparable protein expression Quotation withheld because this record is not cleared for redistribution. 9,500-fold (τ off,dark = 115 s ± 9) — slowing of channel closure produced by the C128A mutation, creating the bistable SwiChR++ Quotation withheld because this record is not cleared for redistribution. 488 nm — peak activation wavelength of iC++, indicating an intact retinal-binding pocket Quotation withheld because this record is not cleared for redistribution. near −65 mV — reversal potential of the first-generation chloride-conducting channelrhodopsins Quotation withheld because this record is not cleared for redistribution. 9 and 10 residues — number of amino acid replacements in iC1C2 and iC++ relative to the parental cation channel C1C2 Quotation withheld because this record is not cleared for redistribution. at least seven times — efficiency advantage of the iC++ channel over the eNpHR3.0 pump in inhibiting current-driven spiking Quotation withheld because this record is not cleared for redistribution. more than 90% — proportion of iC++-expressing cells in which light blocked every current input tested Quotation withheld because this record is not cleared for redistribution. ∼15% — share of lateral amygdala pyramidal neurons transduced before fear conditioning in the memory-engram experiment Quotation withheld because this record is not cleared for redistribution. more than 15-fold, and more than 16 times at matched expression — photocurrent improvement over the first generation Quotation withheld because this record is not cleared for redistribution. reversing near -65 mV, decreased by a further about 15 mV — reversal potential of the first and second generation channels Quotation withheld because this record is not cleared for redistribution. 9 and 10 residues replaced — how heavily engineered the two constructs are Quotation withheld because this record is not cleared for redistribution. about 15% of lateral amygdala pyramidal neurons transduced — the engram experiment's targeting Quotation withheld because this record is not cleared for redistribution.

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