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Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability applied sciences Article Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability Xiaodong Duan, Georg Nagel * and Shiqiang Gao * Botanik I, Julius-Maximilians-Universität Würzburg, Biozentrum, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany; xiaodong.duan@stud-mail.uni-wuerzburg.de * Correspondence: nagel@uni-wuerzburg.de (G.N.); gao.shiqiang@uni-wuerzburg.de (S.G.) Received: 14 January 2019; Accepted: 12 February 2019; Published: 15 February 2019 Featured Application: This study provides optogenetic tools with superior photocurrent + 2+ amplitudes and high Na and Ca conductance. Abstract: (1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly 2+ discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant + 2+ (XXM) showed enhanced Na and Ca conductance, which was not noticed before, while the + 2+ D156C mutation (XXL) influenced the Na and Ca conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a + + much-improved photocurrent, compared to wild type, and even higher Na selectivity to H than 2+ XXM. PsChR D139H also showed a strongly enhanced Ca conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na selectivity and 2+ Ca conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for 2+ Ca manipulation. Keywords: optogenetics; channelrhodopsins; sodium; calcium; DC gate 1. Introduction Channelrhodopsins were first discovered and characterized from C. reinhardtii [1,2]. After the showing of light-switched large passive cation conductance in HEK293 and BHK cells by Nagel et al., the ChR2 (C. reinhardtii channelrhodopsin-2) was immediately applied in neuroscience by several independent groups for studies in hippocampal neurons [3,4], Caenorhabditis elegans [5], inner retinal neurons [6], and PC12 cells [7]. H134R (histidine to arginine mutation at position 134) was the first ChR2 gain-of-function mutant which showed enhanced plasma membrane expression and larger stationary photocurrents in comparison to ChR2 wild type [5]. Other variants came out in rapid sequence, either of natural origin or mutated and engineered. The calcium translocating channelrhodopsin CatCh (a ChR2 leucine to cysteine mutation at position 2+ 132, L132C) showed improved Ca conductivity together with a larger photocurrent and higher light sensitivity [8]. Newly discovered Chronos (Stigeoclonium helveticum channelrhodopsin = ShChR) and Chrimson (CnChR1 from Chlamydomonas noctigama) showed a faster channel closing and a red-shifted action spectrum, respectively [9]. An E90R (glutamate to arginine mutation at position 90) Appl. Sci. 2019, 9, 664; doi:10.3390/app9040664 www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, 664 2 of 11 point mutation could extend the cation conductance of ChR2 to additional anion conductance [10]. Naturally, very specific anion conductive channelrhodopsins, GtACR1 and GtACR2 (Guillardia theta anion channelrhodopsin 1 and 2), were discovered afterwards [11]. Mutation of ChR2 C128 (cysteine at position 128) to threonine (T), alanine (A), and serine (S) slowed the closing kinetics dramatically [12–14]. Mutation of ChR2 D156 (cysteine at position 156) to alanine also decreased the closing kinetics [14]. Spectral studies suggested that a putative hydrogen bond between C128 and D156 could be an important structural determinant of the channel’s closing reaction [14] or might represent the valve of the channel [15]. Thus, hydrogen bond-linked D156 and C128 was proposed as the putative gate buried in the membrane (“DC gate”) [14,15]. But the first channelrhodopsin structure was a chimaera (C1C2) of truncated ChR1 and ChR2, and the distances between C167 (corresponding to C128 in ChR2) and D195 (corresponding to D156 in ChR2) are too far away to be associated by a hydrogen bond [16]. However, the recently solved structure of wild type ChR2 revealed a water molecule between C128 and D156 to bridge them, indeed, by hydrogen bonds [17]. Mutation of the DC gate has a strong effect on the open channel lifetime. The ChR2 D156C (aspartate to cysteine mutation at position 134) mutant (XXL) generated very large photocurrents and is 1000-fold more light-sensitive than wild type ChR2 in Drosophila larvae [18]. The ChR2 D156H (aspartate to histidine mutation at position 134) mutant (XXM) also showed a superior photo stimulation efficiency with faster kinetics than XXL, which made it an ideal optogenetic tool for Drosophila neurobiological studies [19]. The aspartate D156 in ChR2 is located close to the protonated retinal Schiff base (RSBH+). Thus, mutations of D156 logically have strong effects on the open channel lifetime by influencing the protonation state of the retinal Schiff base. However, the water-bridged C128 and D156 are not in the putative ion pore proposed by Volkov et al. [17]. And no attention had been paid to the potential changes of ion selectivity by DC gate mutations. In this study, we compared the ion selectivity of our previously published XXL and XXM + + and found that XXM showed a four-fold increased Na selectivity over H together with a + + two-fold increased K selectivity over H , compared to wild type ChR2. Based on this finding, we made further aspartate to histidine and cysteine mutations of PsChR (Platymonas subcordiformis channelrhodopsin) [20] and Chronos [9]. PsChR wild type was already reported to be highly Na + + + conductive and indeed showed a six-fold increased Na and K selectivity over H compared to wild type ChR2 in our measurements. But the D139H mutation of PsChR further increased the Na selectivity over H five-fold. Furthermore, PsChR D139H showed a 5-fold larger photocurrent than PsChR wt. 2+ 2+ We further compared the Ca permeability of these mutants. XXM showed an increased Ca 2+ current compared to CatCh [8]. PsChR wild type already showed a good Ca current, but the D139H 2+ mutation further increased the Ca current. We concluded that the mutant PsChR D139H would be a 2+ powerful tool for optogenetic Ca manipulation. 2. Materials and Methods 2.1. Plasmids and RNA Generation for Xenopus Laevis Oocyte Expression ChR2, XXM, and XXL in the pGEMHE vector were described in previous studies [18,19,21]. PsChR (from Platymonas subcordiformis, Accession No.: JX983143) and Chronos (from Stigeoclonium helveticum, Accession No.: KF992040) were synthesized by GeneArt Strings DNA Fragments (Life Technologies, Thermo Fisher Scientific), according to the published amino acid sequences, with the codon usage optimized to Mus musculus. The synthesized DNA segment was inserted into the pGEMHE vector with N-terminal BamHI and C-terminal XhoI restriction sites. Yellow fluorescent protein (YFP), together with a plasma membrane trafficking signal (KSRITSEGEYIPLDQIDINV) [22] beforehand and an ER export signal (FCYENEV) [22] afterward, the YFP was attached to the C-terminal Appl. Sci. 2019, 9, 664 3 of 11 end. Mutations were made by QuikChange Site-Directed Mutagenesis. The sequence was confirmed by DNA sequencing. Plasmids were linearized by NheI digestion and used for in vitro generation of cRNA with the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies). 2.2. Two-Electrode Voltage-Clamp Recordings of Xenopus Laevis Oocytes cRNA-injected oocytes were incubated in ND96 solution (96 mM NaCl, 5 mM KCl, 1 mM MgCl , 1 mM CaCl , 5 mM HEPES, pH 7.4) containing 1 M all-trans-retinal at 16 C. Two-electrode 2 2 voltage-clamp (TEVC) recordings were performed with solutions, as indicated in figures, at room 2+ 2+ temperature. For experiments with external Ca , we blocked activation of the Ca -activated 0 0 endogenous chloride channels of oocytes by 1,2-bis(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic 2+ acid (BAPTA) injection. We injected 50 nl 200 mM of the fast Ca chelator BAPTA (potassium–salt) into each oocyte (~10 mM final concentration in the oocyte), incubated for 90 mins at 16 C and then performed the TEVC measurement at room temperature. Twenty nanograms of cRNA were injected into Xenopus oocyte for all the constructs. Photocurrents were measured two days after injection. For Figures 1 and 2, measurements were performed in standard solution with BaCl instead of CaCl 2 2 (110 mM NaCl, 5 mM KCl, 2 mM BaCl , 1 mM MgCl , 5 mM HEPES and pH 7.6). 2 2 2.3. Light Stimulation Illumination conditions were different, considering the published action spectra of ChR2, Chronos, and PsChR: 473 nm for ChR2, XXM, and XXL; 445 nm for PsChR, PsChR D139H, and PsChR D139C; 532 nm for Chronos, Chronos D173H, and Chronos D173C. Lasers were from Changchun New Industries Optoelectronics Technology. Light power was set to 5 mW/mm , except for the light sensitivity measurement. The light intensities were measured with a PLUS 2 Power & Energy Meter (LaserPoint s.r.l). For light sensitivity measurement, applied light intensities ranged from 1.7 to 5000 W/mm . 2.4. Protein Quantification by Fluorescence All expression levels of channelrhodopsin variants in oocytes were quantified by the fluorescence emission values of the YFP-tagged protein. Fluorescence emission was measured at 538 nm by a Fluoroskan Ascent microplate fluorometer (Thermo Scientific) with 485 nm excitation. 