INAUGURAL ARTICLE

Structural foundations of optogenetics: Determinantsof channelrhodopsin ion selectivity
Andre Berndta,1, Soo Yeun Leea,1, Jonas Wietekb,1, Charu Ramakrishnana, Elizabeth E. Steinbergc,d, Asim J. Rashide,
Hoseok Kimf, Sungmo Parke, Adam Santoroe, Paul W. Franklande, Shrivats M. Iyera, Sally Paka, Sofie Ährlund-Richterf,
Scott L. Delpa, Robert C. Malenkac,d, Sheena A. Josselyne, Marie Carlénf, Peter Hegemannb, and Karl Deisserotha,c,g,2
a
Department of Bioengineering, Stanford University, Stanford, CA 94305; bInstitute for Biology, Experimental Biophysics, Humboldt Universität zu Berlin,
D-10115 Berlin, Germany; cDepartment of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA 94305; dNancy Pritzker Laboratory, Stanford
University, Stanford, CA 94305; eProgram in Neurosciences and Mental Health, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada M5G
1X8; fDepartment of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden; and gHoward Hughes Medical Institute, Stanford University,
Stanford, CA 94305

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2012.

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of thisremarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part bydevelopment of a structure-informed electrostatic model for poreselectivity, included both the introduction of amino acids withpositively charged side chains into the ion conduction pathway andthe 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 andfurther 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 bylight-activated chloride pumps. The design and functional featuresof these next-generation chloride-conducting channelrhodopsinsprovide 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.
optogenetics

Converging lines of work recently achieved the latter goal; resolving
the high-resolution structure of channelrhodopsin (7) allowed aprincipled structure-guided approach to engineering for chloride
selectivity by testing an electrostatic model for pore function (8, 9).
Subsequently, by screening the genome of the Guillardia theta microbe, two naturally occurring light-gated chloride-conducting
channelrhodopsins (10) were identified.
Because optogenetic control of behavior has not yet been
demonstrated with chloride channelrhodopsins, and to test further
integrative ideas regarding pore function from structural considerations as shown here, we sought to design and test the next
generation of enhanced chloride channels (iC++ and SwiChR++).
Along the way, we provide the initial test of the hypothesis thatlight-activated channels will be more efficient tools than pumps for
optogenetic neuronal inhibition at the cellular level, demonstrate
the initial utility of light-gated chloride channels in controlling
behavior in freely moving animals, and reveal key principles regarding the functional selectivity of light-gated ion channel pores.
Significance
Channelrhodopsins are membrane proteins that enable cellular
regulation of transmembrane ion conductance through light-gatedpores; these proteins have found application in optogenetics. This
paper tests the hypothesis that selectivity of channelrhodopsins isdetermined by surface potential of the pore region: Cations are
conducted by a negatively charged pore, and chloride ions are
conducted by a pore that has neutral and positively charged residues. In confirming this hypothesis and applying the resulting
principles, we engineer improved chloride-conducting channels
with higher chloride selectivity and conductivity. We also provide
insights into the distinct mechanisms underlying inhibition mediated by higher-efficiency chloride channels compared with ion
pumps. Finally, we demonstrate initial utility of light-gated microbial opsin-based chloride channels in controlling behavior of freely
moving animals.

| channelrhodopsin | structure | chloride | neuronal inhibitionD

iscovery and engineering of the microbial opsin genes not
only has stimulated basic science investigation into thestructure–function relationships of proteins involved in lighttriggered ion flow but also has opened up opportunities for biological investigation (reviewed in ref. 1) via the technique of
optogenetics, which involves targeting these genes and corresponding optical stimuli to control activity within specified types ofcells within intact and functioning biological systems. For example,
optogenetics has been used to identify causally the brain cells andprojections involved in behaviors relevant to memory formation,affective states, and motor function, among many other discoveries(2–4). For the channelrhodopsins, an important member of thisprotein family widely used in optogenetics (5, 6), the light-activatedcation-conducting channel pore has been the subject of structuralinvestigation, both because of curiosity regarding the physicalproperties of its ion conduction and because the creation of inhibitory channels had been sought for optogenetic applications.
www.pnas.org/cgi/doi/10.1073/pnas.1523341113

Author contributions: A.B., S.Y.L., and K.D. designed the project; A.B., S.Y.L., J.W., C.R.,
E.E.S., A.J.R., H.K., S. Park, A.S., S.M.I., S. Pak, and S.Ä.-R. performed research; C.R. contributed new reagents/analytic tools; A.B., S.Y.L., J.W., E.E.S., A.J.R., H.K., S. Park, A.S.,
P.W.F., S.M.I., S.Ä.-R., S.L.D., R.C.M., S.A.J., M.C., and P.H. analyzed data; A.B., S.Y.L., and
K.D. wrote the paper; and K.D. supervised all aspects of the work.
Reviewers: L.Y.J., University of California, San Francisco; and A.K., University of California,
San Francisco.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1A.B., S.Y.L., and J.W. contributed equally to this work.

2

To whom correspondence should be addressed. Email: deissero@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1523341113/-/DCSupplemental.

