Characterization of engineered channelrhodopsin variants with improved properties and kinetics

Abstract

Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile(170) of ChEF to Val (yielding "ChIEF") accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.

Figure 1

Schematic of ChR chimeras and their basic properties. (A) Schematic of ChR showing the sites of chimera crossings at the TIVW sequence of helix D (X1: ChD), VPKG sequence of the helix E-F loop (X2: ChEF), and EGFG sequence at transmembrane helix F (X3: ChF). (B) Confocal images of ChD, ChEF, and ChF expressing HEK cells with mCherry fluorescence at plasma membrane. (C) The typical responses of ChR2 (C1), ChR1 (C1), and the three chimeras (C2) in pH 7.35 saline to 500 ms of 470 nm light. The black bars above indicate the time of the light stimulation. (D) Summaries of the mean maximal response amplitudes (D1) and the plateau/maximum response ratio (D2) with ChR1 (n = 7), ChR2 (n = 11), ChD (n = 9), ChEF (n = 8), and ChF (n = 6). The mean response amplitudes of ChD and ChEF greatly exceed the mean ChR1 response but are identical to ChR2, and the level of inactivation of ChEF is comparable to ChR1 and ChF. (E) Current-voltage relationships of ChR2, and ChEF in the presence of 145 mM extracellular sodium (E1), 5 mM extracellular sodium ions (E2), and 5 mM extracellular sodium ions + 25 mM extracellular potassium (E3), showing the reversal potentials of ChR2 and ChEF at various extracellular sodium and potasium ion concentrations. (F) Fura-2 measurements of calcium in cells expressing ChR2 and ChEF to 5 s of 470 nm light in the presence of 80 mM extracellular calcium showing detectable elevation of intracellular calcium. Scale bar in B: 10 μm.

Figure 2

Spectral and kinetic properties of ChR variants to varying light density and duration. (A) Spectral responses of ChR2 (A1), ChEF (A2), and ChIEF (A3). The vertical lines indicate the estimated peaks. All responses normalized to the maximum response obtained from the cell tested at the various wavelengths (n = 5 for ChR2, ChEF; n = 6 for ChIEF). (B) Examples of ChR2 (B1), ChEF (B2), and ChIEF (B3) responses to 0.11, 0.48, 2.59, 9.64, and 19.81 mW/mm² of light provided by an LED 470 nm light source. Note the faster channel closure after light removal for ChIEF compared to ChEF. (C1) The intensity-amplitude and intensity-onset (C3) relationship of ChR2 (black, n = 8), ChEF (light gray, n = 7), and ChIEF (dark gray, n = 11) for the maximum response (C1) and the plateau component of the response (C2) normalized to projected maximum response of the individual cell tested. Introduction of I170V (ChIEF) reduced the EC50 of ChEF by 2.3× (for the maximum response) and 3× (for the plateau response). (D) Responses of ChR2 (D1), ChEF (D2) and ChIEF (D3) to 1, 2, 3, 4, 5, 10, and 20 ms of light stimulation at ∼19.8 mW/mm².

Figure 3

Nonstationary fluctuation analysis of ChR2, ChEF, and ChIEF. (A) An example of nonstationary fluctuation analysis of ChIEF. The mean (A1) and variance (A2) of ChIEF were obtained from 60 pulses of 470 nm light 10 s apart. (A3) The mean-variance plot and the least-squares fitted curve of ChIEF obtained from the up-slope of the response. (B) The estimated single-channel currents of ChR2 (−0.092 ± 0.022pA; n = 8), ChEF (−0.0965 ± 0.012pA; n = 6), and ChIEF (−0.113 ± 0.020 pA; n = 9) (B1) and estimated single-channel conductance calculated assuming ohmic conductance (1.084 ± 0.258 pS for ChR2, 1.185 ± 0.150 pS for ChEF and 1.463 ± 0.253 pS for ChIEF) (B2). The electromotive force used for estimating single-channel conductance was measured to be ∼87 mV for ChR2 and ∼82 mV for the chimeric channels.

Figure 4

Recovery of ChRs from inactivation and the ChR response to 50 Hz and 100 Hz of burst stimulation. (A) Example of ChIEF-mediated responses to second stimulations after 5 s and 25 s delay. The response after 25 s delay exhibited incomplete recovery of the transient peak and appearance of a slow component (arrow). (B) The recovery of the three ChR variants at different interpulse intervals (ChR2, n = 11; ChEF, n = 10; ChIEF, n = 10). The recovery ratio is obtained by dividing the maximal amplitudes of the second response by the first. ChR2 showed near-complete recovery after 25 s, but ChEF and ChIEF reached only ∼80% of the initial response. (C) Currents resulting from 3 ms 470 nm light pulses (19.8 mW/mm²) delivered at 50 Hz (left column) and 100 Hz (right column) for 100 ms, then repeated 150 ms later, applied to ChR2 (C1), ChEF (C2), ChIEF (C3), ChR2/H134R (C4), and ChD (C5).

Figure 5

Comparisons of action potential inducing fidelity of ChR2 and ChIEF in transfected neurons. Typical responses of ChR2 (A) and ChIEF (B) transfected cultured hippocampal neurons to 25 Hz (A1 and B1), 50 Hz (A2 and B2), and 75 Hz (A3 and B3) of pulsed light stimulation (470 nm, 19.8 mW/mm², 4 ms). (C) Summary of the percentage of successful action potentials induced in ChR2- and ChIEF-transfected neurons. (D) Maximum projection confocal images of ChR2-EGFP and ChIEF-EGFP expressing cultured hippocampal neurons. (E) The integrated fluorescence values of ChR2-EGFP (n = 10) and ChIEF-EGFP (n = 11) expressing neurons measured from a square 21.73μm² area in the soma at the in-focus optical slice of the neurons at the interface between the cell and the coverslip. In C, ^∗ indicates significance at the 0.01% level (ChR2, n = 10; ChIEF, n = 9). Scale bar in D: 20 μm.

Figure 6

Comparisons of action potential inducing fidelity of ChR2/H134R and ChIEF in transfected neurons at high level of expression. Typical responses of ChR2/H134R (A) and ChIEF (B) transfected cultured hippocampal neurons to 25 Hz (A1 and B1), 50 Hz (A2 and B2), and 75 Hz (A3 and B3) of pulsed light stimulation (470 nm, 9.8 mW/mm², 0.5 ms). (C) Summary of the percentage of successful action potentials induced in ChR2/H134R- and ChIEF-transfected neurons at 6.1 mW/mm² (C1), 9.8 mW/mm² (C2), and 17 mW/mm² (C3) of stimulating light intensity. In C1, ^∗∗ indicates significance at the 0.05% level; in C2, ^∗ indicates significance at the 5% level.