2.5. Fluorescence Imaging Fluorescence pictures of Xenopus oocytes were taken under 5x objective with a Leica DM6000 confocal microscope after two days’ expression. Oocytes were put in a 35 x 10 mm petri dish (Greiner GBO) containing ND96 for imaging. Excitation was done using 496 nm laser light. Fluorescence emission was detected from 520 nm to 585 nm. 2.6. Data Processing pClamp 7.0 was used to read out the photocurrent. Figures 1a, 2, 3, 4, 5a and A1a were made with OriginPro 2017. Figures 1b, 5b and A1b were made with GraphPad Prism. Tables 1 and 2 were made with Microsoft Excel. Sequence alignment in Appendix B was performed by BioEdit. Closing time was determined by biexponential fit. Light sensitivity curves were fitted with Hill equation. All values were plotted or presented with mean values, and error bars represent the standard deviations (SD) or standard error mean (SEM), as indicated in each figure. Statistical analysis was done by t test within GraphPad Prism. Differences were considered significant at p < 0.05. *** = p < 0.001, ** = p < 0.01, * = p < 0.05. Appl. Sci. 2019, 9, x 4 of 11 136 compared to ChR2 (Figure a1). The steady-state photocurrents were increased ~30- and ~48-fold for 137 XXM (D156H) and XXL (D156C), respectively, compared to ChR2 (Figure 1, Table 1). The enhanced 138 photocurrent might have been a comprehensive outcome of the higher plasma membrane expression 139 leAppl. vel, h Sci. igh 2019 er ,li9g , h 664 t-sensitivity, or increased single channel conductance. Both XXM and XXL show 4e of d 11 140 much-prolonged closing kinetics, leading to higher light-sensitivity (Figure 1a, Table 1). Appl. Sci. 2019, 9, x 5 of 11 142 Figure 1. Comparison of ChR2, PsChR, and Chronos variants. (a) Representative photocurrent traces Figure 1. Comparison of ChR2, PsChR, and Chronos variants. (a) Representative photocurrent traces 160 density (LPD) for 50% photocurrent (EPD50) of ~ 3.2 µW/mm , which was ~ 250 times more sensitive 143 of ChR2, PsChR, Chronos, and their mutants. (b) Comparison of stationary photocurrents of above of ChR2, PsChR, Chronos, and their mutants. (b) Comparison of stationary photocurrents of above 161 than PsChR (Figure 2, Table 1). The EPD50 for XXL was ~ 5.4 µW/mm , which was ~ 130 times more 144 channelrhodopsin variants. All data points were plotted in the figure and mean ± standard error mean channelrhodopsin variants. All data points were plotted in the figure and mean standard error mean 162 sensitive than ChR2 (Figure 2, Table 1). 145 (SEM) (n = 7-8) was indicated. (SEM) (n = 7–8) was indicated. 146 Table 1. Basic properties of ChR2, PsChR, and Chronos variants. Closing time (ms) EPD50 Expression* Is ǂ ǂ 2 (µW/mm ) τ1 (% ) τ2 (% ) Ϯ Ϯ ChR2 1× 1× 7.5 ± 0.5 (> 98) - 710 XXM 2.7 × 30 × 80 ± 5.5 (8) 1100 ± 110 (92) 90 71000 ± 2900 (> XXL 3 × 48 × - 5.4 96) Ϯ Ϯ Chronos 1 × 1 × 2.8 ± 0.3 (> 99) - 500 Chronos D173H 2.2 × 1.2 × 1400 ± 160 (56) - 84 Figure 2. Comparing the light sensitivities of ChR2, PsChR, and Chronos variants. (a) 5 s, 30 s, Chronos D173C 2.6 × 10 × 22 ± 1.3 (85) 130 ± 16 (15) 190 164 Figure 2. Comparing the light sensitivities of ChR2, PsChR, and Chronos variants. (a) 5 s, 30 s, and and 100 s continuous 473 nm blue light illumination were applied to ChR2, XXM and XXL, respectively. Ϯ Ϯ 165 P1 sC 00h sR c ontinuous 471 3 n ×m blue ligh1 t i× llu mina8 t. i5 o n ± w 0.e6 r e ( > a p 9p 8l)i ed to ChR2, X - XM and XXL, respe 8 c1 ti0 v ely. Photocurrent at maximum light power density of each oocyte was normalized as 1. (b) 5 s continuous 166 PsChP Rh D ot1 o3 cu 9H rre nt at max 2i.m 2 u ×m light pow 6 e ×r densit3 y7 o ± f e 2 a.c 7h ( o 1o 4c ) yte was 8 n1 o0 rm ±a 5 li4 z e (d 86 a)s 1. (b) 5 s con1 ti6 n0 u ous 532 nm blue light illumination were applied to Chronos, Chronos D173H, and Chronos D173C. 167 532 nm blue light illumination were applied to Chronos, Chronos D173H, and Chronos D173C. PsChR D139C 3.1 × 11 × 3.4 ± 0.8 (4) 74000 ± 4300 (96) 3.2 Photocurrent at maximum light power density of each oocyte was normalized as 1. (c) 5 s, 30 s, and 100 168 Photocurrent at maximum light power density of each oocyte was normalized as 1. (c) 5 s, 30 s, and 147 Ϯ, se continuous xpression o 445 r pnm hoto blue currlight ent o illu f cmination orrespond wer ing e applied wild typ to e Ps w ChR, as no Ps rm ChR alizD139H, ed as 1and x. IsPs , s ChR tatioD139C. nary 169 100 s continuous 445 nm blue light illumination were applied to PsChR, PsChR D139H, and PsChR 148 (pPhotocurr lateau) cuent rren at t. maximum ǂ, as most light ChRspower exhibidensity t biphasof ic each off-koocyte inetics was whic normalized h comprised as a1. fa Photocurr st and a s ents low of 170 D139C. Photocurrent at maximum light power density of each oocyte was normalized as 1. 149 coXXM, mpone XXL, nt, h Ps erChR e the D139H, % indand icate Ps d ChR the D139C percentwer agee o measur f the a ed mp atli tu60 demV of because the fast of (τthe 1) olar r s ger low curr (τ2ent ) 171 Photocurrents of XXM, XXL, PsChR D139H, and PsChR D139C were measured at −60 mV because of and slower kinetics; other constructs were measured at 100 mV. Data points were presented as mean 150 component to the whole photocurrent. Data are shown as mean ± SEM, n = 4-5. Values are presented 172 the larger current and slower kinetics; other constructs were measured at −100 mV. Data points were SD, n = 3–4. 151 as approximates. *, expression level was calculated from the data of fluorescence value in Figure A1. 173 presented as mean ± SD, n = 3–4. 3. Results 152 We further synthesized Chronos [9] and PsChR [20] and characterized the corresponding 174 3.2. Mutation of the Aspartate in TM4 Influences the Na Permeability 153 aspartate to cysteine, and the histidine mutations as the aspartate in transmembrane helix 4 (TM4) 3.1. Mutating the Conserved Aspartate of TM Helix 4 Influences the Expression, Photocurrent, and Kinetics 175 The potential influence on ion selectivity by mutating ChR2 D156 was not reported. To 154 were conserved in all three channelrhodopsins (Appendix B). Chronos D173C, D173H, and PsChR 176 invesSimilar tigate th to is,pr w eviously e measur published ed the pho rt esults ocurre[n 18 t ,a 19 t ], dif fluor feren escence t potentmeasur ials and ements calcula of ted whole the re oocyte versal 155 D139H, D139C, all showed an increased expression level, compared to their wild type (Figure 1a). 177 membranes potential shifwith t of th YFP-t ese m agged utants,XXM whenand syste XXL matishowed cally chan ag~ ing thr bee-fold ath soluincr tioneased s with expr diffeession rent pH level, and 156 All mutants also showed increased light-sensitivities along with prolonged off kinetics (Figure 2, + + 178 compar Na or K ed cto onChR2 centra(Figur tions. e A1). The steady-state photocurrents were increased ~30- and ~48-fold for 157 Table 1). Chronos D173C, PsChR D139H and PsChR D139C showed dramatically increased 179 XXMC (D156H) hR2 is a and non XXL -sele (D156C), ctive catr iespectively on channe,l compar which ed is m too ChR2 stly p (Figur ermea eb1 leand to T H able [2]1 . ).BThe oth enhanced changing 158 photocurrents while the Chronos D173H was similar to the wild type Chronos (Figure 1, Table 1). 180 photocurr extracelluent lar p might H from have 7.6 been to 9.6a acompr nd cha ehensive nging exoutcome tracellulaof r N the a c higher oncent plasma ration fmembrane rom 120 mM expr to ession 1 mM 159 Among these variants, PsChR D139C was the most light-sensitive with an effective light power + + 181 level, altered higher the Ch light-sensitivity R2 reversal pot,eor ntiincr al (F eased igure single 3). Thechannel ChR2 pe conductance. rmeability ratBoth io of XXM Na toand H (XXL PNa+/P showed H+) was −7 + 182 much-pr determin olonged ed as P closing Na+/PH+ kinetics, = 3.1×10leading (Tablto e higher 2). Intlight-sensitivity erestingly, D156 (Figur H (Xe X1 M a)and infT lu able ence 1d ). the Na 183 permeability and increased the PNa+/PH+ four times, while D156C (XXL) changed the PNa+/PH+ only 184 slightly (Table 2). 100 + V shift Na (mV) V shift H (mV) ChR2 XXM XXL Chronos Chronos D173H Chro os D173 n C PsChR PsChR D139H PsChR D139C V shift (mV) r Appl. Sci. 2019, 9, x 5 of 11 160 density (LPD) for 50% photocurrent (EPD50) of ~ 3.2 µW/mm , which was ~ 250 times more sensitive 161 than PsChR (Figure 2, Table 1). The EPD50 for XXL was ~ 5.4 µW/mm , which was ~ 130 times more 162 sensitive than ChR2 (Figure 2, Table 1). 164 Figure 2. Comparing the light sensitivities of ChR2, PsChR, and Chronos variants. (a) 5 s, 30 s, and 165 100 s continuous 473 nm blue light illumination were applied to ChR2, XXM and XXL, respectively. 166 Photocurrent at maximum light power density of each oocyte was normalized as 1. (b) 5 s continuous 167 532 nm blue light illumination were applied to Chronos, Chronos D173H, and Chronos D173C. 168 Photocurrent at maximum light power density of each oocyte was normalized as 1. (c) 5 s, 30 s, and 169 100 s continuous 445 nm blue light illumination were applied to PsChR, PsChR D139H, and PsChR 170 D139C. Photocurrent at maximum light power density of each oocyte was normalized as 1. 171 Photocurrents of XXM, XXL, PsChR D139H, and PsChR D139C were measured at −60 mV because of 172 the larger current and slower kinetics; other constructs were measured at −100 mV. Data points were 173 presented as mean ± SD, n = 3–4. 174 3.2. Mutation of the Aspartate in TM4 Influences the Na Permeability 175 The potential influence on ion selectivity by mutating ChR2 D156 was not reported. To 176 investigate this, we measured the photocurrent at different potentials and calculated the reversal 177 potential shift of these mutants, when systematically changing bath solutions with different pH and + + 178 Na or K concentrations. 179 ChR2 is a non-selective cation channel which is mostly permeable to H [2]. Both changing 180 extracellular pH from 7.6 to 9.6 and changing extracellular Na concentration from 120 mM to 1 mM + + 181 altered the ChR2 reversal potential (Figure 3). The ChR2 permeability ratio of Na to H (PNa+/PH+) was −7 + 182 determined as PNa+/PH+ = 3.1×10 (Table 2). Interestingly, D156H (XXM) influenced the Na 183 perm Appl. Sci. eab 2019 ility , 9,a 664 nd increased the PNa+/PH+ four times, while D156C (XXL) changed the PNa+/PH+ 5o of nl11 y 184 slightly (Table 2). 100 + V shift Na (mV) V shift H (mV) + + Figure 3. Comparison of Na and H permeabilities of ChR2, Chronos, PsChR, and their mutants. Reversal potentials (V ) were determined after photocurrent measurements from 90 mV to + 10 mV. Reversal potential shift for Na was calculated by the reversal potential differences in two outside buffers containing 120 mM NaCl pH 7.6 and 1 mM NaCl pH 7.6. Reversal potential shift for H was determined by the reversal potential differences in two outside buffers of pH 7.6 and 9.6 containing Appl. Sci. 2019, 9, x 7 of 11 120 mM NaCl. Data points were presented as mean  SD, n = 4–6. V shift K (mV) V shift H (mV) + + + + 223 F Figure igure 4. 