A user's guide to channelrhodopsin variants: features, limitations and future developments

Abstract

Channelrhodopsins (ChRs) are light-activated channels from algae that provide these organisms with fast sensors to visible light for phototaxis. Since its discovery, channelrhodopsin-2 (ChR2) has been used as a research tool to depolarize membranes of excitable cells with light. Subsequent chimeragenesis, mutagenesis and bioinformatic approaches have introduced additional ChR variants, such as channelrhodopsin-2 with H134R mutation (ChR2/H134R), channelrhodopsin-2 with E123T mutation (ChETA), Volvox carteri channelrhodopsin-1 (VChR1), Volvox carteri channelrhodopsin-2 (VChR2), channelrhodopsin-2 with C128 or D156A mutations (ChR2/C128X/D156A), chimera D (ChD), chimera EF (ChEF) and chimera EF with I170V mutation (I170V). Each of these ChR variuants has unique features and limitations, but there are few resources summarizing and comparing these ChRs in a systematic manner. In this review, the seven following key properties of ChRs that have significant influences on their effectiveness as research tools are examined: conductance, selectivity, kinetics, desensitization, light sensitivity, spectral response and membrane trafficking. Using this information, valuable qualities and deficits of each ChR variant are summarized. Optimal uses and potential future improvements of ChRs as optogenetic tools are also discussed.

Figure 1

Theoretical changes in the membrane potential induced by ChRs with 20 Hz pulsed light stimulation (blue lines) on a passive membrane model. (A) The ideal ChR with no desensitisation and rapid kinetics (labelled ChR (Ideal)), responds well to pulsed stimulation with consistent responses and the repolarisation is primarily determined by the membrane properties. (B) The photocurrents from ChR with ChR2-like channel (ChR(ChR2-like)) desensitise strongly to light pulses, lead to reduced membrane depolarisation with later light pulses in a membrane with low resistance. In a membrane with high resistance, the cell enters ‘depolarisation block’ and repetitive stimulation does not allow repolarisation. (C) Photocurrents from a ChR with slow kinetics (ChR (Slow)) does not allow sufficient repolarisation of the membrane between pulses leading to ‘depolarisation block’. (D) Reduction of ChR conductance and/or level of membrane expression (ChR(Half response)) can result in insufficient depolarisation with light. For modelling parameters see Supplementary Figure 1. Orange lines indicate threshold of −45 mV.

Figure 2

Comparisons of the ‘fast’ ChRs with ChR2. (A) Voltage-clamp recordings of ChR2 (A1), ChETA (A2), ChD (A3), and ChIEF (A4) transfected HEK293 cells to 500 ms continuous light and pulsed light stimulation (50 Hz). ChIEF show the most consistent photocurrents for both stimulation protocols. ChETA and ChD have faster kinetics than ChR2 after the termination of light pulses. All recordings were made in small HEK293 cells to ensure accurate voltage-clamping. (B) The light-intensity response curve for ChR variants for their peak (B1) and the steady-state response (B2) normalised to the projected peak response. The sensitivity to light is ChR2 > ChIEF > ChD > ChETA. (C) Comparison of the off-rate time constant for ChR2, ChETA, ChD and ChIEF. (D1) Examples of ChR2 and ChIEF expressed at low levels in cultured neurons and their light-induced action potential trains at 25 Hz. ChIEF shows superior performance relative to ChR2. (D2) Summary of D1 in multiple neurons. (E1) Similar comparison as in D1, with ChR2/H134R and ChIEF, but at a higher expression level. (E2) Summary of E1 in multiple neurons. * indicates p < 0.01%.

Figure 3

Comparisons of VChR1, ChR2 and ChIEF. (A) Expression of ChR2, ChIEF, and VChR1 in HEK293 cells showing membrane trafficking of the variants. (B1) Typical responses of ChR2 (gray) and VChR1 (black) to different colour lights (same photon flux). (B2) The response spectra of the peak and steady-state responses of the VChR1, ChR2 and ChIEF, normalised to their peak response. (C1) The response of VChR1 to 570 nm light does not completely recover in the dark. (C2) Recovery of ChR2, VChR1 and ChIEF responses at various time points after the initial stimulation. Scale bar in A is 10 µm.