Regulation of Drosophila TRPL channels by immunophilin FKBP59

Transient receptor potential and transient receptor potential-like (TRPL) a re Ca2+-permeable cation channels found in Drosophila photoreceptor cells a ssociated with large multimeric signaling complexes held together by the sc affolding protein, INAD. To identify novel proteins involved in channel reg ulation, Drosophila INAD was used as bait in a yeast two-hybrid screen of a Drosophila head cDNA library. Sequence analysis of one identified clone sh owed it to be identical to the Drosophila homolog of human FK506-binding pr otein, FKBP52 (previously known as FKBP59). To determine the function of dF KBP59, TRPL channels and dFKBP59 were co-expressed in Sf9 cells. Expression of dFKBP59 produced an inhibition of Ca2+ influx via TRPL in fura-2 assays . Likewise, purified recombinant dFKBP59 produced a graded inhibition of TR PL single channel activity in excised inside-out patches when added to the cytoplasmic membrane surface. Immunoprecipitations from Sf9 cell lysates us ing recombinant tagged dFKBP59 and TRPL showed that these proteins directly interact with each other and with INAD. Addition of FK506 prior to immunop recipitation resulted in a temperature-dependent dissociation of dFKBP59 an d TRPL. Immunoprecipitations from Drosophila S2 cells and from fly head lys ates demonstrated that dFKBP59, but not dFKBP12, interacts with TRPL in viv o. Likewise, INAD immunoprecipitates with dFKBP59 from S2 cell and head lys ates. Immunocytochemical evaluation of thin sections of fly heads revealed specific FKBP immunoreactivity associated with the eye. Site-directed mutag enesis showed that mutations of P702Q or P709Q in the highly conserved TRPL sequence (LPPPFN)-L-701-VLP799 eliminated interaction of the TRPL with dFK BP59. These results provide strong support for the hypothesis that immunoph ilin dFKBP59 is part of the TRPL-INAD signaling complex and plays an import ant role in modulation of channel activity via interaction with conserved l eucyl-prolyl dipeptides located near the cytoplasmic mouth of the channel.