4. C Comparison omparison o of f K K a and nd H H p permeabilities ermeabilities oof f C ChR hR22, , C Chr hro onos, nos, P Ps sC ChR, hR, aand nd ttheir heir m mutants. utants. 224 R Reversal eversal p potentials otentials w wer ere e d determined etermined a after fter p photocurr hotocurre ent nt m measur easure ements ments f fr ro om m − 990 0 m mV V tto o + +10 10 m mV V.. 225 R Reversal eversal p potential otential s shift hift f for or K K w was as c calculated alculated b by y tthe he r re eversal versal p potent otentiial al d dif iff fe er re ences nces iin n t two wo outside outside buffers containing 120 mM KCl pH 7.6 and 1 mM KCl pH 7.6. Reversal potential shift for H was 226 buffers containing 120 mM KCl pH 7.6 and 1 mM KCl pH 7.6. Reversal potential shift for H was determined by the reversal potential differences in two outside buffers of pH 7.6 and 9.6 containing 227 determined by the reversal potential differences in two outside buffers of pH 7.6 and 9.6 containing 120 mM KCl. Data points were presented as mean  SD, n = 4–6. 228 120 mM KCl. Data points were presented as mean ± SD, n = 4–6. We further synthesized Chronos [9] and PsChR [20] and characterized the corresponding aspartate 229 Chronos, Chronos D173H, and Chronos D173C had much lower K permeability and the highest to cysteine, and the histidine mutations as the aspartate in transmembrane + helix 4 (TM4) were 230 PNa+/PK+ value among the tested constructs (Figure 4, Table 2). However, the H permeability was the conserved in all three channelrhodopsins (Appendix B). Chronos D173C, D173H, and PsChR D139H, 231 highest for all Chronos variants (Table 2). D139C, all showed an increased expression level, compared to their wild type (Figure 1a). All mutants 2+ also showed increased light-sensitivities along with prolonged off kinetics (Figure 2 and Table 1). 232 3.4. Mutation of the Aspartate in TM4 Influences the Ca Permeability Chronos D173C, PsChR D139H and PsChR D139C showed dramatically increased photocurrents 233 As obvious impacts of mutation of the conserved aspartate in TM4 on ion selectivity were while the Chronos D173H was similar to the wild type Chronos (Figure 1 and Table 1). Among these 2+ 234 observed, we further compared the Ca permeability of these mutants, considering the importance variants, PsChR D139C was the most light-sensitive with an effective light power density (LPD) for 2+ 2+ 235 of Ca in biological systems. Due to the existence of Ca -activated chloride channels in Xenopus 50% photocurrent (EPD ) of ~ 3.2 W/mm , which was ~ 250 times more sensitive than PsChR 2+ 236 oocytes [23], BAPTA was injected into the oocyte to a final concentration of ~10 mM, to block the Ca - (Figure 2 and Table 1). The EPD for XXL was ~ 5.4 W/mm , which was ~ 130 times more sensitive 237 induced chloride current (Figure 5). Then the photocurrents at −100 mV were measured in outside than ChR2 (Figure 2 and Table 1). 238 solution containing 80 mM CaCl2 at pH 9.0. At −100 mV and pH 9, no net H current could be observed 2+ 239 and the inward photocurrent was then only from the Ca influx. 241 Figure 5. Calcium permeabilities of selected channelrhodopsin variants. (a) Photocurrent traces of 242 different channelrhodopsins before (grey) and after (black) 1,2-bis(o-aminophenoxy)ethane- 243 N,N,N′,N′-tetraacetic acid (BAPTA) injection. Measurements were done in 80 mM CaCl2 pH 9.0 at 244 −100mV. Blue bars indicate light illumination. (b) Comparison of calcium permeability of ChR2 (light 245 grey), Chronos (dark grey), PsChR (black), CatCh (light grey), XXM (dark grey), and PsChR D139H 246 (black). All data points were plotted in the figure and mean ± SEM was indicated. 247 ChR2 showed a robust composite photocurrent with 80 mM CaCl2 at pH 9.0 and −100 mV, which 2+ 248 was dramatically reduced to the pure Ca current after injection of 10 mM BAPTA (Figure 5a), as 2+ 249 reported previously [2]. Both Chronos and ChR2 showed small Ca photocurrents (Figure 5a). ChR2 ChR2 XXM XXL Chronos Chronos D173H Chronos D173C PsChR PsChR D139H PsChR D139C ChR2 XXM XXL Chronos Chronos D173H Chronos D173C PsChR PsChR D139H PsChR D139C V shift (mV) V shift (mV) r Appl. Sci. 2019, 9, x 7 of 11 V shift K (mV) V shift H (mV) + + 223 Figure 4. Comparison of K and H permeabilities of ChR2, Chronos, PsChR, and their mutants. 224 Reversal potentials were determined after photocurrent measurements from −90 mV to +10 mV. 225 Reversal potential shift for K was calculated by the reversal potential differences in two outside 226 buffers containing 120 mM KCl pH 7.6 and 1 mM KCl pH 7.6. Reversal potential shift for H was 227 determined by the reversal potential differences in two outside buffers of pH 7.6 and 9.6 containing 228 120 mM KCl. Data points were presented as mean ± SD, n = 4–6. 229 Chronos, Chronos D173H, and Chronos D173C had much lower K permeability and the highest 230 PNa+/PK+ value among the tested constructs (Figure 4, Table 2). However, the H permeability was the 231 highest for all Chronos variants (Table 2). 2+ 232 3.4. Mutation of the Aspartate in TM4 Influences the Ca Permeability 233 As obvious impacts of mutation of the conserved aspartate in TM4 on ion selectivity were 2+ 234 observed, we further compared the Ca permeability of these mutants, considering the importance 2+ 2+ 235 of Ca in biological systems. Due to the existence of Ca -activated chloride channels in Xenopus 2+ 236 oocytes [23], BAPTA was injected into the oocyte to a final concentration of ~10 mM, to block the Ca - 237 induced chloride current (Figure 5). Then the photocurrents at −100 mV were measured in outside 238 solution containing 80 mM CaCl2 at pH 9.0. At −100 mV and pH 9, no net H current could be observed Appl. Sci. 2019, 9, 664 6 of 11 2+ 239 and the inward photocurrent was then only from the Ca influx. Figure 5. Calcium permeabilities of selected channelrhodopsin variants. (a) 241 Figure 5. Calcium permeabilities of selected channelrhodopsin variants. (a) Photocurrent traces of Photocurrent traces of different channelrhodopsins before (grey) and after (black) 242 different channelrhodopsins before (grey) and after (black) 1,2-bis(o-aminophenoxy)ethane- 0 0 1,2-bis(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid (BAPTA) injection. Measurements 243 N,N,N′,N′-tetraacetic acid (BAPTA) injection. Measurements were done in 80 mM CaCl2 pH 9.0 at were done in 80 mM CaCl pH 9.0 at 100mV. Blue bars indicate light illumination. (b) Comparison of 244 −100mV. Blue bars indicate light illumination. (b) Comparison of calcium permeability of ChR2 (light calcium permeability of ChR2 (light grey), Chronos (dark grey), PsChR (black), CatCh (light grey), 245 grey), Chronos (dark grey), PsChR (black), CatCh (light grey), XXM (dark grey), and PsChR D139H XXM (dark grey), and PsChR D139H (black). All data points were plotted in the figure and mean 246 (black). All data points were plotted in the figure and mean ± SEM was indicated. SEM was indicated. 247 ChR2 showed a robust composite photocurrent with 80 mM CaCl2 at pH 9.0 and −100 mV, which Table 1. Basic properties of ChR2, PsChR, and Chronos variants. 2+ 248 was dramatically reduced to the pure Ca current after injection of 10 mM BAPTA (Figure 5a), as Closing Time (ms) EPD 2+ 249 reported previously [2]. Both Chronos and ChR2 showed small Ca photocurrents (Figure 5a). ChR2 Expression * I ‡ ‡ 2 (% )  (% ) (W/mm ) 1 2 † † ChR2 1 1 7.5  0.5 (>98) - 710 XXM 2.7  30  80  5.5 (8) 1100  110 (92) 90 XXL 3  48  - 71000  2900 (>96) 5.4 † † Chronos 1  1  2.8  0.3 (>99) - 500 Chronos 2.2  1.2  1400  160 (56) - 84 D173H Chronos D173C 2.6  10  22  1.3 (85) 130  16 (15) 190 † † PsChR 8.5  0.6 (>98) - 810 1  1 PsChR D139H 2.2  6  37  2.7 (14) 810  54 (86) 160 PsChR D139C 3.1  11  3.4  0.8 (4) 74000  4300 (96) 3.2 † ‡ , expression or photocurrent of corresponding wild type was normalized as 1. Is, stationary (plateau) current. , as most ChRs exhibit biphasic off-kinetics which comprised a fast and a slow component, here the % indicated the percentage of the amplitude of the fast ( ) or slow ( ) component to the whole photocurrent. Data are shown as 1 2 mean  SEM, n = 4-5. Values are presented as approximates. *, expression level was calculated from the data of fluorescence value in Appendix A Figure A1. Table 2. Ion selectivity of ChR2, PsChR, and Chronos variants. Reversal Potential Shift (mV) Permeability Ratio + + † + + ‡ + + + + + + Na K Na /H K /H Na /K H H 7 7 ChR2 24  0.6 20  0.7 21  0.6 22  0.8 3.1  10 2.5  10 1.2 7 7 XXM 49  1.5 8.2  0.6 32  1.0 11  1.1 12  10 5  10 2.2 7 7 XXL 30  2 10  0.8 27  0.8 10  0.4 4.5  10 3.8  10 1.2 7 7 PsChR 58  2.8 8.8  5.5 54  5.7 20  1.3 18  10 16  10 1.2 7 7 PsChR D139H 91  2.2 5.5  0.6 65  5.5 11  0.6 90  10 26  10 3.5 7 7 PsChR D139C 51  2.1 5  0.4 40  1.7 6.0  0.6 13  10 8  10 1.7 7 7 Chronos 19  0.4 21  1.1 3.8  0.6 53  1.8 7.1 2.3  10 0.33  10 7 7 Chronos D173H 21  2.4 14  1.1 3.3  0.6 38  3.6 2.6  10 0.28  10 9.4 7 7 Chronos D173C 31  2.4 9.5  0.6 7.2  0.7 37  2.6 5  10 0.67  10 7.3 Reversal potentials and permeability ratio were determined from stationary currents in the indicated solution. Values represent mean  SD, n = 4–6. Values without SD are presented as approximates. , with the existence of + ‡ + 120 mM Na . , with the existence of 120 mM K . ChR2 XXM XXL Chronos Chronos D173H Chronos D173C PsChR PsChR D139H PsChR D139C V shift (mV) r Appl. Sci. 2019, 9, 664 7 of 11 3.2. Mutation of the Aspartate in TM4 Influences the Na Permeability The potential influence on ion selectivity by mutating ChR2 D156 was not reported. To investigate this, we measured the photocurrent at different potentials and calculated the reversal potential shift of these mutants, when systematically changing bath solutions with different pH and Na or K concentrations. ChR2 is a non-selective cation channel which is mostly permeable to H [2]. Both changing extracellular pH from 7.6 to 9.6 and changing extracellular Na concentration from 120 mM to 1 mM + + altered the ChR2 reversal potential (Figure 3). The ChR2 permeability ratio of Na to H (P /P ) Na+ H+ 7 + was determined as P /P = 3.110 (Table 2). Interestingly, D156H (XXM) influenced the Na Na+ H+ permeability and increased the P /P four times, while D156C (XXL) changed the P /P only Na+ H+ Na+ H+ slightly (Table 2). + 7 Chronos is more permeable to H than ChR2 with a P /P = 2.310 (Table 2). The D173H Na+ H+ mutation did not obviously change this, and D173C increased the P /P slightly to 510 Na+ H+ (Figure 3 and Table 2). PsChR was reported to be highly Na conductive [20]. Changing the outside Na concentration from 120 mM to 1 mM greatly influenced its photocurrent. The inward photocurrent + 7 was nearly abolished at 1 mM Na pH 7.6, and we determined the PsChR P /P to be 1810 , Na+ H+ which was even higher than that of XXM (Figure 3 and Table 2). The D139H mutation increased the P /P even five-fold more, to 9010 , while the D139C mutation decreased the P /P Na+ H+ Na+ H+ slightly (Figure 3 and Table 2). Among the tested constructs, PsChR D139H was the most Na permeable channelrhodopsin with a large photocurrent and, to our knowledge, the most Na -permeable channelrhodopsin ever reported. 