PNAS Early Edition | 1 of 8

BIOPHYSICS ANDCOMPUTATIONAL BIOLOGYContributed by Karl Deisseroth, November 30, 2015 (sent for review November 16, 2015; reviewed by Lily Yeh Jan and Anatol Kreitzer)

Results
Structural Determinants of Chloride Selectivity in Channelrhodopsins.

Previous work showed that iC1C2 (engineered using a crystal
structure-based electrostatic pore model) had higher chloride
selectivity and conductivity at lower pH (8). This pH effect wassuggested to be caused by protonation within the pore and morepositive local electrostatic potential, indicating that it might bepossible to enhance chloride flux at physiological pH further byfurther developing the electrostatic selectivity hypothesis (8). To
test this concept, we began by introducing a number of additional
point mutations to iC1C2, specifically targeting residues in the ionconduction pathway to enhance chloride flux guided by our model;
reversal potential (Vrev) and photocurrent size were tracked for all
variants (Fig. 1 A–D) in cultured hippocampal neurons using wholecell patch-clamp recordings.
The largest shift in Vrev was seen with E83 mutations, but these
also drastically reduced neuronal input resistance (Rin) (Fig. 1C), as
was consistent with creation of a leak current. We observed that
E83 is positioned close to R134 in iC1C2, which had been a histidine in the parental C1C2 chimera (Fig. 1 E and F) and indeed has
been conserved as a histidine in both wild-type channelrhodopsins 1
and 2. Because both E83 and R134 were shown to be inner poregate residues in the C1C2 structure (7), we considered theA[Cl-]ext 147 mM[Cl-]int 4 mMEpossibility that a disruptive interaction had been created here by
joint mutation at these two residues. Indeed, the longer arginineside chain (compared with histidine) could, in principle, interferewith channel closure of iC1C2-E83X mutants, increasing leak currents in the dark and leading to reduced Rin for these otherwiseinteresting E83 variants. Therefore we tested reversion of this arginine back to the histidine that is present in wild-type channelrhodopsins; as hypothesized (and as was consistent with our
structural model), R134H increased Rin and photocurrent size ofthe iC1C2-E83X mutations. E83N then was carried through to our
final construct here along with R134H (Fig. 1 A–C).
Next, chloride selectivity was enhanced further with structureguided pore replacement of two additional lysines by higher-pKa
arginines (K117R and K242R) and replacement of a second
negatively charged amino acid of the inner pore exit with aneutral amino acid (E273S, similar in expected electrostatic effect to E83N). Finally, replacement of serine 90 in iC1C2 withglutamine further hyperpolarized the Vrev; the final construct(the sequence is shown in SI Appendix, Fig. S1) displayed a significantly negatively shifted Vrev, larger photocurrent size at theaction potential (VAP) threshold, and larger Rin compared with
iC1C2 (Fig. 1 A–D). Because this mutant showed substantially
improved properties in all dimensions, we denote the variant

B

C

G

Red residues: new mutations in iC++Blue residues: same mutations in iC1C2 and iC++

E273S (312)

F

D

iC++

H

Pulse width:
30 ms

5 ms

[Cl-]ext 147 mM[Cl-]int 4 mM

E83N (122)

l

na

ti
Re

N258Q (297)

E123S (162)
T246N (285)
V117R (156)

Y70
(109)

E90Q (129)
T59S (98)

E101S (140)

R134
H134
(173)

E83
(122)

V242R (281)

Fig. 1. Design and characterization of iC++ in cultured neurons of rat hippocampus. (A) Vrev of iC1C2 mutations compared with chloride-conducting
channelrhodopsins at an intracellular chloride concentration of 4 mM. EN/Q/C: E83N/Q/C; ES: E273S; RH: R134H; SQ: S90Q; 2KR: K117R/K242R. (B) Photocurrent
amplitudes measured at the VAP (−51 ± 4 mV). (C) Rin of channelrhodopsin-expressing cells. (D) Stationary photocurrents per fluorescence of eYFP-tagged
constructs expressed in cultured neurons. (E) C1C2 structure depicting iC++ mutations. Numbering refers to N-terminal–truncated channel iC++. Corresponding positions in the original C1C2 structure are indicated in parentheses. (F) Structural model of the putative cytosolic ion gate in C1C2. Histidine 134(173) (gray) was replaced by arginine in iC1C2 (orange) but not in iC++. (G) Voltage trace of a cultured neuron expressing iC++. APs were evoked by pulsed
electrical inputs at 10 Hz (dotted line) and were inhibited by continuous light application (blue bar) for 4 s. (H) Inhibition probability of APs evoked by pulsed
current injections (6 s, 10 Hz) with pulse widths of 30 ms (Left), and 5 ms (Right) under 4-s light application. Current inputs were individually titrated to the
VAP. Shorter pulses required stronger current amplitudes. Average: 30 ms: 202 ± 11 pA; 5 ms: 559 ± 40 pA. pHext = 7.3, pHint = 7.2; [Cl−]ext = 147 mM, [Cl−]int = 4 mM for
entire figure. ***P < 0.005, ****P < 0.0001. Error bars indicate SEM; all values and numbers are listed in SI Appendix, Table S1.

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Berndt et al.

