Nat. Methods 11, 338–346 (2014); published online 9 February 2014; addendum published after print 28 August 2014
A trafficking variant of the Chrimson molecule (Fig. 1c) was used for the Drosophila experiments in the original version of the paper (i.e., Fig. 3, Supplementary Figs. 14–16 and Supplementary Videos 2–6). This trafficking variant, called CsChrimson-KGC-GFP-ER2, is a CsChR-Chrimson chimera in which the Chrimson N terminus is replaced with the CsChR N terminus (Fig. 1a and Supplementary Fig. 1), with appended KGC and ER2 trafficking sequences (Fig. 1c).
In the original paper, we found CsChR to have high membrane expression levels (original Supplementary Figs. 5 and 6). We therefore attempted to boost Chrimson expression by swapping the Chrimson N terminus with the CsChR N terminus. As no transmembrane regions were modified, we unsurprisingly found that CsChrimson has the same spectral and kinetic properties as Chrimson in murine cultured neurons (Fig. 1b,d,f,g). We additionally compared CsChrimson with and without KGC and/or ER2 trafficking sequences and found all variants to have similar photocurrents in cultured neurons (Fig. 1d,e). However, we observed more cytosolic aggregates with the KGC version and a reduction of aggregates with the ER2 version (Supplementary Fig. 2). It is therefore likely that CsChrimson will be of use with the ER2 trafficking sequence in some biological contexts.
Whole-cell patch-clamp recordings were made using a Multiclamp 700B amplifier and a Digidata 1550 digitizer (Molecular Devices). All other experimental conditions are the same as previously described.
GenBank/EMBL/DDBJ: CsChrimson is listed under accession code KJ995863.
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Klapoetke, N., Murata, Y., Kim, S. et al. Addendum: Independent optical excitation of distinct neural populations. Nat Methods 11, 972 (2014). https://doi.org/10.1038/nmeth0914-972
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