Channelrhodopsins (ChRs) are the only known ion channels that are directly gated by light. These channels are seven-transmembrane-domain proteins that provide algae with visible light ‘perception’. When excited by light, ChR channels open and depolarize the membrane. Since their first ex vivo characterizations (Nagel et al. 2002, 2003), ChRs have generated much interest as research tools to depolarize neuronal membranes with light (Boyden et al. 2005; Li et al. 2005; Nagel et al. 2005). Although ChRs are applied to neuroscientific research currently, they can potentially be used to activate cardiac myocytes, skeletal muscles and voltage-gated ion channels expressed in cell lines to study other physiological functions. The use of ChRs to control membrane excitabilities of neurons has been extensively reviewed previously (Zhang et al. 2006) and will not be the main focus of this review. Instead, this review aims to provide an overview of the unique biophysical properties and limitations of the different ChR variants and highlight the specific experiments for which each variant is best suited.
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.
Factors that influence the effectiveness of ChR-induced membrane depolarization
Ideally, when using ChRs to manipulate membrane potential, the change should be instantaneous and predictable. However, the intrinsic properties of the membrane (resistance and capacitance) limit the rate and level of achievable depolarization. Assuming instantaneous opening of the ChR and the absence of voltage-gated ion channels in the membrane, the membrane charges or discharges exponentially with the time constant of the membrane, which is dependent on the membrane capacitance and number of open channels. The level of depolarization is determined by the membrane resistance and membrane current (Ohm's law). The unique attributes of each ChR have a significant impact on the ability of the experimenter to control depolarization with light. The following seven properties of ChRs can affect the accuracy of manipulating membrane potentials with this technology.
- 1Channel conductance. The estimated single-channel conductance of ChR2 is below 1 pS (Nagel et al. 2003; Bamann et al. 2008; Feldbauer et al. 2009; Lin et al. 2009a), which is less than the conductance of the common membrane channels. Conductance directly determines the effectiveness of light-induced depolarization. In a cell with 50 MΩ membrane resistance, ∼500,000 functional ChRs need to open simultaneously to depolarize the membrane by 15 mV (assuming 100 fS single-channel conductance, reversal potential of 0 mV and membrane potential −60 mV).
- 2Ion selectivity. All ChRs are cation permeant, non-selective towards H+, Na+, K+ and Ca2+, and have a reversal potential near 0 mV at physiological pH (Nagel et al. 2003; Berthold et al. 2008; Zhang et al. 2008; Lin et al. 2009a; Tsunoda & Hegemann, 2009; Gunaydin et al. 2010). The maximal change in membrane potential that can be achieved with ChRs is near its reversal potential of 0 mV if the cell has minimal basal ‘leak’ current.
- 3Kinetics. Opening and closing rates of ChRs are critical factors in achieving temporally precise manipulation of membrane potentials. The speed of membrane potential change induced by ChR is associated with both the channel kinetics and the intrinsic membrane properties of the cell. Although the ideal kinetics of a ChR should be as fast as is physically allowed, very rapid kinetics must be balanced with sacrifices in light sensitivity.
- 4Desensitization and recovery from the desensitized state. In the presence of continuous, strong illumination or repetetive, pulsed stimulation, the response of ChR2 decays by 80% from a peak response to a steady-state level response (Nagel et al. 2003). This ‘desensitization’ requires the expression of fivefold more functional ChR2 to achieve depolarization above threshold after desensitization. All known ChRs desensitize, and some variants do not recover fully after initial stimulation (Nagel et al. 2002; Lin et al. 2009a,b; Schoenenberger et al. 2009).
- 5Light sensitivity. Channelrhodopsins vary in light sensitivity. Mutations that increase light sensitivity of ChRs often have negative impacts on the kinetics of the channel (Berndt et al. 2009; Lin et al. 2009a). The transition rate from the open to the closed state of the channel reflects the energy difference between the two states of the channel, which directly reflects on the energy required to sustain the channel in the open state.