A

C1C2 - cation selective

TM7

TM3

B

C1C2 with iC++ replacements
chloride selective

iC++: Chloride Selectivity and pH Dependency. We next probed thebiophysical properties of iC++ in HEK293 cells to confirm Vrev
and chloride dependence in a setting independent of neuronal
active conductances. We observed, as expected, outward iC++
currents in the setting of inward-directed chloride gradients([Cl−]ext = 150 mM, [Cl−]int = 10 mM) (Fig. 3A and SI Appendix,
Fig. S2), with an initial peak stabilizing to a stationary current.
We systematically measured Vrev with varying intracellular chloride concentrations (Fig. 3 B and C); the Vrev of the peak andstationary currents closely matched the expected equilibrium potential under various chloride gradients, indicating the high chloride selectivity of the channel. We also found that iC++-expressing
HEK293 cells maintained Rin even under strong chloride gradientsin the dark; in contrast, iC1C2-expressing cells showed decreased
Rin in the setting of strongly shifted chloride gradients, indicatingthat iC1C2, but not iC++, may exhibit slight current in HEK293
cells in the dark under these culture conditions (Fig. 3D). The
peak activation wavelength of iC++ was measured to be 488 nm(SI Appendix, Fig. S2), suggesting that the properties of the retinalbinding pocket environment were maintained.
We previously reported that iC1C2 exhibited a degree of pH
dependency, with increased chloride conductivity and selectivity
at lower intracellular and lower extracellular pH (8). The newly
introduced mutations of iC++ were found here to reduce thispH-dependent increase in photocurrent size, confirming a stable,highly chloride-conducting state in the iC++ pore (Fig. 3 E and
F). Moreover, the Vrev of iC++ was independent of the proton
gradient, in contrast to iC1C2, indicating that iC++ does notconduct protons (Fig. 3G). Additional characterization with ionsubstitution experiments revealed that iC++ can conduct otheranions, such as iodide and bromide, but not small organic anions

C1C2 with iC1C2 replacements
chloride selective

C C1C2 with GtACR2 replacements
chloride selective

TM1
TM2

electrostatic potential:-1 kT/e, +1 kT/eFig. 2. Model for ion selectivity in channelrhodopsins. (A) Structure of the nonselective cation channel C1C2 (Protein Data Bank ID code: 3UG9) (7) withcalculated electrostatic potentials of transmembrane helices 1 (TM1) and 7 (TM7), which form the ion-conducting pore together with TM2 and TM3. The
electrostatic potential is predominantly negatively charged because of the large number of glutamates facing the pore interior (red: −1 kT/e). Charged groups
were considered fully protonated for a simplified first approximation. (B) Structures of C1C2 with replacements for iC++ (Left) and iC1C2 (Right) mutations.
The predominantly positive charge of the electrostatic potential of the pore presumably attracts anions and excludes cations (blue: +1 kT/e). (C) C1C2
structure with four replacements that are equivalently found in GtACR2: C1C2-E122S (GtACR2-S57), C1C2-E136T (GtACR2-T67), C1C2-E140A (GtACR2-A71),
and C1C2-E162S (GtACR2-S93). These replacements presumably contribute to the formation of a positively charged, anion-conducting pore.

Berndt et al.

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INAUGURAL ARTICLE

GtACR2-A71 (C1C2/iC++: E140/S101), and GtACR2-S93(C1C2/iC++: E162/S123) (SI Appendix, Fig. S1). This striking
convergence reveals that the engineering of iC1C2 and iC++ for
chloride conductance based entirely on the structure-derived
model of electrostatic pore selectivity (7, 8) resulted in a solution
very similar to that arrived at by nature over the long timescales of
evolution; the overall model for ion selectivity is consistent in bothnatural and engineered channelrhodopsins (Fig. 2).

BIOPHYSICS ANDCOMPUTATIONAL BIOLOGY

here as “iC++” (with the “++” notation both indicating the next
increment in the development of inhibitory channelrhodopsins
and referencing the expected increase in approximately two
positive charges in the pore resulting from the replacement of
two additional glutamate residues). Protein expression levels ofiC++ were comparable to those of iC1C2 (SI Appendix, Fig. S2),
but the effective photocurrent size of iC++ was more than 16 times
larger (Fig. 1D), whereas membrane properties of cells expressing
iC++ remained comparable to those of eYFP-expressing cells(SI Appendix, Fig. S2).
We next carried out in vitro verification of the capability of iC++
to inhibit electrically evoked action potentials (APs) elicited bycurrent injection. Because the use of continuously varying current
ramps can cause confounds from use-dependent alteration in bothchannelrhodopsin function and neuronal membrane properties, we
instead tested different current injection parameters independently,
beginning with separate delivery of strong and fast (5-ms) ormoderate and slow (30-ms) current injections, in each case titrated
to the level needed to drive trains of APs faithfully in the recorded
cell. iC++ demonstrated a robust spike inhibition capability, withparticular advantages over iC1C2 in the setting of short 5-ms pulse
widths where strong current amplitudes of >500 pA were required
to induce spiking (Fig. 1 G and H).
iC1C2 and iC++ are highly engineered (9 and 10 residues
were replaced, respectively) variants of the cation-selective parental construct C1C2, with mutations chosen for the predictedability to alter the electrostatic potential of the ion-conducting
pore from predominantly negative to predominantly positive (7, 8,
11). From the crystal structure we had predicted the channelrhodopsin pore to be relatively large, disordered, and without highaffinity ion-binding sites (7, 8, 11); the iC1C2/iC++ engineering
was designed accordingly and was predicted to exclude positively
charged cations and to conduct negatively charged ions such as
chloride (Fig. 2). Interestingly, the newly discovered natural lightgated chloride channel GtACR2 (10) exhibits finely detailed similarity in predicted pore electrostatics to the iC1C2 and iC++
mutations (Fig. 2), which were generated before knowledge of
GtACR2 and in which specific side chains facing the pore are
replaced by less polar or nonpolar residues, just as seen in
GtACR2: for example, GtACR2-S57 (C1C2/iC++: E122/N83),

