Journal: bioRxiv

Article Title: Temporally extended and reversible neuronal silencing with Aion

doi: 10.1101/2022.02.25.481932

Figure Lengend Snippet: (A) Left: Engineering strategy yielding Aion and Phobos CADN , two new blue-shifted, step-function ACRs with temporally extended conducting states. Mutation of the D156 residue to either C or N (yielding Aion and Phobos CADN , respectively) greatly slows down the closing kinetics of the ACRs compared to its parental construct Phobos CA . The generated variants are highlighted by a black frame. Right: Schematic of Aion indicating point mutations (triangles) at the relative position within the respective transmembrane helix. (B) Representative photocurrent traces of the slow-cycling ACRs Phobos CA , Aion, Phobos CADN , and Gt ACR1 CA activated by a short 20 ms light pulse at indicated wavelengths and irradiance of 3.35 mW/mm 2 (460nm) and 3.14 mW/mm 2 (505 nm). (C) Left: quantification of peak photocurrent ( I p ) and photocurrent 30 s post illumination ( I 30s ) for each ACR. Right: photocurrent ratio at 30 s compared to the peak. (D) Normalized photocurrent after light shutoff. (E) Closing time constants (τ off ) for each ACR. For (C) and (E), black lines correspond to mean values ± SEM, and circles are single measurement data points (n PhobosCA = 5 -6 cells, nAion = 5 - 10 cells, n PhobosCADN = 8 - 9 cells, n Gt ACR1CA = 6 cells). Kruskal- Wallis test with Dunn’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Phobos CA mutants (D156C/Aion, D156N, D156S, D156H) were generated by site-directed mutagenesis of our previously reported plasmid Phobos CA -citrine ( ) (Addgene #98218).

Techniques: Mutagenesis, Residue, Construct, Generated

Journal: bioRxiv

Article Title: Temporally extended and reversible neuronal silencing with Aion

doi: 10.1101/2022.02.25.481932

Figure Lengend Snippet: Maximum-intensity projection images of two-photon stacks showing expression of Phobos CA -D156C ( A ), Phobos CA -D156N ( B ), Phobos CA -D156S ( C ), and Phobos CA -D156H ( D ) in CA1 pyramidal neurons after single-cell electroporation in organotypic hippocampal slice cultures. Fluorescence intensity is shown as inverted gray values. Opsin-citrine fluorescence (top row) was mainly localized at the plasma membrane in Phobos CA -D156C (A) and Phobos CA -D156N (B) expressing cells. However, Phobos CA -D156S (C) and Phobos CA -D156H (D) expressing neurons showed poor trafficking and strong protein accumulation in the soma. mCerulean was co-electroporated together with all opsin variants and served as a morphology marker (bottom row).

Article Snippet: Phobos CA mutants (D156C/Aion, D156N, D156S, D156H) were generated by site-directed mutagenesis of our previously reported plasmid Phobos CA -citrine ( ) (Addgene #98218).

Techniques: Expressing, Electroporation, Fluorescence, Clinical Proteomics, Membrane, Marker

Journal: bioRxiv

Article Title: Temporally extended and reversible neuronal silencing with Aion

doi: 10.1101/2022.02.25.481932

Figure Lengend Snippet: ( A ) Representative current traces of kinetically modified Phobos CA variants and Gt ACR1 CA . ACR variants were illuminated with 500 ms (row 1-4) or 20 ms (last row) actinic light at indicated wavelengths, while termination of photocurrents was accelerated with orange or red light for 10 s as indicated. ( B ) and ( C ) temporally expanded photocurrent traces as shown in (A) showing different modes of inactivation (B) and accelerated closing kinetics of Phobos CA variants (C). ( D ) Quantification of the residual photocurrent after orange or red-light application. While Gt ACR1 CA showed a 30 % photocurrent reduction when red light was applied 2 s post initial activation, application after 30 s caused partial re-opening of the channel. Phobos CA variants could be fully closed by orange light. (E) Quantification of the accelerated channel closing of Phobos CA variants with orange light.

Article Snippet: Phobos CA mutants (D156C/Aion, D156N, D156S, D156H) were generated by site-directed mutagenesis of our previously reported plasmid Phobos CA -citrine ( ) (Addgene #98218).

Techniques: Modification, Activation Assay