- 6Spectral response. The maximal and steady-state components of the photocurrents may have different spectral excitation peaks (Lin et al. 2009a,b). Many ChRs can be ‘preconditioned’ with a second light pulse of a different wavelength to speed up the recovery of the desensitized component (Lin et al. 2009a,b).
- 7Membrane trafficking and expression. Low expression and/or intracellular aggregation of ChRs reduce their effectiveness of membrane depolarization (Lin et al. 2009b; Tsunoda & Hegemann, 2009). Overexpression of an exogenous membrane protein can be toxic and/or adversely affect the membrane properties.
Figure 1 and Fig. S1 demonstrate the effects of changes in ChR conductance, expression, desensitization and kinetics on achieving temporally precise membrane depolarization by light pulses on a passive membrane. Increased desensitization reduces the consistency of depolarization, whereas the changes in kinetics alter the repolarization rate. Improved membrane trafficking and/or channel conductance can increase the amplitudes of membrane depolarization, but also reduce effective repolarization. Some shortcomings of ChRs (low conductance, desensitization) can be partly compensated by overexpression. High expression is not ideal because this can change the native membrane environment. The level of membrane depolarization and rate of repolarization are also heavily influenced by the intrinsic properties of the membrane, in addition to the expression of voltage-gated channels in the membrane.
Unique properties and limitations of ChR variants
Channelrhodopsin-2. Channelrhodopsin-2 was the first channelrhodopsin used to excite neurons with light (Boyden et al. 2005; Li et al. 2005; Nagel et al. 2005). When stimulated with high-intensity light, ChR2 has a rapid on-rate and moderate channel closing rate (Fig. 2; Nagel et al. 2003; Ishizuka et al. 2006; Lin et al. 2009a). Channelrhodopsin-2 is maximally excited by 470 nm light (Fig. 3; Nagel et al. 2003; Ishizuka et al. 2006; Lin et al. 2009a). The main deficiency of ChR2 is the high level of desensitization (Fig. 2), which reduces the current by ∼80% at physiological pH (Nagel et al. 2003; Ishizuka et al. 2006; Lin et al. 2009a). The desensitized response fully recovers after 25 s in the dark (Fig. 3; Lin et al. 2009a). Channelrhodopsin-2 trafficks to the membrane well when expressed at low levels but forms intracellular aggregates at high levels (Lin et al. 2009a,b). The conductance of ChR2 is estimated to be 50–250 fS (Nagel et al. 2003; Bamann et al. 2008; Feldbauer et al. 2009; Lin et al. 2009a).
Channelrhodopsin-2/H134R. Channelrhodopsin-2 with H134R mutation (ChR2/H134R) has a modest reduction in desensitization, a slight increase in light sensitivity and slower channel closing compared with ChR2 (Nagel et al. 2005; Lin et al. 2009a,b). These changes increase photocurrents, but the slower kinetics makes ChR2/H134R less temporally precise than ChR2. Although not tested, ChR2/H134R is assumed to have the same ion permeability and recovery from desensitization as ChR2. Channelrhodopsin-2/H134R has no advantages over the newer Chimera EF (ChEF) and ChEF with I170V mutation (ChIEF) variants (Lin et al. 2009a).
Channelrhodopsin-2/C128X (X being threonine (T), alanine (A) or serine (S)) and ChR2/D156A. The introduction of C128X (X being threonine (T), alanine (A) or serine (S)) (Berndt et al. 2009) or D156A mutation (Bamann et al. 2010) to ChR2 increases light sensitivity, but slows kinetics (off-rate 2–100 s) and reduces photocurrent. The channel closure rate can be modestly sped up with orange light. The first published study described ChR2/C128X as ‘step-function’ or ‘bistable’ opsins, and these variants were used to induce prolonged subthreshold depolarization (Berndt et al. 2009). A recent study reveals that ChR2/C128X recovers little from the strong desensitization in the dark, which is not ‘bistable’ as initially described (Schoenenberger et al. 2009). Despite these complications, ChR2/C128X or ChR2/D156A is useful for inducing prolonged depolarization when temporal precision and depolarization level are not critical (Schoenenberger et al. 2009).