A iC++

B

C

D

E

F

G

490 nm, 3 mW/mm2

I0[Cl-]ext 150 mM[Cl-]int 10 mM

IS
300 pA+ 40 mV

50 ms

0 pA
- 80 mV

Fig. 3. Biophysical characterization of iC++ in HEK293 cells. (A) iC++ photocurrents recorded at membrane potentials from −80 to +40 mV during lightapplication (blue bar). Peak currents (I0) decay to smaller stationary level (IS). (B and C) Amplitudes (B) and Vrev (C) of iC++ peak and stationary currents with150 mM external ([Cl−]ext) and varying internal [Cl−]int chloride concentrations. The Nernst equilibrium for chloride (ECl) is indicated for each condition (greenbar). (D) Change in Rin in iC1C2- and iC++-expressing HEK cells in the dark when [Cl−]ext was increased from 10 to 150 mM and [Cl−]int was fixed at 10 mM.
Values are normalized to [Cl−]ext = 10 mM (black line). iC++ does not conduct ions in the dark. (E) pH-dependent photocurrents of iC1C2 and iC++ at neutral(black, pHext 7.2) and acidic (red, pHext 5.0) external pH values, pHint 7.2. Amplitudes at different pH values were recorded from the same cells. (F) Photocurrent at pHext 5.0/0 mV normalized to values at pHext 7.2/0 mV (black line). (G) Vrevs from E. ****P < 0.0001. Error bars indicate SEM; all values and numbers
are listed in SI Appendix, Table S2.

that could be encountered in physiological systems, such as aspartate, bicarbonate, and gluconate (SI Appendix, Fig. S2).
iC++ Performance Reflects Applied Chloride Gradients. Next, we
moved to acute slice recordings to challenge iC++ over a rangeof physiological chloride gradients within intact tissue. To ourknowledge, no previous studies of chloride-conducting channelrhodopsins have varied chloride gradients, but photocurrents
and consequently the inhibition of APs depend not only on theexpression level in the plasma membranes but also on cytosolicand extracellular chloride concentrations, which vary among cell
types, subcellular regions, and developmental stages (12–15). We
constructed an adeno-associated viral vector (AAV) carrying thegene encoding iC++ fused to YFP and TS (the membrane-trafficking sequence used for optimizing microbial opsin expression inneurons; ref. 16), all under the control of the CaMKIIα promoter.
At 4 wk postinjection into mouse medial prefrontal cortex (mPFC),
robust fusion protein expression was observed (Fig. 4A). Cell
membrane properties were not significantly different from those ofcontrol cells expressing only eYFP (SI Appendix, Fig. S3).
For physiological relevance to the chloride-driving forces encountered during optogenetic experimentation, photocurrents
were measured at the membrane VAP for each cell. As expectedfrom the HEK cell data (Fig. 3 B and C), channel function(assessed by Vrev and photocurrent magnitude) faithfully tracked
chloride gradients (Fig. 4 B and C). To explore the ability of iC++
to inhibit spiking, distinct electrical current-injection protocols(titrated in strength to drive spiking in each individual cell reliably)
were tested under different chloride gradients. APs elicited bysteady current injections (which elicit more physiological, irregularly spaced spikes than do pulsed current injections) (Fig. 4D)
were especially susceptible to complete inhibition by iC++ over abroad range of intracellular chloride levels (Fig. 4E). When APs
were evoked by pulsed current injections, nearly complete spike
inhibition was observed even with intracellular chloride elevatedbeyond the typical physiological range of 4–20 mM, although the
inhibition in such cases could be partially overcome with extremely
strong (0.8-nA) current injections (SI Appendix, Fig. S3).
Next-Generation Step-Function Inhibitory Channelrhodopsin: SwiChR++.

