Fig. 8

Fig. 8 KCRs with improved K⁺ selectivity have increased potency.

A, B Representative confocal images of fly brains carrying AMB247 > KCR1-C29D or BMB247 > WiChR. Both opsins are strongly expressed in the MB lobe axonal regions. Anti-Brp staining is shown in magenta and anti-GFP staining is shown in green. Scale bar = 50 μm. For both genotypes, n = 1 biologically independent sample over 1 independent experiment. C Quantifications of anti-GFP intensity in posterior (P) and anterior (A) brain regions for KCR1-C29D and WiChR crossed with MB247-Gal4. Top: individual brain hemispheres are shown as slope plots. The height of the gray bars shows average intensity values. Bottom: posterior–anterior mean difference effect sizes of anti-GFP intensities; error bars represent the 95% CI. MB247 > KCR1-C9D, n = 4 biologically independent samples over 4 independent experiments. MB247 > WiChR, n = 6 biologically independent samples over 6 independent experiments. D Representative images of the opsins in N2a cells. (Left) KCR1-C29D and (Right) WiChR showed expression at the membrane, along with some intracellular puncta. Scale bar = 10 µm. For both genotypes, n = 1 biologically independent sample over 1 independent experiment. E Climbing performance of WiChR (E1 and E3) and KCR1-C29D (E2 and E4) flies in the presence of light. The opsins were expressed pan-neuronally (elav-Gal4, E1-2) or in motor neurons (OK371-Gal4, E3-4). The performance of opsin-expressing flies was compared with the averaged performance of the corresponding Gal-4 driver and UAS responder controls (gray) in the presence of light. The last 10 s of the experiment were used for effect-size comparisons. Error bars represent the 95% CI. Green illumination intensity was 11 μW/mm². Blue illumination was 85 μW/mm². Genotypic controls for elav > WiChR, n = 209 biologically independent animals over 14 independent experiments. Elav > WiChR, n = 193 biologically independent animals over 13 independent experiments. Genotypic controls for elav > KCR1-C29D, n = 250 biologically independent animals over 16 independent experiments. Elav > KCR1-C29D, n = 156 biologically independent animals over 11 independent experiments. Genotypic controls for OK371 > WiChR, n = 211 biologically independent animals over 14 independent experiments. OK371 > WiChR, n = 176 biologically independent animals over 11 independent experiments. Genotypic controls for OK371 > KCR1-C29D, n = 278 biologically independent animals over 18 independent experiments. OK371 > KCR1-C29D, n = 176 biologically independent animals over 10 independent experiments. F, G Inhibiting MB neurons with MB247 > KCR1-C29D (F) or MB247 > WiChR (G) impaired shock-odor memory. Retesting the same animals in the absence of illumination restored conditioned odor avoidance (PI) in KCR1-C29D flies. The performance of WiChR-expressing flies remained low during retest. Green light illumination intensity was 58 µW/mm². Blue light illumination was 21 μW/mm². Error bands show 95% CI. Genotypic controls for MB247 > KCR1-C29D, n = 600 biologically independent animals over 12 independent experiments. MB247 > KCR1-C29D, n = 354 biologically independent animals over 7 independent experiments. Genotypic controls for MB247 > WiChR, n = 528 biologically independent animals over 11 independent experiments. MB247 > WiChR, n = 312 biologically independent animals over 6 independent experiments. H The top panel displays the averaged paired comparisons of feeding volume between the lights off and on testing epochs for Gr64f > KCR1-C29D flies and genotypic controls. The bottom panel shows the averaged mean difference in feeding volume effect size for the light off and on epochs. Green light illumination intensity was 24 µW/mm². Error bars show 95% CI. KCR1-C29D/+, n = 27 biologically independent animals over 3 independent experiments. Gr64f/+, n = 29 biologically independent animals over 3 independent experiments. Gr664f > KCR1-C29D, n = 33 biologically independent animals over 3 independent experiments. I The top panel (I1) shows the averaged horizontal walking speed of OK371 > WiChR flies and genotypic controls before, during (indicated by blue and green boxes), and after light actuation. The bottom panel (I2) shows the speed of the same OK371 > WiChR flies and speeds for flies expressing OK371 > ACR1 and OK371 > KCR1-ET. Error bands show a 95% CI. Green light illumination intensity was 24 µW/mm². Blue light illumination was 24 μW/mm². Genotypic controls for WiChR, n = 126 biologically independent animals over 7 independent experiments. OK371 > WiChR, n = 80 biologically independent animals over 5 independent experiments. OK371 > ACR1, n = 72 biologically independent animals over 8 independent experiments. OK371 > KCR1-ET, n = 58 biologically independent animals over 6 independent experiments. J Kaplan–Meier post-actuation recovery plots for flies expressing opsins in motor neurons (OK371-Gal4, J1) or pan-neuronally (elav-Gal4, J2). All flies expressing ACR1 recovered in the first 2 s. The majority of KCR1-ET flies recovered in the first 10 s and the majority of WiChR-expressing flies remained immobile >60 s after illumination. OK371 > WiChR, n = 80 biologically independent animals over 1 independent experiment. OK371 > ACR1, n = 72 biologically independent animals over 1 independent experiment. OK371 > KCR1-ET, n = 58 biologically independent animals over 1 independent experiment. Elav > WiChR, n = 55 biologically independent animals over 1 independent experiment. Elav > ACR1, n = 64 biologically independent animals over 1 independent experiment. Elav > KCR1-ET, n = 66 biologically independent animals over 1 independent experiment. K Climbing performance of NKCC > ACR1 (K1), NKCC > KCR1-C29D (K2), and NKCC > WiChR (K3) flies and their respective genotypic controls (gray) during light illumination. Exposing NKCC > ACR1 flies to light did not impair climbing performance. Light exposure induced twitching behavior and occasional falls in NKCC > KCR1-C29D flies. Overall, the light effect was not sufficiently strong to induce substantial climbing impairment. During illumination, NKCC > WiChR flies displayed twitching, falls, and partial paralysis which led to a strong reduction in climbing. The last 10 s of the experiment were used for effect size comparisons. Error bands represent the 95% CI. Green light illumination intensity was 11 μW/mm². Blue light illumination was 85 μW/mm². Genotypic controls for ACR, n = 201 biologically independent animals over 12 independent experiments. NKCC > ACR1, n = 41 biologically independent animals over 6 independent experiments. Genotypic controls for KCR1-C29D, n = 204 biologically independent animals over 12 independent experiments. NKCC > KCR1-C29D, n = 102 biologically independent animals over 6 independent experiments. Genotypic controls for WiChR, n = 204 biologically independent animals over 12 independent experiments. NKCC > WiChR,n = 97 biologically independent animals over 6 independent experiments. Additional statistical information for all panels is presented in Supplementary Dataset 1. Source data are provided as a Source Data file.