Channelrhodopsin-2/E123T (ChETA). The E123T mutation in ChR2 (ChETA) creates faster kinetics but reduces photocurrent amplitude (Gunaydin et al. 2010). ChETA has strong desensitization in our hands and much reduced light sensitivity (Fig. 2 and Table 1). The reduced photocurrent might be beneficial, as it decreases the incidence of depolarization block associated with overexpression of ChR2. The first published result with ChETA in neurons incorporates the H134R mutation to increase the amplitude of the photocurrent, but the H134R mutation also partly negates the increase in kinetics. The first publication (Gunaydin et al. 2010) described ChETA as useful for high-frequency stimulation, but whether ChETA provides any advantages over ChD or ChIEF is unclear. The reduced photocurrent, red-shifted spectral response and reduced sensitivity of ChETA may be ideal for performing simultaneous calcium imaging experiments with fura-2 (Zhang et al. 2006).
|Response spectra peak||Level of desensitization||Light sensitivity/EC50||Opening rate τ (ms)||Closing rate τ (ms)|
|Channel variant||Peak response||Steady-state response||I steady-state/Ipeak||Peak response||Steady-state response||19.8 mW mm−2 light intensity|
|ChR2||∼470 nm||∼450 nm||∼0.22 (470 nm)||∼1.10 mW mm−2||∼1.05 mW mm−2||∼1.21 ms||∼13.5 ms|
|ChR2/H134R||∼450 nm||∼450 nm||∼0.39 (470 nm)||∼1.07 mW mm−2||∼0.98 mW mm−2||∼1.92 ms||∼17.9 ms|
|ChETA||∼490 nm*||∼0.24 (470 nm)‡||∼5.02 mW mm−2‡||∼0.62 mW mm−2‡||∼0.86 ms‡||∼7.9–8.5 ms*‡|
|VChR1||∼570 nm||∼550 nm||∼0.48 (570 nm)||Not tested||Not tested||∼2.8 ms||>90 ms†|
|(15 mW mm−2**)|
|ChD||∼450 nm||∼450 nm||∼0.31 (470 nm)||∼3.23 mW mm−2||∼1.02 mW mm−2||∼1.49 ms||∼7.82 ms|
|ChEF||∼470 nm||∼490 nm||∼0.70 (470 nm)||∼0.72 mW mm−2||∼0.46 mW mm−2||∼1.56 ms||∼24.9 ms|
|ChIEF||∼450 nm||∼450 nm||∼0.80 (470 nm)||∼1.65 mW mm−2||∼1.38 mW mm−2||∼1.62 ms||∼12.0 ms|
Volvox carteri channelrhodopsin-1 (VChR1). This Volvox cateri VChR1 was initially characterized as a red-shifted ChR variant with peak response of ∼530 nm (Zhang et al. 2008). We found, with the same photon flux for stimulation and 410 nm preconditioning light to induce full recovery, that the peak response occurs at ∼570 nm and the steady-state response at ∼550 nm (Fig. 3; Lin et al. 2009b). Despite the red shift, VChR1 is excited strongly by 400 nm light, and simultaneous use with a second ChR or calcium imaging is not possible without cross-excitation. Volvox cateri VChR1 also has the following limitations: (1) slow channel kinetics, leading to temporal imprecision with photostimulation (Zhang et al. 2008); (2) incomplete recovery from the desensitized state (Lin et al. 2009b); and (3) poor membrane trafficking and expression (Fig. 3; Lin et al. 2009b; Tsunoda & Hegemann, 2009). Introducing the E123T (ChETA mutation) into VChR1 modestly improves the kinetics of VChR1 (∼35 ms off-rate; J.Y. Lin, unpublished observation). Currently, VChR1 has limited utility but is a useful template to engineer improved red-shifted ChR variants.