3.3. Mutation of the Aspartate in TM4 Influences the K Permeability As tools for light-induced depolarization, ideal cation-permeable channelrhodopsins should be + + + more Na conductive and less K conductive, because K efflux across the plasma membrane would lead to a more hyperpolarized membrane potential. To test the potential influences on K permeability of different mutations, we measured photocurrents and calculated the reversal potential shift of these + + mutants when systematically changing bath solutions from 120 mM K to 1 mM K in comparison to changing pH from 7.6 to 9.6. + + ChR2 had a slightly weaker K conductance in comparison to Na with a P /P = 1.2. XXL Na+ K+ + + + increased the Na and K permeability slightly and equally. XXM increased the Na permeability more than that for K , and the P /P of XXM reached 2.2 (Figure 4 and Table 2). PsChR showed a Na+ K+ + + + higher Na permeability, together with an enhanced K permeability in comparison to that for H , with a similar P /P as ChR2 (Figure 4 and Table 2). Interestingly, the D139H mutation increased Na+ K+ + + the Na permeability five-fold, while changing the K permeability only 1.6-fold, thus the P /P Na+ K+ of PsChR D139H increased to 3.5 (Figure 4 and Table 2). The increased P /P makes PsChR D139H Na+ K+ even more suitable as a depolarization tool. Chronos, Chronos D173H, and Chronos D173C had much lower K permeability and the highest P /P value among the tested constructs (Figure 4 and Table 2). However, the H permeability Na+ K+ was the highest for all Chronos variants (Table 2). 2+ 3.4. Mutation of the Aspartate in TM4 Influences the Ca Permeability As obvious impacts of mutation of the conserved aspartate in TM4 on ion selectivity were 2+ observed, we further compared the Ca permeability of these mutants, considering the importance 2+ 2+ of Ca in biological systems. Due to the existence of Ca -activated chloride channels in Xenopus oocytes [23], BAPTA was injected into the oocyte to a final concentration of ~10 mM, to block the 2+ Ca -induced chloride current (Figure 5). Then the photocurrents at 100 mV were measured in outside solution containing 80 mM CaCl at pH 9.0. At 100 mV and pH 9, no net H current could 2+ be observed and the inward photocurrent was then only from the Ca influx. Appl. Sci. 2019, 9, 664 8 of 11 ChR2 showed a robust composite photocurrent with 80 mM CaCl at pH 9.0 and100 mV, which 2+ was dramatically reduced to the pure Ca current after injection of 10 mM BAPTA (Figure 5a), as 2+ reported previously [2]. Both Chronos and ChR2 showed small Ca photocurrents (Figure 5a). ChR2 2+ L132C (CatCh) showed an increased Ca photocurrent, compared to ChR2 (Figure 5), as previously 2+ reported [8]. Astonishingly, XXM also showed an increased Ca photocurrent, even higher than that 2+ of CatCh (Figure 5). PsChR D139H showed the highest Ca photocurrent, which on average was more than two times higher than that of CatCh (Figure 5b). 4. Discussion Channelrhodopsins, originating from different organisms, show quite different properties with respect to kinetics, action spectrum, and ion selectivity. Such changes can also be engineered by point mutations. In this study we compared the properties of ChR2, Chronos [9], PsChR [20], and their corresponding mutants of the aspartate in TM4 (DC gate aspartate). Generally, the aspartate to histidine or cysteine mutations of the three channelrhodopsins increased the expression level (probably because the mutant became more stable against degradation [21]) and slowed the closing kinetics. Nearly all mutants showed a much-increased photocurrent, probably because of a much-prolonged open state or enhanced single channel conductance, with only Chronos D173H as an exception. The tools with slowed kinetics are unfavorable for ultra-fast multiple stimulation but preferred for experiments which require low light and longtime stimulation. The prolonged open times were accompanied by elevated light sensitivities. Among the tested constructs, PsChR D139C and XXL became ~ 220 times and ~ 130 times more sensitive than ChR2. If slow closing would have not been a problem nor even desired, the more light-sensitive channelrhodopsins would have been ideal for efficient deep brain stimulation with infrared light via upconversion nanoparticles (UCNPs) [24]. These tools need to be further tested in mammalian systems for a broader field application. Furthermore, we investigated the influence of mutation of the aspartate in TM4 on ion selectivity. + 2+ We found that aspartate to histidine mutation of ChR2 and PsChR increased the Na and Ca 2+ 2+ permeability dramatically. To test the Ca current, we used BAPTA to block the Ca -activated 2+ endogenous chloride channels of oocytes. The fast Ca chelator BAPTA may have been altering the ion currents in more ways [25]. However, as we could see from the kinetics in Figure 5a that the Cl current (which shows a slower off kinetics) was well-blocked. Then we could reliably compare only the photocurrent of our channelrhodopsins. + 2+ With the large photocurrent, increased Na permeability, and bigger Ca current, PsChR D139H 2+ is a novel powerful optogenetic tool for depolarization and Ca manipulation. Channelrhodopsins 2+ 2+ with higher Ca currents have the advantage of being “direct” light-gated Ca channels, in contrast 2+ to the highly Ca -conductive CNG (cyclic nucleotide-gated) channels which became light-gated channels when fused with bPAC (photoactivated adenylyl cyclase) [26]. In summary, we found that mutating the conserved aspartate in TM4 influenced not only the expression level and kinetics of channel closing but also the ion selectivity; with appropriate mutations, we provided novel optogenetic tools with superior photocurrent amplitudes and high Na and 2+ Ca conductance. Author Contributions: Conceptualization, S.G. and G.N.; methodology, X.D., S.G. and G.N.; software, X.D. and S.G.; validation, X.D., S.G. and G.N.; formal analysis, X.D. and S.G.; investigation, X.D. and S.G.; resources, X.D., S.G. and G.N.; data curation, X.D. and S.G.; writing—original draft preparation, S.G.; writing—review and editing, X.D., S.G. and G.N.; visualization, X.D. and S.G.; supervision, S.G. and G.N.; project administration, G.N.; funding acquisition, G.N. Funding: This research was funded by grants from the German Research Foundation to GN (TRR 166/A03 and TR 240/A04). GN acknowledges support provided by the Prix-Louis-Jeantet. Acknowledgments: We are grateful to Shang Yang for help with some of the cloning work. This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding program Open Access Publishing. Appl. Sci. 2019, 9, 664 9 of 11 Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appl. Sci. 2019, 9, x 9 of 11 Appl. Sci. 2019, 9, x 9 of 11 300 Figure A1. Expression level of ChR2, PsChR, and Chronos variants in Xenopus oocyte. (a) 301 Representative confocal images of all the constructs, scale bar = 500 µm. (b) Yellow fluorescent protein 302 (YFP) fluorescence emission values from oocytes expressing different channelrhodopsins. Data was Figure A1. Expression level of ChR2, PsChR, and Chronos variants in Xenopus oocyte. (a) 300 Figure A1. Expression level of ChR2, PsChR, and Chronos variants in Xenopus oocyte. (a) 303 shown as mean ± SEM, n = 5–6. Pictures and fluorescence emission values were taken and measured Representative confocal images of all the constructs, scale bar = 500 m. (b) Yellow fluorescent 301 Representative confocal images of all the constructs, scale bar = 500 µm. (b) Yellow fluorescent protein 304 2 days after 20 ng cRNA injection. protein (YFP) fluorescence emission values from oocytes expressing different channelrhodopsins. Data 302 (YFP) fluorescence emission values from oocytes expressing different channelrhodopsins. Data was was shown as mean  SEM, n = 5–6. Pictures and fluorescence emission values were taken and 303 shown as mean ± SEM, n = 5–6. Pictures and fluorescence emission values were taken and measured 305 Appendix B measured 2 days after 20 ng cRNA injection. 304 2 days after 20 ng cRNA injection. 306 Sequence alignment of ChR2, Chronos, and PsChR. Conserved cysteine and aspartate of the DC gate Appendix B 305 Appendix B 307 were marked in the red box. 306 Sequence alignment of ChR2, Chronos, and PsChR. Conserved cysteine and aspartate of the DC gate 307 were marked in the red box. Figure A2. Sequence alignment of ChR2, Chronos, and PsChR. Conserved cysteine and aspartate of 309 References the DC gate were marked in the red box. 310 1. 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Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Sciences Multidisciplinary Digital Publishing Institute

Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

Applied Sciences , Volume 9 (4) – Feb 15, 2019

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applied sciences Article Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability Xiaodong Duan, Georg Nagel * and Shiqiang Gao * Botanik I, Julius-Maximilians-Universität Würzburg, Biozentrum, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany; xiaodong.duan@stud-mail.uni-wuerzburg.de * Correspondence: nagel@uni-wuerzburg.de (G.N.); gao.shiqiang@uni-wuerzburg.de (S.G.) Received: 14 January 2019; Accepted: 12 February 2019; Published: 15 February 2019 Featured Application: This study provides optogenetic tools with superior photocurrent + 2+ amplitudes and high Na and Ca conductance. Abstract: (1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly 2+ discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant + 2+ (XXM) showed enhanced Na and Ca conductance, which was not noticed before, while the + 2+ D156C mutation (XXL) influenced the Na and Ca conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a + + much-improved photocurrent, compared to wild type, and even higher Na selectivity to H than 2+ XXM. PsChR D139H also showed a strongly enhanced Ca conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na selectivity and 2+ Ca conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for 2+ Ca manipulation. Keywords: optogenetics; channelrhodopsins; sodium; calcium; DC gate 1. Introduction Channelrhodopsins were first discovered and characterized from C. reinhardtii [1,2]. After the showing of light-switched large passive cation conductance in HEK293 and BHK cells by Nagel et al., the ChR2 (C. reinhardtii channelrhodopsin-2) was immediately applied in neuroscience by several independent groups for studies in hippocampal neurons [3,4], Caenorhabditis elegans [5], inner retinal neurons [6], and PC12 cells [7]. H134R (histidine to arginine mutation at position 134) was the first ChR2 gain-of-function mutant which showed enhanced plasma membrane expression and larger stationary photocurrents in comparison to ChR2 wild type [5]. Other variants came out in rapid sequence, either of natural origin or mutated and engineered. The calcium translocating channelrhodopsin CatCh (a ChR2 leucine to cysteine mutation at position 2+ 132, L132C) showed improved Ca conductivity together with a larger photocurrent and higher light sensitivity [8]. Newly discovered Chronos (Stigeoclonium helveticum channelrhodopsin = ShChR) and Chrimson (CnChR1 from Chlamydomonas noctigama) showed a faster channel closing and a red-shifted action spectrum, respectively [9]. An E90R (glutamate to arginine mutation at position 90) Appl. Sci. 2019, 9, 664; doi:10.3390/app9040664 www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, 664 2 of 11 point mutation could extend the cation conductance of ChR2 to additional anion conductance [10]. Naturally, very specific anion conductive channelrhodopsins, GtACR1 and GtACR2 (Guillardia theta anion channelrhodopsin 1 and 2), were discovered afterwards [11]. Mutation of ChR2 C128 (cysteine at position 128) to threonine (T), alanine (A), and serine (S) slowed the closing kinetics dramatically [12–14]. Mutation of ChR2 D156 (cysteine at position 156) to alanine also decreased the closing kinetics [14]. Spectral studies suggested that a putative hydrogen bond between C128 and D156 could be an important structural determinant of the channel’s closing reaction [14] or might represent the valve of the channel [15]. Thus, hydrogen bond-linked D156 and C128 was proposed as the putative gate buried in the membrane (“DC gate”) [14,15]. But the first channelrhodopsin structure was a chimaera (C1C2) of truncated ChR1 and ChR2, and the distances between C167 (corresponding to C128 in ChR2) and D195 (corresponding to D156 in ChR2) are too far away to be associated by a hydrogen bond [16]. However, the recently solved structure of wild type ChR2 revealed a water molecule between C128 and D156 to bridge them, indeed, by hydrogen bonds [17]. Mutation of the DC gate has a strong effect on the open channel lifetime. The ChR2 D156C (aspartate to cysteine mutation at position 134) mutant (XXL) generated very large photocurrents and is 1000-fold more light-sensitive than wild type ChR2 in Drosophila larvae [18]. The ChR2 D156H (aspartate to histidine mutation at position 134) mutant (XXM) also showed a superior photo stimulation efficiency with faster kinetics than XXL, which made it an ideal optogenetic tool for Drosophila neurobiological studies [19]. The aspartate D156 in ChR2 is located close to the protonated retinal Schiff base (RSBH+). Thus, mutations of D156 logically have strong effects on the open channel lifetime by influencing the protonation state of the retinal Schiff base. However, the water-bridged C128 and D156 are not in the putative ion pore proposed by Volkov et al. [17]. And no attention had been paid to the potential changes of ion selectivity by DC gate mutations. In this study, we compared the ion selectivity of our previously published XXL and XXM + + and found that XXM showed a four-fold increased Na selectivity over H together with a + + two-fold increased K selectivity over H , compared to wild type ChR2. Based on this finding, we made further aspartate to histidine and cysteine mutations of PsChR (Platymonas subcordiformis channelrhodopsin) [20] and Chronos [9]. PsChR wild type was already reported to be highly Na + + + conductive and indeed showed a six-fold increased Na and K selectivity over H compared to wild type ChR2 in our measurements. But the D139H mutation of PsChR further increased the Na selectivity over H five-fold. Furthermore, PsChR D139H showed a 5-fold larger photocurrent than PsChR wt. 2+ 2+ We further compared the Ca permeability of these mutants. XXM showed an increased Ca 2+ current compared to CatCh [8]. PsChR wild type already showed a good Ca current, but the D139H 2+ mutation further increased the Ca current. We concluded that the mutant PsChR D139H would be a 2+ powerful tool for optogenetic Ca manipulation. 2. Materials and Methods 2.1. Plasmids and RNA Generation for Xenopus Laevis Oocyte Expression ChR2, XXM, and XXL in the pGEMHE vector were described in previous studies [18,19,21]. PsChR (from Platymonas subcordiformis, Accession No.: JX983143) and Chronos (from Stigeoclonium helveticum, Accession No.: KF992040) were synthesized by GeneArt Strings DNA Fragments (Life Technologies, Thermo Fisher Scientific), according to the published amino acid sequences, with the codon usage optimized to Mus musculus. The synthesized DNA segment was inserted into the pGEMHE vector with N-terminal BamHI and C-terminal XhoI restriction sites. Yellow fluorescent protein (YFP), together with a plasma membrane trafficking signal (KSRITSEGEYIPLDQIDINV) [22] beforehand and an ER export signal (FCYENEV) [22] afterward, the YFP was attached to the C-terminal Appl. Sci. 2019, 9, 664 3 of 11 end. Mutations were made by QuikChange Site-Directed Mutagenesis. The sequence was confirmed by DNA sequencing. Plasmids were linearized by NheI digestion and used for in vitro generation of cRNA with the AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies). 2.2. Two-Electrode Voltage-Clamp Recordings of Xenopus Laevis Oocytes cRNA-injected oocytes were incubated in ND96 solution (96 mM NaCl, 5 mM KCl, 1 mM MgCl , 1 mM CaCl , 5 mM HEPES, pH 7.4) containing 1 M all-trans-retinal at 16 C. Two-electrode 2 2 voltage-clamp (TEVC) recordings were performed with solutions, as indicated in figures, at room 2+ 2+ temperature. For experiments with external Ca , we blocked activation of the Ca -activated 0 0 endogenous chloride channels of oocytes by 1,2-bis(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic 2+ acid (BAPTA) injection. We injected 50 nl 200 mM of the fast Ca chelator BAPTA (potassium–salt) into each oocyte (~10 mM final concentration in the oocyte), incubated for 90 mins at 16 C and then performed the TEVC measurement at room temperature. Twenty nanograms of cRNA were injected into Xenopus oocyte for all the constructs. Photocurrents were measured two days after injection. For Figures 1 and 2, measurements were performed in standard solution with BaCl instead of CaCl 2 2 (110 mM NaCl, 5 mM KCl, 2 mM BaCl , 1 mM MgCl , 5 mM HEPES and pH 7.6). 2 2 2.3. Light Stimulation Illumination conditions were different, considering the published action spectra of ChR2, Chronos, and PsChR: 473 nm for ChR2, XXM, and XXL; 445 nm for PsChR, PsChR D139H, and PsChR D139C; 532 nm for Chronos, Chronos D173H, and Chronos D173C. Lasers were from Changchun New Industries Optoelectronics Technology. Light power was set to 5 mW/mm , except for the light sensitivity measurement. The light intensities were measured with a PLUS 2 Power & Energy Meter (LaserPoint s.r.l). For light sensitivity measurement, applied light intensities ranged from 1.7 to 5000 W/mm . 2.4. Protein Quantification by Fluorescence All expression levels of channelrhodopsin variants in oocytes were quantified by the fluorescence emission values of the YFP-tagged protein. Fluorescence emission was measured at 538 nm by a Fluoroskan Ascent microplate fluorometer (Thermo Scientific) with 485 nm excitation. 