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Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling.

ABSTRACT: Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H⁺, Na⁺, K⁺ and anions have been discovered or engineered, Ca²⁺-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca²⁺ permeability and improve their Ca²⁺ affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca²⁺ permeation at negative voltage and low extracellular Ca²⁺ concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca²⁺ concentrations. Moreover, CapChR2 can robustly trigger Ca²⁺ signalling in hippocampal neurons. When expressed together with genetically encoded Ca²⁺ indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca²⁺ entry in brain explants.

SUBMITTER: Fernandez Lahore RG

PROVIDER: S-EPMC9772239 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling.

Fernandez Lahore Rodrigo G RG Pampaloni Niccolò P NP Schiewer Enrico E Heim M-Marcel MM Tillert Linda L Vierock Johannes J Oppermann Johannes J Walther Jakob J Schmitz Dietmar D Owald David D Plested Andrew J R AJR Rost Benjamin R BR Hegemann Peter P

Nature communications 20221221 1

Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H+, Na+, K+ and anions have been discovered or engineered, Ca2+-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca2+ permeability and improve their Ca2+ affinity by targeted mutagenesis at the ...[more]

PMID: 36543773

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Project description:The brain helps us survive by forming internal representations of the external world1,2. Excitatory cortical neurons are often precisely tuned to specific external stimuli3,4. However, inhibitory neurons, such as parvalbumin-positive (PV) interneurons, are generally less selective5. PV interneurons differ from excitatory neurons in their neurotransmitter receptor subtypes, including AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs)6,7. Excitatory neurons express calcium-impermeable AMPARs that contain the GluA2 subunit (encoded by GRIA2), whereas PV interneurons express receptors that lack the GluA2 subunit and are calcium-permeable (CP-AMPARs). Here we demonstrate a causal relationship between CP-AMPAR expression and the low feature selectivity of PV interneurons. We find low expression stoichiometry of GRIA2 mRNA relative to other subunits in PV interneurons that is conserved across ferrets, rodents, marmosets and humans, and causes abundant CP-AMPAR expression. Replacing CP-AMPARs in PV interneurons with calcium-impermeable AMPARs increased their orientation selectivity in the visual cortex. Manipulations to induce sparse CP-AMPAR expression demonstrated that this increase was cell-autonomous and could occur with changes beyond development. Notably, excitatory-PV interneuron connectivity rates and unitary synaptic strength were unaltered by CP-AMPAR removal, which suggested that the selectivity of PV interneurons can be altered without markedly changing connectivity. In Gria2-knockout mice, in which all AMPARs are calcium-permeable, excitatory neurons showed significantly degraded orientation selectivity, which suggested that CP-AMPARs are sufficient to drive lower selectivity regardless of cell type. Moreover, hippocampal PV interneurons, which usually exhibit low spatial tuning, became more spatially selective after removing CP-AMPARs, which indicated that CP-AMPARs suppress the feature selectivity of PV interneurons independent of modality. These results reveal a new role of CP-AMPARs in maintaining low-selectivity sensory representation in PV interneurons and implicate a conserved molecular mechanism that distinguishes this cell type in the neocortex.

Dataset Information

Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling.

Publications

Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling.

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