One of the most useful properties of channel-type microbial opsins(compared with pumps) for optogenetics is the opportunity tocreate step-function opsin bistability (17), which allows ordersof-magnitude greater light sensitivity in expressing cells as wellas lasting modulation without continuous light delivery (17,4 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1523341113

18). Step-function iC1C2 variants, denoted “SwiChRCA” and“SwiChRCT,” were reported previously (17), although thecurrents were low and efficacy was tested only in vitro. Here,building from the iC++ backbone, we introduced the CA mutation C128A (8), which decelerated iC++ closure (τoff,fast =12.1 ms ± 0.4 SEM) by 9,500-fold (resulting in SwiChR++ with
τoff,dark = 115 s ± 9 SEM) (Fig. 5 A–D). SwiChR++ could bedeactivated immediately by red light (τoff,600nm = 0.15 s ± 0.02
SEM), enabling temporally delimited bistable control over chlorideA

iC++-eYFP in mouse mPFC

D

B

C

E

[Cl-]int: 4 mM

20 mM

[Cl-]ext 132 mM[Cl-]int 4 mM

Fig. 4. Chloride-dependent inhibition of pyramidal neurons in mouse mPFC.
(A) Confocal image of iC++ expression in mPFC 5 wk after virus injection. (Scalebar: 100 μm.) (B and C) Vrevs (B) and photocurrents (C) of iC++ at the VAP(−52 ± 1 mV) measured for 5 s with light application under varying internal
chloride concentrations ([Cl−]int: 4, 12, and 20 mM). Average Vrest = −80 mV ± 2.
(D) Voltage trace of an iC++-expressing neuron showing AP generation bycontinuous current injections (black bar) and inhibition during 10-s light application (blue bar). (E) Spike frequency before (0.5 s), during (10 s), and after (1 s)light application under varying internal chloride concentrations. Continuous
current injections were titrated individually to reach the VAP; average: 229 ±24 pA. n.s.: P > 0.05; ****P < 0.0001. Error bars indicate SEM; all values andnumbers are listed in SI Appendix, Table S3.

Berndt et al.

iC++ Inhibition of Neuronal Activity Across Diverse Cell Types andBrain Regions. To examine iC++-mediated inhibition of fast-

spiking interneurons, we performed in vivo extracellular recordings within the mPFC of anesthetized PV (parvalbumin)::
Cre mice injected with the Cre recombinase-dependent vector
AAV8-EF1a::DIO-iC++-EYFP (SI Appendix, Fig. S5). Spontaneous activity of PV+ interneurons was inhibited by light (SIAppendix, Fig. S5), with no reduction in the spike frequency ofprincipal (excitatory) mPFC neurons, as expected. To explore
the application range of iC++ further, we tested the ability ofiC++ to inhibit evoked APs within intact tissue, in this caseelicited by the potent neuromodulator oxytocin, which has beenshown to depolarize PV+ interneurons in the hippocampus selectively (19). We injected Cre-dependent AAV carrying iC++
into the hippocampus of PV::Cre mice, observing strong expression >4 wk postinjection (SI Appendix, Fig. S6). After confirming
iC++ inhibition of electrically evoked APs in these cells (SIAppendix, Fig. S6), we applied [Thr4,Gly7]-oxytocin (TGOT) to
depolarize iC++-expressing PV+ cells and drive spiking (SIAppendix, Fig. S6); light delivery during TGOT application

A 490 nm / 10 ms

SwiChR++iC++

490 nm / 10 ms

B

490 nmDirect Comparison of iC++ and eNpHR3.0 in Vitro: Higher Inhibition
Efficiency of iC++. We next carried out a direct comparison withthe commonly used chloride pump halorhodopsin engineered
from Natromonas pharaonis (eNpHR3.0). We delivered AAV8
encoding iC++, eNpHR3.0 (16), or eYFP into the mPFC of miceand assayed expression and activity 4 wk postinjection using
patch-clamp electrophysiology in acute slices. The mean Rin
observed in each condition was similar (SI Appendix, Fig. S3).
We began by testing inhibition efficiency in response to continuous current injection titrated in a stepwise manner (SI Appendix, SI Methods) and challenged with 2-s light pulses to drive
opsin expression (SI Appendix, Fig. S7 A and B).

C

600 nm

D

τfast

ext

[Cl-]int 10 mM
100 s

E

SwiChR++-eYFP in mouse mPFC

τslow

F

2s

100 ms

SwiChR++

100 pA

0 mV

100 [Clµm] 150 mM

G[Cl-]int: 4 mM

20 mM

[Cl-]ext 132 mM[Cl-]int 4 mM

Fig. 5. SwiChR++ characterization in HEK293 cells and neurons from acute mPFC slices. (A) Photocurrent traces of iC++ (red) and SwiChR++ (black)showing the time course of the channel closure. (Left) Traces were recorded in HEK293 cells at 0 mV in response to brief application of blue light (bluearrow). (Right) The same recordings at higher time resolution, depicting the biexponential channel closure of iC++ with a dominant fast component (τfast)
and a slow component (τslow). (B) Photocurrent of SwiChR++ upon the application of blue light (blue bar) and delayed red light (red bar), which accelerates channel deactivation. (C, Left) Tau values for biexponential channel closure of iC++. (Right) Amplitude ratios for the fast (τfast) and slow (τslow)
components. (D) Tau values for the monoexponential channel closure of SwiChR++ in the dark and with the application of light (600 nm). (E) Confocal
image of SwiChR++ expression in mouse mPFC 5 wk after virus injection. (Scale bar: 100 μm.) (F ) Voltage trace of an SwiChR++-expressing pyramidal
neuron showing APs evoked by continuous current injections (black bar) and inhibition for 10 s upon light application for 1 s (blue bar). Spiking recovers
upon red light stimulation (red bar). (G) Spike frequency before (0.5 s), during (10 s), and after (2 s) SwiChR activation at varying intracellular chloride
concentrations. Input currents were titrated individually to reach the VAP; average: 205 ± 27 pA. n.s.: P > 0.05; ****P < 0.0001. Error bars indicate SEM; allvalues and numbers are listed in SI Appendix, Table S4.

