CheRiff acidifies polarized cells.

A) Genetic constructs for simultaneous optogenetic stimulation and pH imaging. B) Calibration of pHoran4 pH sensor in HEK cells and neurons. Error bars represent S.D. of n = 8 measurements in HEK cells, 42 measurements in neurons. C) Protocol for measuring pH responses to optogenetic stimulation. Stimulation (blue) and measurement (yellow) were interleaved for 150 s; then pH recovery was measured for 150 s without optogenetic stimulation. D) Example images of cultured neurons showing (left) GFP fluorescence, a marker for CheRiff expression, (middle) -ΔF/F in the pHoran4 channel after 150 s of the protocol shown in (C), (right) merge. Scale bars 100 μm. E) Time-course of pH in cultured neurons. Cells expressing pHoran4 but not CheRiff did not acidify. Bold lines show population average. F) CheRiff-expressing neurons acidified to a pH of 6.76 ± 0.35 (mean ± S.D., n = 34 cells). Neurons not expressing CheRiff had significantly less acidification, pH = 7.3 ± 0.08 (mean ± S.D., n = 26 cells, p = 5e-8 Wilcoxon rank sum test). Box plots show inter-quartile ranges, tick-marks show data range, + shows outlier. G) Half-time of (left) acidification or (right) recovery for neuron somas vs dendrites stimulated with the protocol in (C). Black line shows equal kinetics.

CheRiff exhibits high proton conductance.

A) Genetic constructs for simultaneous optogenetic stimulation and pH imaging in polarized HEK293T cells. B) Diagram of HEK cell monolayer connected by gap junctions. C) Experimental paradigm for measuring pH responses to optogenetic stimulation. Stimulation (488 nm) and measurement (561 nm) were interleaved to avoid optical crosstalk. D) Time-course of pH in HEK cells. Expression of Kir2.1 increased the driving force for proton influx, substantially enhancing the acidification. E) Images of HEK cell monolayers showing (top) GFP fluorescence, a marker for CheRiff expression and (bottom) -ΔF/F in the pHoran4 channel after protocol shown in (C). Scale bars 100 μm. F) Quantification of the data in (D-E). pHoran4 alone: pH = 7.31 ± 0.02 (mean ± S.D., n = 13 cells); CheRiff and pHoran4 pH = 7.21 ± 0.05 (n = 70 cells); CheRiff, pHoran4 and Kir2.1: pH = 6.96 ± 0.15 (n = 75 cells). Statistical comparisons via Wilcoxon signed rank test. G) Protocol for mapping voltage responses to patterned optogenetic stimulation (488 nm) via fluorescence of BeRST1 (640 nm exc.). H) Images of HEK cell monolayers showing (left) fluorescence of GFP with patterned blue illumination, (right) ΔF/F of BeRST1. Scale bars 100 μm. I) Time-course of BeRST1 fluorescence in HEK cells inside (Blue On) and outside (Blue Off) the optogenetic stimulus regions. J) Protocol for measuring pH responses to patterned optogenetic stimulation. Stimulation (488 nm) and measurement (561 nm) were interleaved to avoid optical crosstalk. K) (Left) Fluorescence of GFP with patterned blue illumination, (right) ΔF/F in the pHoran4 channel after protocol shown in (J). Scale bars 100 μm. L) Time-course of pH inside (Blue On) and outside (Blue Off) the optogenetic stimulus regions. M) Quantification of the data in (L). Directly stimulated cells acidified to pH = 7.27 ± 0.016 (mean ± S.D., n = 26 cells), indirectly depolarized cells (Blue Off) did not acidify: pH = 7.31 ± 0.003 (n = 19 cells, p = 1.5e-8 Wilcoxon signed-rank test).

ChR2-3M and PsCatCh2.0 are potent non-acidifying channelrhodopsins.

