Blue-light-responsive GtACR2 shows gating distinct from green-light-responsive GtACR1 and voltage-dependent closure
Background: Channel gating differs as well as light color
The blue-light-absorbing anion channelrhodopsin GtACR2 has a different channel-gating mechanism from the green-light-absorbing GtACR1, and its rapid channel closure was found to depend strongly on the holding voltage. Published in the Proceedings of the National Academy of Sciences (PNAS), this study compared the two proteins not only by their light-response wavelengths but also by their electrical and photochemical properties.
Optogenetics uses light-responsive proteins to regulate the electrical activity of cells. Guillardia-derived anion channelrhodopsins GtACR1 and GtACR2 are being studied as molecular tools for suppressing neuronal firing with light. GtACR2 has faster channel kinetics, which offers an advantage for optogenetic control of neurons that fire at high frequencies, but its mechanism of action was less well understood than that of GtACR1. The proteins’ original functions in algae are also unclear.
Key findings: A single gating mechanism and changes in variants
The researchers recorded currents by patch clamp in mammalian cells and analyzed the photochemical properties of purified proteins and amino-acid substitution variants. The study examined functional differences between the channels by assessing cellular electrical responses alongside the proteins’ light responses.
A single channel-gating mechanism was identified in GtACR2. This distinguishes it from GtACR1, for which the researchers had previously identified two mechanisms. The observation of one gating mechanism does not mean that it operates independently of voltage. GtACR2’s rapid channel closure depended strongly on the voltage maintained during the experiments. During the photocycle, in which light-activated proteins pass through multiple intermediate states, substantial reversibility between intermediates was also observed. Photocurrents induced by successive light pulses likewise revealed differences between the channels that reflect secondary photochemical reactions.
Variant analysis narrowed down the amino acids involved in these differences. When the researchers replaced five GtACR2 amino-acid residues that differ between the proteins with the corresponding residues from GtACR1, the spectral sensitivity and slow, biphasic channel closure of GtACR1 were reproduced. Biphasic closure means that two components appear during the channel-closing process. Among the individual variants, only R129C restored the characteristic GtACR1 gating mechanism. R129C also caused the largest red shift—that is, a shift toward longer wavelengths—in sensitivity among the individual substitutions.
Differences also emerged in ion permeability. During a single photocycle, the relative permeability of GtACR2 to fluoride and chloride ions changed, whereas no such change was observed for GtACR1. The abstract does not specify the direction or magnitude of this change, making it difficult to assess its implications for neuronal suppression.
Meaning and outlook: A basis for comparing optogenetic tools
These findings provide a basis for evaluating inhibitory optogenetic tools by considering the voltage dependence of channel closure, gating mechanism, and permeability during the photocycle alongside absorption wavelength. Based on comparisons with cation channelrhodopsins from Chlamydomonas and anion channelrhodopsins from other protists, the researchers proposed that the differences between blue- and green-light-absorbing channels may reflect a broader pattern. This is the researchers’ interpretation, which could guide future tool development.
The findings fall within the scope of mechanistic studies using mammalian cells and purified proteins. The available abstract does not provide specific voltage conditions, closure time constants, or effect sizes. It also does not support claims that neuronal suppression without delay, complete elimination of interference in multicolor control, or therapeutic efficacy for epilepsy has been demonstrated.
The methods and main findings can be checked in the PNAS article’s abstract and bibliographic record.
PNAS, Vol. 123, Issue 20, May 2026. DOI: 10.1073/pnas.2603129123
Summary: This structural biophysics study unravels the operational kinetics of Guillardia theta anion channelrhodopsin 2 (GtACR2), a premier tool for optogenetic neuronal silencing with a 50-nm blue-shifted absorption spectrum relative to GtACR1. Integrating time-resolved optical spectroscopy with whole-cell patch-clamp electrophysiology, researchers identified a distinct photointermediate trajectory during retinal isomerization that accelerates channel gating kinetics over two-fold. Mechanistically, while GtACR1 undergoes voltage-dependent structural shifts, GtACR2 employs a voltage-independent electrostatic switch directly coupled to photon absorption, minimizing signal latency and establishing a high-fidelity sequence framework for multi-color optogenetic circuit engineering.
Researchers selecting optogenetic tools to suppress high-frequency neuronal firing should compare not only response wavelength but also how quickly a channel closes at the voltage used in their experiments. The finding that GtACR2’s rapid closure depends strongly on holding voltage provides a reason to evaluate candidate tools under the same voltage conditions. The five-residue substitution and R129C results offer a starting point for follow-up variant studies of the relationship between spectral sensitivity and gating properties. For example, protein-design studies aimed at shifting response wavelength could assess changes in variant sensitivity and channel closure together. The abstract alone, however, does not establish the neuronal circuit-control performance or therapeutic suitability of any particular variant.