Berndt et al.

INAUGURAL ARTICLE

hyperpolarized iC++-expressing cells and abolished all induced
firing (SI Appendix, Fig. S6).
We broadened the validation to additional cell types, investigating iC++ function in dopaminergic neurons of acute slices
from substantia nigra pars compacta and in cultured peripheral
dorsal root ganglia neurons. APs were evoked by pulsed current
injections (10-ms pulse width). Internal chloride concentrations
were kept equal (12 mM) to allow study of inhibition independentof varying chloride gradients. Photocurrent amplitudes werecomparable, indicating similar expression levels across all cell
types. As before, inputs were titrated to reach the VAP reliably
to account for differences between individual cells and cell
types. Dopaminergic, dorsal root ganglia, and PV cells all
spiked with high reliability even in response to moderate current injection [<360 pA, consistent with relatively depolarized
resting membrane potential (Vrest)], and this spiking was
blocked with high efficiency by iC++ (SI Appendix, Fig. S6).

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BIOPHYSICS ANDCOMPUTATIONAL BIOLOGY

conductance and neuronal inhibition (Fig. 5 B and D). SwiChR++
expressed in cultured hippocampal neurons exhibited higher photocurrents and a more negatively shifted Vrev than SwiChRCA,
which resulted in more effective AP inhibition (SI Appendix,
Fig. S4). SwiChR++ also showed robust in vivo expression andbistable properties over long timescales in mPFC slices, in which
Vrev, photocurrents, and AP inhibition also depended, as expected,
on chloride gradients (Fig. 5 E–G).

A

B

AAV-DIO-iC++-eYFP
AAV-DIO-eNpHR3.0-eYFP
AAV-Arch3.0-eYFP

Optical fiberiC++

eNpHR3.0

Arch3.0
DAT:Cre mouse

C

Fig. 6. Place aversion induced by inhibition of dopaminergic VTA neurons
in mice. (A) An AAV encoding a Cre-dependent opsin (iC++, eNpHR3.0, or
eArch3.0 n = 4 per group) was injected into the VTA of DAT:Cre mice, andoptical fibers were implanted dorsal to this area. (B, Upper) Schematic of thereal-time place-aversion task. Mice were allowed to explore a two-compartment chamber freely. Entry into one compartment triggered continuous
illumination (473 nm for iC++; 532 nm for eNpHR3.0 and eArch3.0) as longas the mouse remained in the light-paired compartment. The test was repeated, with distinct contextual cues to minimize generalization, at multiple
light intensities. (Lower) Representative tracking data from the 5-mW experiment demonstrating robust avoidance of the light-paired compartment.
(C) Percent time spent in the light-paired compartment at various light intensities. No side preference was observed when the light intensity was
0 mW (light-off control), but at 0.5 mW and 5 mW all groups avoided thelight-paired compartment.***P < 0.005. Error bars indicate SEM; all values
and numbers are listed in SI Appendix, Table S5.

Pyramidal cells are especially susceptible to becoming refractory to spiking when large inputs are applied, because ofdepolarization block. Although pyramidal cells are common
targets of optogenetic intervention, this property complicates theassessment of inhibition mediated by opsins when challenged
with strong inputs; therefore during titration we did not increase theamplitude of the current input further when the depolarizationblocking potential was reached under light. We found that lightdelivered to more than 90% of iC++-expressing cells could inhibit

A iC++/CREB-eYFP in B
mouse lateral amygdala

Non-allocated, inactive neuron
Allocated, active engram neuron
Allocation

any current input tested; conversely, all the eNpHR3.0-expressing
cells started to spike at some current amplitude under light (SIAppendix, Fig. S7 D and E). Moreover, eNpHR3.0-expressing cells
could inhibit only responses to input currents that were equal to themagnitude of eNpHR3.0 photocurrent; in contrast, iC++-expressing
cells were at least seven times more efficient and exhibited aprominent light-induced reduction in Rin (SI Appendix, Fig. S7F).
As a result, iC++-expressing cells weakened the impact of current
inputs more efficiently than pump-expressing cells, with larger
current inputs necessary to evoke APs during illumination (SI Appendix, Fig. S7 G and H). Similar results were observed with pulsed
current injection; iC++-expressing cells inhibited a much wider
range of input current amplitudes (SI Appendix, Fig. S7I). The main
mechanism of this efficient inhibition from iC++ is likely via
shunting inhibition, because the Vrev of iC++ lies between the VAP
and Vrest (revealing that the inhibition is not driven by strong hyperpolarization). Reduction in Rin through iC++ activation will
reduce excitability, just as seen with endogenous chloride-conducting channels such as GABAA receptors, providing efficient inhibition via shunting of excitatory inputs (20).
Behavioral Comparison of iC++, eNpHR3.0, and eArch3.0: Real-Time
Place-Aversion Test. We next sought to determine if iC++-medi-

