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. 2017 Aug 30;7:9928. doi: 10.1038/s41598-017-09600-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Chrimson glutamate mutants along the ion-conducting pathway. (a) Representative photocurrents of Chrimson glutamate mutants at different voltages and different extracellular solutions of 110 mM NaCl and pH_e 7.2 (left), 1 mM NaCl and pH_e 7.2 (middle) and 110 mM NaCl and pH_e 9.0 (right) with intracellular 110 mM NaCl pH_i 7.2. (b) Corresponding photocurrent amplitudes at −60 mV normalized to symmetric standard conditions of 110 mM NaCl pH_e 7.2 (dashed line) (Mean ± SD; LJP corrected; blue: 1 mM NaCl and pH_e 7.2; red: 110 mM NaCl and pH_e 9.0; compared to WT by a two sample t-test for “low NaCl pH_e 7.2”/“high NaCl pH_e 9.0” conditions respectively; WT: n = 8/10 cells; E1’A: n = 6/6, p = 0.7/0.4; E2’A: n = 5/7 p = 0.6/0.0002; E3’A: n = 5/8,p = 0.6/0.9; E4’A: n = 5/8, p = 0.0002/< 0.0001; E5’A: n = 6/9, p < 0.0001/0.0001) (c) Related reversal potential shifts upon extracellular buffer exchange from symmetric 110 mM NaCl and pH_e 7.2 to low extracellular sodium (left, blue, 1 mM NaCl and pH_e 7.2) or low extracellular proton (right, red, 110 mM NaCl and pH_e 9.0) concentration (mean ± SD; LJP corrected; compared to WT by a two sample t-test for “low NaCl pH_e 7.2”/“high NaCl pH_e 9.0” respectively; WT: n = 8/10 cells; E1’A: n = 6/6, p = 0.2/0.3; E2’A: n = 5/7 p = 0.45/0.0004; E3’A: n = 5/8, p = 0.11/0.06; E4’A: n = 5/8, p < 0.0001/0.0001; E5’A: n = 6/9, p < 0.0001/0.0001). (d) Photocurrent amplitudes at −60 mV in symmetric 110 mM NaCl and pH_i,e 7.2 (mean ± SD; WT n = 16 cells; E1’A n = 9; E2’A n = 10; E3’A n = 14; E4’A n = 11; and E5’A n = 10). (e) Apparent off-kinetics (τ_off,apparent) at pH_e/i 7.2 and −60 mV or +30 mV (mean ± SD; WT n = 18 cells; E1’A n = 9; E2’A n = 8; E3’A n = 14; E4’A n = 9 cells; E5’A n = 9). (f) Normalized peak photocurrents after 10 ms excitation at different wavelengths of equal photon count (mean ± SD; symmetric 110 mM NaCl, pH_e,i 7.2 and −60 mV; WT n = 10 cells; E1’A n = 7; E2’A n = 5; E3’A = 8; E4’A n = 6; E5’A n = 5).