Chimera EF (ChEF) and Chimera EF with I170V mutation (ChIEF). Chimera EF and ChIEF variants were engineered by chimeragenesis of ChR1 and ChR2 (Lin et al. 2009a). Both ChEF and ChIEF have increased steady-state phase responses without reduction of the peak photocurrent. ChEF and ChIEF have the most consistent photocurrent responses when stimulated with continuous light or high-frequency pulsed light (Fig. 2; Lin et al. 2009a). The kinetics of ChIEF is faster than ChEF but comparable to ChR2 (Fig. 2). ChIEF has slightly reduced light sensitivity relative to ChR2 and should not interfere with its use. The desensitized responses of ChEF and ChIEF do not fully recover in the dark, but recovery of the photoresponse can be induced by 570 nm light (Lin et al. 2009a). The greater steady-state responses of CHEF and ChIEF compensate for the desensitized peak response despite incomplete recovery. ChEF and ChIEF express better than ChR2 in mammalian cells and traffick efficiently to the membrane (Lin et al. 2009b; Wang et al. 2009). Efficient membrane trafficking may result in toxicity when ChEF and ChIEF are expressed at high levels. ChIEF is the best ChR for conducting experiments with high-frequency, repetitive stimulation or continuous illumination, because the response amplitudes are most consistent with millisecond time scale kinetics (Fig. 2).
Other ChR variants: ChR1, VChR2 and Chimera D (ChD). Channelrhodopsin-1 was the first ChR characterized ex vivo (Nagel et al. 2002; Berthold et al. 2008; Tsunoda & Hegemann, 2009). The low conductance of ChR1 at pH 7 limits its use as a scientific tool.
The ChD variant was generated by the chimeragenesis of ChR1 and ChR2 (Wang et al. 2009; Lin et al. 2009a). ChD is comparable to ChETA in kinetics, but has better membrane trafficking/expression (Wang et al. 2009; Lin et al. 2009a). ChD may be used in a similar manner to ChETA (Fig. 2) but does not require the H134R mutation to increase the photocurrent.
The properties of the ChR variants are summarized in Table 1.
Future developments of ChR variants as research tools
The following improvements should enhance the utility of the ChR technology.
- 1Higher conductance. An increased conductance would enhance efficiency and reduce the need for overexpression.
- 2Change ion selectivity. Channelrhodopsins that have selective permeability for chloride or potassium ions would theoretically hyperpolarize the membrane when activated. The theoretical efficiency of these ChRs would be higher than the current microbial opsin pumps (Zhang et al. 2007). Calcium-selective and calcium-impermeable ChRs would be useful in signal transduction and imaging experiments, respectively.
- 3Improving kinetics without sacrificing light sensitivity. The ideal ChR would have a rectangular response with rapid kinetics, a true ‘step-function opsin’, and these mutations would not reduce light sensitivity.
- 4Narrowing VChR1 spectral responses. Reducing the response of VChR1 to blue light (∼470 nm) will allow for the simultaneous use with calcium imaging dyes and other ChRs in the same preparation without cross-excitation.
- 5Red shift the spectral response above 600 nm. Peak and steady-state responses above 600 nm would allow for stimulation in deep tissue with minimal light absorption and scattering. Volvox carteri VChR1 can respond to light above 600 nm, but its kinetics and properties at this wavelength greatly limit its usefulness (Fig. 3).
The use of ChRs allows for the manipulation of membrane potential in genetically defined cells with light. Channelrhodopsins can be invaluable research tools of the future, as indicated by the increase in literature based on use of this technology. While many ChR variants have been developed, many characteristics still need to be optimized to generate better ChRs. Care should be taken to characterize new variants and compare them with known variants to ensure adequate information for the users of these tools.
J. Y. Lin received funding from the Foundation of Research, Science and Technology of New Zealand. Research is supported by grants to Roger Y. Tsien from NIH (NS027177) and Howard Hughes Medical Institute. I thank Drs S. B. Sann and E. A. Rodriguez for editorial assistance. The ChD, ChEF and ChIEF constructs can be requested from http://www.tsienlab.ucsd.edu or requested from John Y. Lin (firstname.lastname@example.org).