By screening the Zuker collection of EMS-mutagenized Drosophila ( Koundakjian et al., buy INK1197 2004), we identified a mutant, xport1, which displayed an abnormal electroretinogram (ERG) compared to wild-type ( Figure 1A). The xport1 mutant had a transient response during prolonged light stimulation, which was indistinguishable in amplitude
and time course from the transient receptor potential (trp) phenotype observed in the trp343 null mutant. Photoreceptor cells that lack TRP protein are unable to sustain a steady-state current, via TRPL, due to reduced Ca2+ influx. More specifically, low levels of Ca2+ result in a failure of Ca2+ and PKC dependent inhibition of PLC. Consequently, uncontrolled PLC activity depletes its Doxorubicin substrate (microvillar PIP2) leading to premature closure of TRPL channels ( Gu et al., 2005 and Hardie et al., 2001). Consistent with the transient light response, xport1 displayed a severe reduction in TRP protein levels compared to wild-type ( Figure 1B). Interestingly, Rh1 protein levels were also severely reduced in the xport1 mutant ( Figure 1C). The mutation in xport1 is recessive, as the heterozygotes were normal for both TRP and Rh1 protein ( Figures 1B and 1C). Therefore, xport is required for the proper expression of both TRP
and Rh1. To identify the xport locus, we first narrowed the cytogenetic location by deficiency mapping to 92B3-92C1 on the third chromosome, corresponding to 26 loci spanning 145 kb of DNA ( Figures 1B, 1C, and see Figure S2A available online). We identified a C to T substitution at nucleotide position 145 within the coding region of CG4468, causing a premature stop codon at glutamine49 ( Figure S2A, arrow). To confirm Carnitine palmitoyltransferase II that CG4468 was the xport locus, we restored wild-type function by introducing a wild-type copy of CG4468 into the genome of the xport1 mutant. TRP and Rh1 protein expression were restored to wild-type levels in the rescue line ( Figures
1B and 1C). These data confirm that CG4468 is, indeed, the xport locus. In addition, we found that eye-specific expression of three independent CG4468 RNAi transgenes leads to a severe reduction in TRP by western blot analysis ( Figure S1A). We analyzed electrical responses to light in the xport1 mutants by whole cell voltage-clamp recordings of dissociated ommatidia. In wild-type photoreceptors, brief flashes elicited rapid macroscopic inward currents mediated by TRP and TRPL channels ( Figure 1D). In xport1 mutants, response amplitudes were ∼20-fold reduced ( Figure 1E), consistent with a severe reduction in TRP and Rh1. Sensitivity was restored to wild-type levels in xport1 flies expressing the wild-type xport cDNA rescue construct ( Figure 1D). If TRP channel expression was completely eliminated in the xport1 mutants, then we would predict that the residual response in xport1 would be eliminated in a trpl302;xport1 double mutant.