2.5. Fluorescence Imaging Fluorescence pictures of Xenopus oocytes were taken under 5x objective with a Leica DM6000 confocal microscope after two days’ expression. Oocytes were put in a 35 x 10 mm petri dish (Greiner GBO) containing ND96 for imaging. Excitation was done using 496 nm laser light. Fluorescence emission was detected from 520 nm to 585 nm. 2.6. Data Processing pClamp 7.0 was used to read out the photocurrent. Figures 1a, 2, 3, 4, 5a and A1a were made with OriginPro 2017. Figures 1b, 5b and A1b were made with GraphPad Prism. Tables 1 and 2 were made with Microsoft Excel. Sequence alignment in Appendix B was performed by BioEdit. Closing time was determined by biexponential fit. Light sensitivity curves were fitted with Hill equation. All values were plotted or presented with mean values, and error bars represent the standard deviations (SD) or standard error mean (SEM), as indicated in each figure. Statistical analysis was done by t test within GraphPad Prism. Differences were considered significant at p < 0.05. *** = p < 0.001, ** = p < 0.01, * = p < 0.05. Appl. Sci. 2019, 9, x 4 of 11 136 compared to ChR2 (Figure a1). The steady-state photocurrents were increased ~30- and ~48-fold for 137 XXM (D156H) and XXL (D156C), respectively, compared to ChR2 (Figure 1, Table 1). The enhanced 138 photocurrent might have been a comprehensive outcome of the higher plasma membrane expression 139 leAppl. vel, h Sci. igh 2019 er ,li9g , h 664 t-sensitivity, or increased single channel conductance. Both XXM and XXL show 4e of d 11 140 much-prolonged closing kinetics, leading to higher light-sensitivity (Figure 1a, Table 1). Appl. Sci. 2019, 9, x 5 of 11 142 Figure 1. Comparison of ChR2, PsChR, and Chronos variants. (a) Representative photocurrent traces Figure 1. Comparison of ChR2, PsChR, and Chronos variants. (a) Representative photocurrent traces 160 density (LPD) for 50% photocurrent (EPD50) of ~ 3.2 µW/mm , which was ~ 250 times more sensitive 143 of ChR2, PsChR, Chronos, and their mutants. (b) Comparison of stationary photocurrents of above of ChR2, PsChR, Chronos, and their mutants. (b) Comparison of stationary photocurrents of above 161 than PsChR (Figure 2, Table 1). The EPD50 for XXL was ~ 5.4 µW/mm , which was ~ 130 times more 144 channelrhodopsin variants. All data points were plotted in the figure and mean ± standard error mean channelrhodopsin variants. All data points were plotted in the figure and mean standard error mean 162 sensitive than ChR2 (Figure 2, Table 1). 145 (SEM) (n = 7-8) was indicated. (SEM) (n = 7–8) was indicated. 146 Table 1. Basic properties of ChR2, PsChR, and Chronos variants. Closing time (ms) EPD50 Expression* Is ǂ ǂ 2 (µW/mm ) τ1 (% ) τ2 (% ) Ϯ Ϯ ChR2 1× 1× 7.5 ± 0.5 (> 98) - 710 XXM 2.7 × 30 × 80 ± 5.5 (8) 1100 ± 110 (92) 90 71000 ± 2900 (> XXL 3 × 48 × - 5.4 96) Ϯ Ϯ Chronos 1 × 1 × 2.8 ± 0.3 (> 99) - 500 Chronos D173H 2.2 × 1.2 × 1400 ± 160 (56) - 84 Figure 2. Comparing the light sensitivities of ChR2, PsChR, and Chronos variants. (a) 5 s, 30 s, Chronos D173C 2.6 × 10 × 22 ± 1.3 (85) 130 ± 16 (15) 190 164 Figure 2. Comparing the light sensitivities of ChR2, PsChR, and Chronos variants. (a) 5 s, 30 s, and and 100 s continuous 473 nm blue light illumination were applied to ChR2, XXM and XXL, respectively. Ϯ Ϯ 165 P1 sC 00h sR c ontinuous 471 3 n ×m blue ligh1 t i× llu mina8 t. i5 o n ± w 0.e6 r e ( > a p 9p 8l)i ed to ChR2, X - XM and XXL, respe 8 c1 ti0 v ely. Photocurrent at maximum light power density of each oocyte was normalized as 1. (b) 5 s continuous 166 PsChP Rh D ot1 o3 cu 9H rre nt at max 2i.m 2 u ×m light pow 6 e ×r densit3 y7 o ± f e 2 a.c 7h ( o 1o 4c ) yte was 8 n1 o0 rm ±a 5 li4 z e (d 86 a)s 1. (b) 5 s con1 ti6 n0 u ous 532 nm blue light illumination were applied to Chronos, Chronos D173H, and Chronos D173C. 167 532 nm blue light illumination were applied to Chronos, Chronos D173H, and Chronos D173C. PsChR D139C 3.1 × 11 × 3.4 ± 0.8 (4) 74000 ± 4300 (96) 3.2 Photocurrent at maximum light power density of each oocyte was normalized as 1. (c) 5 s, 30 s, and 100 168 Photocurrent at maximum light power density of each oocyte was normalized as 1. (c) 5 s, 30 s, and 147 Ϯ, se continuous xpression o 445 r pnm hoto blue currlight ent o illu f cmination orrespond wer ing e applied wild typ to e Ps w ChR, as no Ps rm ChR alizD139H, ed as 1and x. IsPs , s ChR tatioD139C. nary 169 100 s continuous 445 nm blue light illumination were applied to PsChR, PsChR D139H, and PsChR 148 (pPhotocurr lateau) cuent rren at t. maximum ǂ, as most light ChRspower exhibidensity t biphasof ic each off-koocyte inetics was whic normalized h comprised as a1. fa Photocurr st and a s ents low of 170 D139C. Photocurrent at maximum light power density of each oocyte was normalized as 1. 149 coXXM, mpone XXL, nt, h Ps erChR e the D139H, % indand icate Ps d ChR the D139C percentwer agee o measur f the a ed mp atli tu60 demV of because the fast of (τthe 1) olar r s ger low curr (τ2ent ) 171 Photocurrents of XXM, XXL, PsChR D139H, and PsChR D139C were measured at −60 mV because of and slower kinetics; other constructs were measured at 100 mV. Data points were presented as mean 150 component to the whole photocurrent. Data are shown as mean ± SEM, n = 4-5. Values are presented 172 the larger current and slower kinetics; other constructs were measured at −100 mV. Data points were SD, n = 3–4. 151 as approximates. *, expression level was calculated from the data of fluorescence value in Figure A1. 173 presented as mean ± SD, n = 3–4. 3. Results 152 We further synthesized Chronos [9] and PsChR [20] and characterized the corresponding 174 3.2. Mutation of the Aspartate in TM4 Influences the Na Permeability 153 aspartate to cysteine, and the histidine mutations as the aspartate in transmembrane helix 4 (TM4) 3.1. Mutating the Conserved Aspartate of TM Helix 4 Influences the Expression, Photocurrent, and Kinetics 175 The potential influence on ion selectivity by mutating ChR2 D156 was not reported. To 154 were conserved in all three channelrhodopsins (Appendix B). Chronos D173C, D173H, and PsChR 176 invesSimilar tigate th to is,pr w eviously e measur published ed the pho rt esults ocurre[n 18 t ,a 19 t ], dif fluor feren escence t potentmeasur ials and ements calcula of ted whole the re oocyte versal 155 D139H, D139C, all showed an increased expression level, compared to their wild type (Figure 1a). 177 membranes potential shifwith t of th YFP-t ese m agged utants,XXM whenand syste XXL matishowed cally chan ag~ ing thr bee-fold ath soluincr tioneased s with expr diffeession rent pH level, and 156 All mutants also showed increased light-sensitivities along with prolonged off kinetics (Figure 2, + + 178 compar Na or K ed cto onChR2 centra(Figur tions. e A1). The steady-state photocurrents were increased ~30- and ~48-fold for 157 Table 1). Chronos D173C, PsChR D139H and PsChR D139C showed dramatically increased 179 XXMC (D156H) hR2 is a and non XXL -sele (D156C), ctive catr iespectively on channe,l compar which ed is m too ChR2 stly p (Figur ermea eb1 leand to T H able [2]1 . ).BThe oth enhanced changing 158 photocurrents while the Chronos D173H was similar to the wild type Chronos (Figure 1, Table 1). 180 photocurr extracelluent lar p might H from have 7.6 been to 9.6a acompr nd cha ehensive nging exoutcome tracellulaof r N the a c higher oncent plasma ration fmembrane rom 120 mM expr to ession 1 mM 159 Among these variants, PsChR D139C was the most light-sensitive with an effective light power + + 181 level, altered higher the Ch light-sensitivity R2 reversal pot,eor ntiincr al (F eased igure single 3). Thechannel ChR2 pe conductance. rmeability ratBoth io of XXM Na toand H (XXL PNa+/P showed H+) was −7 + 182 much-pr determin olonged ed as P closing Na+/PH+ kinetics, = 3.1×10leading (Tablto e higher 2). Intlight-sensitivity erestingly, D156 (Figur H (Xe X1 M a)and infT lu able ence 1d ). the Na 183 permeability and increased the PNa+/PH+ four times, while D156C (XXL) changed the PNa+/PH+ only 184 slightly (Table 2). 100 + V shift Na (mV) V shift H (mV) ChR2 XXM XXL Chronos Chronos D173H Chro os D173 n C PsChR PsChR D139H PsChR D139C V shift (mV) r Appl. Sci. 2019, 9, x 5 of 11 160 density (LPD) for 50% photocurrent (EPD50) of ~ 3.2 µW/mm , which was ~ 250 times more sensitive 161 than PsChR (Figure 2, Table 1). The EPD50 for XXL was ~ 5.4 µW/mm , which was ~ 130 times more 162 sensitive than ChR2 (Figure 2, Table 1). 164 Figure 2. Comparing the light sensitivities of ChR2, PsChR, and Chronos variants. (a) 5 s, 30 s, and 165 100 s continuous 473 nm blue light illumination were applied to ChR2, XXM and XXL, respectively. 166 Photocurrent at maximum light power density of each oocyte was normalized as 1. (b) 5 s continuous 167 532 nm blue light illumination were applied to Chronos, Chronos D173H, and Chronos D173C. 168 Photocurrent at maximum light power density of each oocyte was normalized as 1. (c) 5 s, 30 s, and 169 100 s continuous 445 nm blue light illumination were applied to PsChR, PsChR D139H, and PsChR 170 D139C. Photocurrent at maximum light power density of each oocyte was normalized as 1. 171 Photocurrents of XXM, XXL, PsChR D139H, and PsChR D139C were measured at −60 mV because of 172 the larger current and slower kinetics; other constructs were measured at −100 mV. Data points were 173 presented as mean ± SD, n = 3–4. 174 3.2. Mutation of the Aspartate in TM4 Influences the Na Permeability 175 The potential influence on ion selectivity by mutating ChR2 D156 was not reported. To 176 investigate this, we measured the photocurrent at different potentials and calculated the reversal 177 potential shift of these mutants, when systematically changing bath solutions with different pH and + + 178 Na or K concentrations. 179 ChR2 is a non-selective cation channel which is mostly permeable to H [2]. Both changing 180 extracellular pH from 7.6 to 9.6 and changing extracellular Na concentration from 120 mM to 1 mM + + 181 altered the ChR2 reversal potential (Figure 3). The ChR2 permeability ratio of Na to H (PNa+/PH+) was −7 + 182 determined as PNa+/PH+ = 3.1×10 (Table 2). Interestingly, D156H (XXM) influenced the Na 183 perm Appl. Sci. eab 2019 ility , 9,a 664 nd increased the PNa+/PH+ four times, while D156C (XXL) changed the PNa+/PH+ 5o of nl11 y 184 slightly (Table 2). 100 + V shift Na (mV) V shift H (mV) + + Figure 3. Comparison of Na and H permeabilities of ChR2, Chronos, PsChR, and their mutants. Reversal potentials (V ) were determined after photocurrent measurements from 90 mV to + 10 mV. Reversal potential shift for Na was calculated by the reversal potential differences in two outside buffers containing 120 mM NaCl pH 7.6 and 1 mM NaCl pH 7.6. Reversal potential shift for H was determined by the reversal potential differences in two outside buffers of pH 7.6 and 9.6 containing Appl. Sci. 2019, 9, x 7 of 11 120 mM NaCl. Data points were presented as mean  SD, n = 4–6. V shift K (mV) V shift H (mV) + + + + 223 F Figure igure 4. 