Perfecting Chr2

RESEARCH HIGHLIGHTS NEUROSCIENCE Perfecting ChR2 Two new reports describe variants of chan- but in a typical neurobiology experiment nelrhodopsin 2 with improved properties. the channel is thought to transport mostly Channelrhodopsin 2 (ChR2) has been a sodium ions. If one were to slightly increase godsend tool to study brain function. This the number of calcium ions transported, protein—originally found in tiny algae— the group reasoned, this could result in is a membrane-ion channel that opens up improvements in the channel’s performance in response to pulses of light, producing a for neuronal activation. change in the membrane potential of charged By modifying one residue in wild-type cells. Algae use ChR2 to signal the presence ChR2, the group generated a mutant with of light and trigger their swimming away or higher calcium permeability, called ‘CatCh’ toward it in the pond; neuroscientists, after (Kleinlogel et al., 2011). In nonneuronal ‘transplanting’ ChR2 into neurons, use it to Image of a neuron expressing the TC mutant, and cells, CatCh’s modest preference for cal- provoke light-triggered action potentials in its spiking trace. Image courtesy of T. Oertner. cium ions elicits approximately three times cells embedded deep in brain tissue. Not sur- higher currents and a slight slowdown of its prisingly, some of ChR2’s natural properties As with previous higher-current ChR2 kinetics compared to wild-type ChR2. But are not exactly ideal for this purpose. mutants, however, the closure of the TC surprisingly, when expressed in neurons, In particular, the channel’s small cur- mutant’s ion channel after a light stimulus is the group saw a nearly 70-fold increase in rents and slow kinetics still limit the poten- slightly slowed down. “A fast closure is very the cell’s light sensitivity and a surprisingly tial applications of ChR2 in neuroscience. important because after an action potential rapid and complete repolarization of its Improving these properties would enable you want to repolarize the neurons to their membrane after each spike. researchers to more reliably induce action initial, baseline membrane potential; if the Behind these properties, Bamberg explains, potentials (‘spikes’) in cells located farther channelrhodopsin is still open, you get all are the indirect effects triggered by the local Nature America, Inc. All rights reserved. All rights Inc. America, Nature 1 away from the applied light source, use sorts of problems,” explains Oertner. To increases in calcium produced by CatCh at lower light powers to stimulate them or get tackle this, the group decided to combine the the neuron’s membrane. “CatCh can be seen © 201 away with weaker transgene expression. TC mutation with a mutation already known as a light-gated membrane-bound calcium One way to improve ChR2’s performance to accelerate the closure of wild-type ChR2, source,” he says. For one, local increases in is by mutagenesis. Although this strategy the E123T mutation or ‘ChETA’. The double calcium result in the activation of voltage- has already yielded several ChR2 variants E123T,T159C (ET-TC) mutant exhibits both gated sodium channels and result in the fact that exhibit faster kinetics or larger cur- increased photocurrents and faster kinetics that you need much less light to get a depo- rents, so far one thing has always come at compared to wild-type ChR2. larization event. Secondly, calcium activates the expense of the other. When expressed in neurons, the ET-TC channels that are responsible for the mem- The search for the ‘perfect ChR2’ continues mutant elicits spikes with high fidelity across brane’s repolarization, accelerating this pro- in many laboratories around the world, and a wide range of light intensities and stimula- cess. CatCh could also be used to modulate two independent teams have now reported tion frequencies. During these studies, the calcium levels in subcellular compartments several improved ChR2 variants. A joint group also found a previously unknown role in response to light in any kind of cell. three-laboratory team composed of the labs for the ET mutation. “We found that [the One exciting lesson from these studies of Peter Hegemann at Humboldt University, mutation that created ChETA] is the voltage- is the potential of combining mutations Karl Deisseroth at Stanford University and sensing position for ChR2, so by introducing to refine the properties of channelrhodop- Thomas Oertner at the Friedrich Miescher this mutation the channel no longer slows sin, promising yet better tools to come for Institute have developed a ChR2(T159C) down at low voltage,” explains Hegemann. optogenetics. mutant, the ‘TC’ mutant, which, when This property enables rapid repolarization of Erika Pastrana expressed in neurons, elicits photocurrents the membrane after a spike. RESEARCH PAPERS almost twofold larger than those of wild- A second study, by members of Ernst Berndt, A. et al. High-efficiency channelrhodopsins type ChR2 (Berndt et al., 2011). Researchers Bamberg’s lab at the Max Planck Institute for fast neuronal stimulation at low light levels. Proc. Natl. Acad. Sci. USA 108, 7595–7600 (2011). can use the TC mutant to spike neurons with of Biophysics, used mutagenesis to alter Kleinlogel, S. et al. Ultra light-sensitive and fast neuronal dimmer light pulses, which will be handy ChR2’s preference for certain ions over oth- activation with the Ca2+-permeable channelrhodopsin when performing experiments in vivo. ers. ChR2 is a nonselective cation channel, CatCh. Nat. Neurosci. 14, 513–518 (2011). NATURE METHODS | VOL.8 NO.6 | JUNE 2011 | 447.

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