(A) CRISPR targeted site in the carT gene and resulting mutations. (B) Primary structure and predicted transmembrane domains (yellow) of CarT. Red arrows indicate location of premature stops within mutant alleles. Gray and black letters indicate conserved and highly conserved amino acids among SLC22 transporters (Eraly et al., 2004; Koepsell, 2013), respectively. The SLC22 family is characterized by a large extracellular loop containing four cysteines (C) and a glycosylation site (blue) at conserved positions. (C) ERGs recorded from female flies carrying wild type (+) or the indicated CarT alleles (16A, 16B, 43, or tdTomato-CarT) with or without expression of a wild-type or a Myc-tagged UAS-CarT transgene driven by longGMR-Gal4 or repo-Gal4 as indicated. (D) Quantifications of ON and OFF transients, and SNPs of all genotypes in panel C averaged from three replicate experiments, including at least 45 traces from 15 flies (G: longGMR-Gal4; R: repo-Gal4; -: not present). (E) Phototactic behavior of OreR and carT43 mutant flies compared with other mutants that disrupt the histamine–carcinine cycle (HdcMB07212, ebony1, or tan1) presented as a light preference index. (F) Phototactic behavior of flies expressing a UAS-CarT transgene in photoreceptor neurons under control of the longGMR-Gal4 driver in carT43 mutants compared with controls shows the restoration of wild type behavior. For each graph, the box outlines the upper and lower quartiles, and the whiskers show minimum and maximum recorded values. ns, not significant; ****p < 0.0001 compared with carT43 (D) or OreR (E and F). CRISPR, clustered regularly-interspaced short palindromic repeats; ERG, electroretinograms; SNPs, sustained negative potentials.