ated inhibition would be capable of modifying behavior in freely
moving, nonanesthetized animals. We injected AAVs encoding
iC++ or, for comparison, eNpHR3.0 or eArch3.0 (21), into theventral tegmental area (VTA) of DAT::Cre mice for expression
in dopaminergic neurons (Fig. 6A and SI Appendix, Fig. S8. Fourweeks later, we optogenetically inhibited VTA dopamine neurons via implanted optical fibers to assess the ability of each opsin
to drive behavioral changes in a real-time place-aversion assay.
Consistent with prior work (22, 23), we observed robust avoidanceof the light-paired compartment at multiple light intensities in allgroups (two-way repeated-measures ANOVA, main effect of light
power, F2, 18 = 64.529, P < 0.001; Holm–Sidak post hoc tests,
0 mW vs. 0.5 or 5 mW, P < 0.005) (Fig. 6 B and C). Under theseexperimental conditions, each opsin was equally effective indriving real-time place aversion (two-way repeated-measures
ANOVA, no effect of group or group × light power interaction,
P > 0.474). Thus, iC++ is suitable for use in vivo to achieve
behaviorally relevant alteration of neural activity.
Inhibition of Memory Retrieval: Potent Behavioral Efficacy of iC++.

To test behavioral efficacy further, we used a well-validated assay
for memory (involving neuronal allocation to participation in

C

Allocated, inactive engram neuron
Allocated, inhibited engram neuron

Encoding

Retrieval(light off)

Retrieval(light on)

Shock / Tone

Tone

Tone
Freezing

Freezing

Home Cage

Freezing

AmygdalaFig. 7. Using inhibitory channelrhodopsins to silence neurons involved in the engram supporting fear memory. (A) Robust, localized transgene expression (green) following microinjection of HSV-CREB/iC++-YFP vector into the LA. (Scale bar: 100 μm.). (B) Cartoon of experimental methods used totest the behavioral effects of inhibitory opsins. Mice were microinjected with viral vectors expressing CREB/iC++, CREB/iC1C2, or CREB/eNpHR3.0 into the
LA before auditory fear conditioning. Neurons overexpressing CREB are preferentially allocated (red) to the engram supporting the resulting auditory
fear memory. Using light to silence these neurons optogenetically results in decreased freezing, indicating impaired memory retrieval. (C ) Neurons
overexpressing CREB before training are preferentially allocated to the engram that supported fear memory. Silencing these neurons using iC++, iC1C2,
or eNpHR3.0 reversibly disrupted memory retrieval. Disrupting the activity of the same number of neurons that are not allocated to the fear engram(those not overexpressing CREB) did not impair memory retrieval. n.s.: P > 0.05; ***P < 0.005. Error bars indicate SEM; all values and numbers are listed in
SI Appendix, Table S6.

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Berndt et al.

Discussion
Here we describe the engineering of enhanced light-gated chloride
channels (iC++ and SwiChR++) by structure-guided modificationof ion selectivity, show improved efficiency for optical inhibition ofneurons with these proteins, and demonstrate initial behavioral
control in freely moving mammals. The earlier iC1C2 channel had
shown increased chloride conductivity at low pH values, suggestingthat more improvement might be seen under more neutral pH
values. We hypothesized that this pH dependency resulted frombasic residues in the pore region of iC1C2, with higher probabilityof protonation under acidic conditions that established favorable
electrostatic interactions for chloride ions. By removing more negatively charged residues from the ion-conducting pore and introducing two arginines, we increased the chloride selectivity andalso strongly reduced the pH dependency of chloride conductivity.
The enhancements comprising iC++ support the model of channelrhodopsin selectivity (8) in which ions are selected primarily byelectrostatic interactions between pore and ions. Strikingly, thismodel is validated further by the recently reported naturally anionconducting GtACR channelrhodopsins (10), which contain amino
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Berndt et al.

Materials and Methods
Data analysis and all in vitro methods, including cloning of vectors, virus production, preparation and transfection of cultured neurons and HEK cells, histology, and in vitro electrophysiology, are described in detail in SI Appendix, SI
Methods. Likewise all methods related to animal subject experimentation, including acute slice preparation and physiology, stereotactic surgery, in vivo
electrophysiology, and behavioral tests in freely moving animals, are described in
SI Appendix, SI Methods. All animal procedures were approved by the Stanford
Administrative Panel on Laboratory Animal Care and are in accordance with the
National Institutes of Health guidelines for animal research (37).
ACKNOWLEDGMENTS. We thank the entire K.D. laboratory for helpful
discussions. K.D. is supported by the National Institute of Mental Health, theSimons Foundation Autism Research Initiative, the National Institute on
Drug Abuse, the Defense Advanced Research Projects Agency, the Gatsby
Charitable Foundation, and the Wiegers Family Fund. A.B. received support
from the German Academic Exchange Service (DAAD) and S.Y.L. received
support from the Fidelity Foundation. The M.C. laboratory is supported by
the Ragnar Söderbergs Foundation, the Knut and Alice Wallenbergs Foundation, the Swedish Brain Foundation (Hjärnfonden), and the European Research Council. The P.H. laboratory is supported by German Research
Foundation (DFG) (SFB1078 B2, FOR1279 SPP1665 to P.H.). The S.A.J. laboratory
is supported by the Canadian Institutes of Health Research (CIHR). The S.L.D.
laboratory is supported by National Institutes of Health Grant R01NS080954 (toS.L.D. and K.D.). The R.C.M. laboratory is supported by National Institutes of
Health Grant 5P50MH086403 (to R.C.M. and K.D.). Both acute and chronictimescale optogenetic tools for reversible inhibition and other methods reported
in this paper are distributed and supported freely (www.optogenetics.org).

