Diminishing neuronal acidification by channelrhodopsins with low proton conduction
- School of Engineering and Applied Sciences, Harvard University, United States
- Department of Chemistry, Harvard University, United States
- Department of Neurophysiology, University of Wurzburg, Germany
- Department of Physics, Harvard University, United States
Figures
Figure 1 with 1 supplement
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 standard deviation (SD) of n = 8 …
Figure 1—figure supplement 1
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 …
Figure 2
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 …
Figure 3 with 2 supplements
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, …
Figure 3—figure supplement 1
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 to 1 mM (mean ± standard deviation [SD], n = 3–4 cells). (B) Photocurrent …
Figure 3—figure supplement 2
Depolarization of HEK cell monolayers via patterned stimulation of channelrhodopsins.
(A) Blue light stimulation patterns. (B) Fluorescence of GFP or YFP tags on the opsins expressed in confluent monolayers of HEK cells, under patterned blue light excitation. (C) BeRST1 ΔF/F showing …
Figure 4
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 …
Tables
Table 1
Comparison of channelrhodopsin gating properties.
EPD50 is the effective power density for 50% activation. CheRiff data are from Fig. S9 and Table S4 of Hochbaum et al., 2014. CheRiff reversal potential is from Zhang et al., 2016.
| Reversal potential (mV) | ton fastest (ms) | ton at EPD50 (ms) | toff (ms) | EPD50 (mW cm−2) | Steady-state photocurrent (pA) | Ipeak/ISS | |
|---|---|---|---|---|---|---|---|
| CheRiff | 4 | 4.5 ± 0.3 | - | 16 ± 0.8 | 22 ± 4 | 1300 ± 80 | 0.65 |
| ChR2-3M (n = 4) | 16.6 ± 3.6 | 57 ± 21 | 800 ± 550 | 1950 ± 500 | 11.6 ± 8.7 | 1378 ± 618 | 1.00 |
| PsCatCh2.0 (n = 6) | 12.3 ± 4.7 | 4.2 ± 3.5 | 9.3 ± 1.2 | 17.6 ± 3.4 | 116 ±1 3 | 847 ± 359 | 0.92 |