A) Genetic constructs for simultaneous optogenetic stimulation using channelrhodopsin variants and pH imaging in polarized HEK cells. B) Images of HEK cells showing (top) GFP or YFP fluorescence, a marker for channelrhodopsin expression and (bottom) -ΔF/F in the pHoran4 channel, measured after protocol shown in (2C). Scale bars 100 μm. C) Time-course of pH in HEK cells expressing the three opsins. D) Quantification of the data in (C). CheRiff: pH = 6.98 ± 0.15 (mean ± S.D., n = 170 cells); ChR2-3M: pH = 7.31 ± 0.10 (n = 63 cells); PsCatCh2.0: pH = 7.30 ± 0.03 (n = 74 cells); p = 4e-31, p = 4e-35, Wilcoxon signed-rank test. E-I) Whole-cell voltage clamp measurements on HEK cells expressing channelrhodopsins. E) Steady-state photocurrents as a function of blue illumination intensity. F) Opening and closing kinetics as a function of blue light intensity. G) Steady-state photocurrents as a function of holding voltage. H) Normalized photocurrents from stimulation with light at 488 nm, 532 nm, 561 nm, 594 nm, and 640 nm (50 mW cm-2 in all cases). I) Normalized photocurrents from combinations of blue (488 nm, 240 mW cm-2) and orange (594 nm, 1 W cm-2) or red (640 nm, 8 W cm-2) light corresponding to intensities typical for all-optical electrophysiology.

Comparison of channelrhodopsin gating properties.

EPD50 is the effective power density for 50% activation. CheRiff data are from Ref. 25, Fig. S9 and Table S4. CheRiff reversal potential is from Ref.26.

Chr2-3M and PsCatCh2.0 acidify neurons less than CheRiff.

A) Genetic constructs for simultaneous optogenetic stimulation and pH imaging. B) Experimental paradigm for measuring pH responses to optogenetic stimulation. Stimulation (blue) and measurement (yellow) are interleaved for 60 s to avoid optical crosstalk. C) Images of cultured neurons showing (top) GFP or YFP fluorescence, a marker for Channelrhodopsin expression, (bottom) DF/F in the pHoran4 channel after the protocol shown in (B). (left) CheRiff-GFP, (middle) ChR2-3M-YFP, (right) PsCatCh2.0. Scale bars xx mm. D) Time-course of pH dynamics in cultured neurons. Cells expressing ChR2-3M and PsCatCh2.0 acidify less than CheRiff. E) Neurons expressing ChR2-3M, pH = 7.13 ± 0.19 (mean ± S.D., n = 31 cells), and PsCatCh2.0, pH = 7.14 ± 0.11 (mean ± S.D., n = 25 cells) had significantly less acidification (p = 2.5e-4, p = 4e-5, respectively, Wilcoxon signed-rank test) than CheRiff-expressing neurons, pH of 6.87 ± 0.27 (mean ± S.D., n = 24 cells). F) Genetic constructs for simultaneous optogenetic stimulation and Voltage imaging. G) Experimental paradigm for measuring voltage responses to optogenetic stimulation. Stimulation (blue) and measurement (red). H) Time-course of optogenetically activated spiking in cultured neuron expressing (top) CheRiff, (middle) ChR2-3M, or (bottom) PsCatCh2.0.

Procedure for calibrating pHoran4 pH measurements.

A) Representative image of cultured neurons expressing pHoran4. The cells have been permeabilized with Nigericin and are in a high K+ extracellular medium (Methods). B) Example fluorescence traces of individual cells as the dish is perfused with buffers of different pH values.

Engineering of ChR2-3M, a channelrhodopsin with high Na+ and K+ selectivity and high photocurrent amplitude.

(A) Shifts in reversal potential (Vr) of ChR2 variants upon changing extracellular Na+ or K+ concentration from 120 mM to 1 mM (mean ± S.D., n = 3-4 cells). (B) Photocurrent amplitudes of ChR2 variants (mean ± S.D., n = 8 cells). The triple mutant E101N/H134Q/D156H was modified with trafficking, ER export, and signal peptides and designated ChR2-3M. Experiments were performed with Xenopus oocytes expressing different Channelrhodopsin variants.

Depolarization of HEK cell monolayers with patterned stimulation of channelrhodopsins.

(A) GFP or YFP expression showing opsin expression in patterned stimulation region of gap junction coupled monolayer. (B) BeRST ΔF/F of Hek cell monolayer showing depolarization from patterned stimulation.