4. C Comparison omparison o of f K K a and nd H H p permeabilities ermeabilities oof f C ChR hR22, , C Chr hro onos, nos, P Ps sC ChR, hR, aand nd ttheir heir m mutants. utants. 224 R Reversal eversal p potentials otentials w wer ere e d determined etermined a after fter p photocurr hotocurre ent nt m measur easure ements ments f fr ro om m − 990 0 m mV V tto o + +10 10 m mV V.. 225 R Reversal eversal p potential otential s shift hift f for or K K w was as c calculated alculated b by y tthe he r re eversal versal p potent otentiial al d dif iff fe er re ences nces iin n t two wo outside outside buffers containing 120 mM KCl pH 7.6 and 1 mM KCl pH 7.6. Reversal potential shift for H was 226 buffers containing 120 mM KCl pH 7.6 and 1 mM KCl pH 7.6. Reversal potential shift for H was determined by the reversal potential differences in two outside buffers of pH 7.6 and 9.6 containing 227 determined by the reversal potential differences in two outside buffers of pH 7.6 and 9.6 containing 120 mM KCl. Data points were presented as mean  SD, n = 4–6. 228 120 mM KCl. Data points were presented as mean ± SD, n = 4–6. We further synthesized Chronos [9] and PsChR [20] and characterized the corresponding aspartate 229 Chronos, Chronos D173H, and Chronos D173C had much lower K permeability and the highest to cysteine, and the histidine mutations as the aspartate in transmembrane + helix 4 (TM4) were 230 PNa+/PK+ value among the tested constructs (Figure 4, Table 2). However, the H permeability was the conserved in all three channelrhodopsins (Appendix B). Chronos D173C, D173H, and PsChR D139H, 231 highest for all Chronos variants (Table 2). D139C, all showed an increased expression level, compared to their wild type (Figure 1a). All mutants 2+ also showed increased light-sensitivities along with prolonged off kinetics (Figure 2 and Table 1). 232 3.4. Mutation of the Aspartate in TM4 Influences the Ca Permeability Chronos D173C, PsChR D139H and PsChR D139C showed dramatically increased photocurrents 233 As obvious impacts of mutation of the conserved aspartate in TM4 on ion selectivity were while the Chronos D173H was similar to the wild type Chronos (Figure 1 and Table 1). Among these 2+ 234 observed, we further compared the Ca permeability of these mutants, considering the importance variants, PsChR D139C was the most light-sensitive with an effective light power density (LPD) for 2+ 2+ 235 of Ca in biological systems. Due to the existence of Ca -activated chloride channels in Xenopus 50% photocurrent (EPD ) of ~ 3.2 W/mm , which was ~ 250 times more sensitive than PsChR 2+ 236 oocytes [23], BAPTA was injected into the oocyte to a final concentration of ~10 mM, to block the Ca - (Figure 2 and Table 1). The EPD for XXL was ~ 5.4 W/mm , which was ~ 130 times more sensitive 237 induced chloride current (Figure 5). Then the photocurrents at −100 mV were measured in outside than ChR2 (Figure 2 and Table 1). 238 solution containing 80 mM CaCl2 at pH 9.0. At −100 mV and pH 9, no net H current could be observed 2+ 239 and the inward photocurrent was then only from the Ca influx. 241 Figure 5. Calcium permeabilities of selected channelrhodopsin variants. (a) Photocurrent traces of 242 different channelrhodopsins before (grey) and after (black) 1,2-bis(o-aminophenoxy)ethane- 243 N,N,N′,N′-tetraacetic acid (BAPTA) injection. Measurements were done in 80 mM CaCl2 pH 9.0 at 244 −100mV. Blue bars indicate light illumination. (b) Comparison of calcium permeability of ChR2 (light 245 grey), Chronos (dark grey), PsChR (black), CatCh (light grey), XXM (dark grey), and PsChR D139H 246 (black). All data points were plotted in the figure and mean ± SEM was indicated. 247 ChR2 showed a robust composite photocurrent with 80 mM CaCl2 at pH 9.0 and −100 mV, which 2+ 248 was dramatically reduced to the pure Ca current after injection of 10 mM BAPTA (Figure 5a), as 2+ 249 reported previously [2]. Both Chronos and ChR2 showed small Ca photocurrents (Figure 5a). ChR2 ChR2 XXM XXL Chronos Chronos D173H Chronos D173C PsChR PsChR D139H PsChR D139C ChR2 XXM XXL Chronos Chronos D173H Chronos D173C PsChR PsChR D139H PsChR D139C V shift (mV) V shift (mV) r Appl. Sci. 2019, 9, x 7 of 11 V shift K (mV) V shift H (mV) + + 223 Figure 4. Comparison of K and H permeabilities of ChR2, Chronos, PsChR, and their mutants. 224 Reversal potentials were determined after photocurrent measurements from −90 mV to +10 mV. 225 Reversal potential shift for K was calculated by the reversal potential differences in two outside 226 buffers containing 120 mM KCl pH 7.6 and 1 mM KCl pH 7.6. Reversal potential shift for H was 227 determined by the reversal potential differences in two outside buffers of pH 7.6 and 9.6 containing 228 120 mM KCl. Data points were presented as mean ± SD, n = 4–6. 229 Chronos, Chronos D173H, and Chronos D173C had much lower K permeability and the highest 230 PNa+/PK+ value among the tested constructs (Figure 4, Table 2). However, the H permeability was the 231 highest for all Chronos variants (Table 2). 2+ 232 3.4. Mutation of the Aspartate in TM4 Influences the Ca Permeability 233 As obvious impacts of mutation of the conserved aspartate in TM4 on ion selectivity were 2+ 234 observed, we further compared the Ca permeability of these mutants, considering the importance 2+ 2+ 235 of Ca in biological systems. Due to the existence of Ca -activated chloride channels in Xenopus 2+ 236 oocytes [23], BAPTA was injected into the oocyte to a final concentration of ~10 mM, to block the Ca - 237 induced chloride current (Figure 5). Then the photocurrents at −100 mV were measured in outside 238 solution containing 80 mM CaCl2 at pH 9.0. At −100 mV and pH 9, no net H current could be observed Appl. Sci. 2019, 9, 664 6 of 11 2+ 239 and the inward photocurrent was then only from the Ca influx. Figure 5. Calcium permeabilities of selected channelrhodopsin variants. (a) 241 Figure 5. Calcium permeabilities of selected channelrhodopsin variants. (a) Photocurrent traces of Photocurrent traces of different channelrhodopsins before (grey) and after (black) 242 different channelrhodopsins before (grey) and after (black) 1,2-bis(o-aminophenoxy)ethane- 0 0 1,2-bis(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid (BAPTA) injection. Measurements 243 N,N,N′,N′-tetraacetic acid (BAPTA) injection. Measurements were done in 80 mM CaCl2 pH 9.0 at were done in 80 mM CaCl pH 9.0 at 100mV. Blue bars indicate light illumination. (b) Comparison of 244 −100mV. Blue bars indicate light illumination. (b) Comparison of calcium permeability of ChR2 (light calcium permeability of ChR2 (light grey), Chronos (dark grey), PsChR (black), CatCh (light grey), 245 grey), Chronos (dark grey), PsChR (black), CatCh (light grey), XXM (dark grey), and PsChR D139H XXM (dark grey), and PsChR D139H (black). All data points were plotted in the figure and mean 246 (black). All data points were plotted in the figure and mean ± SEM was indicated. SEM was indicated. 247 ChR2 showed a robust composite photocurrent with 80 mM CaCl2 at pH 9.0 and −100 mV, which Table 1. Basic properties of ChR2, PsChR, and Chronos variants. 2+ 248 was dramatically reduced to the pure Ca current after injection of 10 mM BAPTA (Figure 5a), as Closing Time (ms) EPD 2+ 249 reported previously [2]. Both Chronos and ChR2 showed small Ca photocurrents (Figure 5a). ChR2 Expression * I ‡ ‡ 2 (% )  (% ) (W/mm ) 1 2 † † ChR2 1 1 7.5  0.5 (>98) - 710 XXM 2.7  30  80  5.5 (8) 1100  110 (92) 90 XXL 3  48  - 71000  2900 (>96) 5.4 † † Chronos 1  1  2.8  0.3 (>99) - 500 Chronos 2.2  1.2  1400  160 (56) - 84 D173H Chronos D173C 2.6  10  22  1.3 (85) 130  16 (15) 190 † † PsChR 8.5  0.6 (>98) - 810 1  1 PsChR D139H 2.2  6  37  2.7 (14) 810  54 (86) 160 PsChR D139C 3.1  11  3.4  0.8 (4) 74000  4300 (96) 3.2 † ‡ , expression or photocurrent of corresponding wild type was normalized as 1. Is, stationary (plateau) current. , as most ChRs exhibit biphasic off-kinetics which comprised a fast and a slow component, here the % indicated the percentage of the amplitude of the fast ( ) or slow ( ) component to the whole photocurrent. Data are shown as 1 2 mean  SEM, n = 4-5. Values are presented as approximates. *, expression level was calculated from the data of fluorescence value in Appendix A Figure A1. Table 2. Ion selectivity of ChR2, PsChR, and Chronos variants. Reversal Potential Shift (mV) Permeability Ratio + + † + + ‡ + + + + + + Na K Na /H K /H Na /K H H 7 7 ChR2 24  0.6 20  0.7 21  0.6 22  0.8 3.1  10 2.5  10 1.2 7 7 XXM 49  1.5 8.2  0.6 32  1.0 11  1.1 12  10 5  10 2.2 7 7 XXL 30  2 10  0.8 27  0.8 10  0.4 4.5  10 3.8  10 1.2 7 7 PsChR 58  2.8 8.8  5.5 54  5.7 20  1.3 18  10 16  10 1.2 7 7 PsChR D139H 91  2.2 5.5  0.6 65  5.5 11  0.6 90  10 26  10 3.5 7 7 PsChR D139C 51  2.1 5  0.4 40  1.7 6.0  0.6 13  10 8  10 1.7 7 7 Chronos 19  0.4 21  1.1 3.8  0.6 53  1.8 7.1 2.3  10 0.33  10 7 7 Chronos D173H 21  2.4 14  1.1 3.3  0.6 38  3.6 2.6  10 0.28  10 9.4 7 7 Chronos D173C 31  2.4 9.5  0.6 7.2  0.7 37  2.6 5  10 0.67  10 7.3 Reversal potentials and permeability ratio were determined from stationary currents in the indicated solution. Values represent mean  SD, n = 4–6. Values without SD are presented as approximates. , with the existence of + ‡ + 120 mM Na . , with the existence of 120 mM K . ChR2 XXM XXL Chronos Chronos D173H Chronos D173C PsChR PsChR D139H PsChR D139C V shift (mV) r Appl. Sci. 2019, 9, 664 7 of 11 3.2. Mutation of the Aspartate in TM4 Influences the Na Permeability The potential influence on ion selectivity by mutating ChR2 D156 was not reported. To investigate this, we measured the photocurrent at different potentials and calculated the reversal potential shift of these mutants, when systematically changing bath solutions with different pH and Na or K concentrations. ChR2 is a non-selective cation channel which is mostly permeable to H [2]. Both changing extracellular pH from 7.6 to 9.6 and changing extracellular Na concentration from 120 mM to 1 mM + + altered the ChR2 reversal potential (Figure 3). The ChR2 permeability ratio of Na to H (P /P ) Na+ H+ 7 + was determined as P /P = 3.110 (Table 2). Interestingly, D156H (XXM) influenced the Na Na+ H+ permeability and increased the P /P four times, while D156C (XXL) changed the P /P only Na+ H+ Na+ H+ slightly (Table 2). + 7 Chronos is more permeable to H than ChR2 with a P /P = 2.310 (Table 2). The D173H Na+ H+ mutation did not obviously change this, and D173C increased the P /P slightly to 510 Na+ H+ (Figure 3 and Table 2). PsChR was reported to be highly Na conductive [20]. Changing the outside Na concentration from 120 mM to 1 mM greatly influenced its photocurrent. The inward photocurrent + 7 was nearly abolished at 1 mM Na pH 7.6, and we determined the PsChR P /P to be 1810 , Na+ H+ which was even higher than that of XXM (Figure 3 and Table 2). The D139H mutation increased the P /P even five-fold more, to 9010 , while the D139C mutation decreased the P /P Na+ H+ Na+ H+ slightly (Figure 3 and Table 2). Among the tested constructs, PsChR D139H was the most Na permeable channelrhodopsin with a large photocurrent and, to our knowledge, the most Na -permeable channelrhodopsin ever reported. 