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INAUGURAL ARTICLE

acids at key positions within the ion pore equivalent to those in theearlier designed and reported iC1C2 and in iC++ (11).
In addition to testing the hypothesized mechanisms underlying
ion selectivity of channelrhodopsins, we also explored the utility ofthese tools for neuronal inhibition. We observed that, because ofstronger shunting, the chloride channel iC++ inhibited APs more
efficiently than the chloride pump eNpHR3.0. Although optogenetic inhibitory pumps display more extreme Vrev than the inhibitory channels described here, the pumps can hyperpolarize cells
far beyond physiological levels, causing unstable membrane properties (21). This effect can be reduced by lowering light intensity,
although this strategy also severely reduces the volume of tissue
inhibited by the pumps, which are less light sensitive than channels
and, unlike the channels, cannot be converted to a more lightsensitive bistable step-function form. In addition, microbial opsin
pump currents will not show the influence of ion gradients reported
here for channels; such gradients can differ across cell types, subcellular regions, developmental stages, and pathological states (12–15, 30), even giving rise to excitation under some conditions. For thesame reason, iC++ currents will mirror the physiological effects ofendogenous open chloride channels more precisely (31, 32).
iC++ and related channels (33) represent a different class of
optogenetic tool, which, like any reagent in biology, should be selected based on suitability for preparation and hypothesis. Here wedemonstrate the initial use of chloride-conducting channelrhodopsins in the behavior of freely moving animals performing tasks
involving the VTA and LA. Beyond this generation of these
optogenetic inhibitors that take advantage of physiological ion gradients rather than pumping ions unidirectionally, iC++ and related
tools (8–10, 33) also lay the groundwork for a deeper understandingof structure–function relationships involving the light-gated ion
conductance pathway of channelrhodopsins. Along with prior work
on chloride channel engineering (11, 34–36), this second round of
predictive validation of the crystal structure-guided electrostatic
model for the channelrhodopsin pore (7, 8, 11) provides basic insight
into the operation of the microbial opsin channel proteins.

BIOPHYSICS ANDCOMPUTATIONAL BIOLOGY

memory engrams). Neurons in the lateral nucleus of the amygdala (LA) are allocated by local circuitry to become part of theengram supporting a discrete cued fear memory (24, 25) in whicha tone is paired with a footshock (26–28). Increasing the functionof the transcription factor CREB (Ca2+/cAMP response element-binding protein) in individual pyramidal (principal) LA
neurons increases the likelihood that the CREB-enhanced neuron will be allocated to a memory engram (24, 25, 29); posttraining genetic ablation of LA neurons overexpressing CREB
(but not ablation of a comparable number of random LA neurons) impairs memory expression (29).
Optogenetic inhibition of neurons overexpressing CREB wastested for the elicitation of reversible impairment of memory
expression (Fig. 7A). The short time course of this manipulation
allowed comparison of tone-induced freezing behavior in individual mice in the presence and absence of optical inhibition(Fig. 7B). We used viral vectors expressing vCREB and iC++,
vCREB and iC1C2, or vCREB and eNpHR3.0 (or each inhibitory opsin on its own) to transduce a small portion (∼15%) of
the LA pyramidal neurons before fear conditioning and tested
mice in both the presence and absence of light. Optogenetic inhibition of vCREB neurons using iC++, iC1C2, or eNpHR3.0 significantly reduced tone-induced freezing, whereas silencing a similar
number of neurons not overexpressing CREB had no effect [light(on, off) × virus ANOVA yielded a significant interaction, F6,52 =11.83, P < 0.001] (Fig. 7C). Post hoc analyses on the significant
interaction showed that light significantly decreased freezing in thevCREB/iC++, vCREB/iC1C2, and vCREB/eNpHR3.0 groups (P <0.001) but not in the iC++, iC1C2, eNpHR3.0, or vCREB-onlygroups (P > 0.05). Of interest, the total freezing scores in the vCREB/
iC++ group were even lower than those of the vCREB/iC1C2 groupin the light-on condition (P < 0.05). However, considering thebaseline freezing when light was off, the reduction in freezing was
not significantly different among the vCREB/iC++, vCREB/iC1C2,
and vCREB/eNpHR3.0 groups (P > 0.05). These results support
the interpretation that silencing neurons overexpressing CREB at
the time of training disrupts memory expression and demonstrate theinitial behavioral efficacy of chloride-conducting channelrhodopsinmediated optogenetic inhibition.

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