Abstract
Channelrhodopsins are light-gated cation channels that have been widely used for optogenetic stimulation of electrically excitable cells. Replacement of a glutamic acid in the central gate with a positively charged amino acid residue reverses the ion selectivity and produces chloride-conducting ChRs (ChloCs). Expressed in neurons, published ChloCs produced a strong shunting effect but also a small, yet significant depolarization from the resting potential. Depending on the state of the neuron, the net result of illumination might therefore be inhibitory or excitatory with respect to action potential generation. Here we report two additional amino acid substitutions that significantly shift the reversal potential of improved ChloC (iChloC) to the reversal potential of endogenous GABAA receptors. As a result, light-evoked membrane depolarization was strongly reduced and spike initiation after current injection or synaptic stimulation was reliably inhibited in iChloC-transfected neurons in vitro. In the primary visual cortex of anesthetized mice, activation of iChloC suppressed spiking activity evoked by visual stimulation. Due to its high operational light sensitivity, iChloC makes it possible to inhibit neurons in a large volume of brain tissue from a small, point-like light source.
Highlights
Off-kinetics of some ChloC variants, sustaining inhibition for several seconds
Threonine 159 was mutated to Cysteine (T159C) to increase photocurrents[12] and Aspartic acid 156 was substituted with Asparagine (D156N) to stabilize the open state of the channel and increase its operational light sensitivity[13]
When we introduced the three pore mutations of improved ChloC (iChloC) into CoChR (E63Q, E70R, E81S), the reversal potential of photocurrents in HEK 293 cells was very negative (Erev = − 62 ± 3 mV; n = 8 ), indicating successful conversion into a chloride-conducting CoChR
Summary
Off-kinetics of some ChloC variants, sustaining inhibition for several seconds. High light sensitivity is an important feature if large volumes of neuronal tissue are to be addressed from a localized light source such as a fiber-coupled laser or LED. Given the dramatic developmental changes in [Cl−]1i 0 and its dependence on the local concentration of impermeant anions[11], the net effect of published ChloC variants on neuronal excitability in vivo is difficult to predict. Photocurrents of our improved ChloC (iChloC) reverse at nearly identical membrane potentials than GABAergic IPSCs, suggesting a high selectivity for Cl− ions. IChloC no longer shows depolarizing activity in patch-clamped neurons and reliably inhibits synaptically evoked spikes in undisturbed CA1 pyramidal cells. When expressed in primary visual cortex, iChloC strongly suppressed visually evoked spiking of pyramidal neurons in vivo. Due to its high Cl−-selectivity and operational light sensitivity, iChloC is an ideal tool to silence neurons in vivo with very low light exposure
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