3.3. Mutation of the Aspartate in TM4 Influences the K Permeability As tools for light-induced depolarization, ideal cation-permeable channelrhodopsins should be + + + more Na conductive and less K conductive, because K efflux across the plasma membrane would lead to a more hyperpolarized membrane potential. To test the potential influences on K permeability of different mutations, we measured photocurrents and calculated the reversal potential shift of these + + mutants when systematically changing bath solutions from 120 mM K to 1 mM K in comparison to changing pH from 7.6 to 9.6. + + ChR2 had a slightly weaker K conductance in comparison to Na with a P /P = 1.2. XXL Na+ K+ + + + increased the Na and K permeability slightly and equally. XXM increased the Na permeability more than that for K , and the P /P of XXM reached 2.2 (Figure 4 and Table 2). PsChR showed a Na+ K+ + + + higher Na permeability, together with an enhanced K permeability in comparison to that for H , with a similar P /P as ChR2 (Figure 4 and Table 2). Interestingly, the D139H mutation increased Na+ K+ + + the Na permeability five-fold, while changing the K permeability only 1.6-fold, thus the P /P Na+ K+ of PsChR D139H increased to 3.5 (Figure 4 and Table 2). The increased P /P makes PsChR D139H Na+ K+ even more suitable as a depolarization tool. Chronos, Chronos D173H, and Chronos D173C had much lower K permeability and the highest P /P value among the tested constructs (Figure 4 and Table 2). However, the H permeability Na+ K+ was the highest for all Chronos variants (Table 2). 2+ 3.4. Mutation of the Aspartate in TM4 Influences the Ca Permeability As obvious impacts of mutation of the conserved aspartate in TM4 on ion selectivity were 2+ observed, we further compared the Ca permeability of these mutants, considering the importance 2+ 2+ of Ca in biological systems. Due to the existence of Ca -activated chloride channels in Xenopus oocytes [23], BAPTA was injected into the oocyte to a final concentration of ~10 mM, to block the 2+ Ca -induced chloride current (Figure 5). Then the photocurrents at 100 mV were measured in outside solution containing 80 mM CaCl at pH 9.0. At 100 mV and pH 9, no net H current could 2+ be observed and the inward photocurrent was then only from the Ca influx. Appl. Sci. 2019, 9, 664 8 of 11 ChR2 showed a robust composite photocurrent with 80 mM CaCl at pH 9.0 and100 mV, which 2+ was dramatically reduced to the pure Ca current after injection of 10 mM BAPTA (Figure 5a), as 2+ reported previously [2]. Both Chronos and ChR2 showed small Ca photocurrents (Figure 5a). ChR2 2+ L132C (CatCh) showed an increased Ca photocurrent, compared to ChR2 (Figure 5), as previously 2+ reported [8]. Astonishingly, XXM also showed an increased Ca photocurrent, even higher than that 2+ of CatCh (Figure 5). PsChR D139H showed the highest Ca photocurrent, which on average was more than two times higher than that of CatCh (Figure 5b). 4. Discussion Channelrhodopsins, originating from different organisms, show quite different properties with respect to kinetics, action spectrum, and ion selectivity. Such changes can also be engineered by point mutations. In this study we compared the properties of ChR2, Chronos [9], PsChR [20], and their corresponding mutants of the aspartate in TM4 (DC gate aspartate). Generally, the aspartate to histidine or cysteine mutations of the three channelrhodopsins increased the expression level (probably because the mutant became more stable against degradation [21]) and slowed the closing kinetics. Nearly all mutants showed a much-increased photocurrent, probably because of a much-prolonged open state or enhanced single channel conductance, with only Chronos D173H as an exception. The tools with slowed kinetics are unfavorable for ultra-fast multiple stimulation but preferred for experiments which require low light and longtime stimulation. The prolonged open times were accompanied by elevated light sensitivities. Among the tested constructs, PsChR D139C and XXL became ~ 220 times and ~ 130 times more sensitive than ChR2. If slow closing would have not been a problem nor even desired, the more light-sensitive channelrhodopsins would have been ideal for efficient deep brain stimulation with infrared light via upconversion nanoparticles (UCNPs) [24]. These tools need to be further tested in mammalian systems for a broader field application. Furthermore, we investigated the influence of mutation of the aspartate in TM4 on ion selectivity. + 2+ We found that aspartate to histidine mutation of ChR2 and PsChR increased the Na and Ca 2+ 2+ permeability dramatically. To test the Ca current, we used BAPTA to block the Ca -activated 2+ endogenous chloride channels of oocytes. The fast Ca chelator BAPTA may have been altering the ion currents in more ways [25]. However, as we could see from the kinetics in Figure 5a that the Cl current (which shows a slower off kinetics) was well-blocked. Then we could reliably compare only the photocurrent of our channelrhodopsins. + 2+ With the large photocurrent, increased Na permeability, and bigger Ca current, PsChR D139H 2+ is a novel powerful optogenetic tool for depolarization and Ca manipulation. Channelrhodopsins 2+ 2+ with higher Ca currents have the advantage of being “direct” light-gated Ca channels, in contrast 2+ to the highly Ca -conductive CNG (cyclic nucleotide-gated) channels which became light-gated channels when fused with bPAC (photoactivated adenylyl cyclase) [26]. In summary, we found that mutating the conserved aspartate in TM4 influenced not only the expression level and kinetics of channel closing but also the ion selectivity; with appropriate mutations, we provided novel optogenetic tools with superior photocurrent amplitudes and high Na and 2+ Ca conductance. Author Contributions: Conceptualization, S.G. and G.N.; methodology, X.D., S.G. and G.N.; software, X.D. and S.G.; validation, X.D., S.G. and G.N.; formal analysis, X.D. and S.G.; investigation, X.D. and S.G.; resources, X.D., S.G. and G.N.; data curation, X.D. and S.G.; writing—original draft preparation, S.G.; writing—review and editing, X.D., S.G. and G.N.; visualization, X.D. and S.G.; supervision, S.G. and G.N.; project administration, G.N.; funding acquisition, G.N. Funding: This research was funded by grants from the German Research Foundation to GN (TRR 166/A03 and TR 240/A04). GN acknowledges support provided by the Prix-Louis-Jeantet. Acknowledgments: We are grateful to Shang Yang for help with some of the cloning work. This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding program Open Access Publishing. Appl. Sci. 2019, 9, 664 9 of 11 Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appl. Sci. 2019, 9, x 9 of 11 Appl. Sci. 2019, 9, x 9 of 11 300 Figure A1. Expression level of ChR2, PsChR, and Chronos variants in Xenopus oocyte. (a) 301 Representative confocal images of all the constructs, scale bar = 500 µm. (b) Yellow fluorescent protein 302 (YFP) fluorescence emission values from oocytes expressing different channelrhodopsins. Data was Figure A1. Expression level of ChR2, PsChR, and Chronos variants in Xenopus oocyte. (a) 300 Figure A1. Expression level of ChR2, PsChR, and Chronos variants in Xenopus oocyte. (a) 303 shown as mean ± SEM, n = 5–6. Pictures and fluorescence emission values were taken and measured Representative confocal images of all the constructs, scale bar = 500 m. (b) Yellow fluorescent 301 Representative confocal images of all the constructs, scale bar = 500 µm. (b) Yellow fluorescent protein 304 2 days after 20 ng cRNA injection. protein (YFP) fluorescence emission values from oocytes expressing different channelrhodopsins. Data 302 (YFP) fluorescence emission values from oocytes expressing different channelrhodopsins. Data was was shown as mean  SEM, n = 5–6. Pictures and fluorescence emission values were taken and 303 shown as mean ± SEM, n = 5–6. Pictures and fluorescence emission values were taken and measured 305 Appendix B measured 2 days after 20 ng cRNA injection. 304 2 days after 20 ng cRNA injection. 306 Sequence alignment of ChR2, Chronos, and PsChR. Conserved cysteine and aspartate of the DC gate Appendix B 305 Appendix B 307 were marked in the red box. 306 Sequence alignment of ChR2, Chronos, and PsChR. Conserved cysteine and aspartate of the DC gate 307 were marked in the red box. Figure A2. Sequence alignment of ChR2, Chronos, and PsChR. Conserved cysteine and aspartate of 309 References the DC gate were marked in the red box. 310 1. 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