(2006) Vision Res

(2006) Vision Res. the effector enzymes in the vertebrate phototransduction cascade (1,C5). Pole PDE6 is composed of homologous catalytic -subunit (PDE6A) and -subunit (PDE6B) and two copies of a small inhibitory -subunit (P) (3). Cone PDE6 is usually a catalytic dimer of two identical -subunits (PDE6C) (3). A cone-specific inhibitory P-subunit is usually highly homologous to the rod P (6). In rod photoreceptors, PDE6 Tolterodine tartrate (Detrol LA) is located in the specialized compartments called rod outer segments (ROS), where it associates with disc membranes. The membrane attachment of PDE6 is usually mediated by farnesylation of the PDE6A C terminus and geranylgeranylation of the PDE6B C terminus (7). In cones, geranylgeranylated PDE6C resides on infoldings of the cone outer segment plasma membrane (8). Following photoexcitation of rod or cone photoreceptor cells, PDE6 is activated by the GTP-bound transducin -subunit (GtGTP) that relieves the P inhibition of the enzyme. cGMP hydrolysis by active PDE6 leads to a cellular response due to a closure of cGMP-gated channels in the photoreceptor plasma membrane (2,C5). Although PDE6 plays a prominent role in vertebrate vision, the structure-function associations of PDE6 are poorly comprehended in comparison with other key phototransduction proteins. The lack of an expression system for PDE6 has become a major impediment for PDE6 research. Importantly, an expression system for PDE6 is required to elucidate the mechanisms of visual diseases linked to mutations in PDE6 catalytic subunits. Mutations in the and genes are responsible for 3C4% and 4% of cases of recessive retinitis pigmentosa, respectively (9, 10). Retinitis pigmentosa is usually a common hereditary disease of retinal degeneration that results in vision loss caused by the death of the rod and cone photoreceptors. Recently, mutations in the gene have been identified in human patients with achromatopsia (11). Achromatopsia results from a loss of cone function and is characterized by low visual acuity and lack of color discrimination. The problem of PDE6 expression was partially resolved through generation and characterization of chimeras between PDE6 and PDE5 (cGMP-binding, cGMP-specific PDE) (12,C15). The two PDE enzymes share similar domain name organization, a relatively high homology of the catalytic domains, specificity for cGMP relative to cAMP, and sensitivity to common catalytic site inhibitors (1, 3). The chimeric PDE5/PDE6 approach facilitated delineation of the P-binding sites of PDE6, but it has severe limitations in identification of the unique catalytic determinants of the visual effector (15). The reason for the inability of PDE6 to fold correctly in various cell types, besides photoreceptor cells, is usually unknown. One possibility is that expression of functional PDE6 requires photoreceptor specific chaperone proteins, such as AIPL1 (aryl hydrocarbon receptor-interacting protein-like 1). AIPL1 was shown to be a specialized chaperone obligatory for expression and stability of PDE6 in rod photoreceptors (16, 17). Mice lacking or expressing reduced levels of AIPL1 show reduced expression and destabilization of PDE6 and develop retinal degeneration (16, 17). In humans, mutations in cause Leber congenital amaurosis, a severe early onset Tolterodine tartrate (Detrol LA) retinopathy (18), apparently by compromising PDE6 expression. Thus, expression of PDE6 in living photoreceptor cells may represent a single approach to produce and mutagenize PDE6. In this study, we demonstrate the power of transgenic frogs for expression and studies of PDE6 by ectopically expressing EGFP fusion protein of human cone PDE6C in rods of embryos. and and expressing EGFP-PDE6C Tolterodine tartrate (Detrol LA) in rods were produced using the method of Tolterodine tartrate (Detrol LA) restriction enzyme-mediated integration (20). Transgenic expressing EGFP-Gt with EGFP insertion within the helical domain name of Gt were generated similarly as described previously (21). Transgenic animals were identified 6 days postinjection by visual examination for EGFP fluorescence using a fluorescence microscope MZ16 Leica equipped with a GFP filter. Several transgenic tadpoles were maintained through metamorphosis into adult frogs. Transgenic male adult frogs at age 10 months were mated with wild-type female to produce a large number of transgenic tadpoles for biochemical characterization of PDE6C. At about stage 50, retinas from transgenic tadpoles were dissected into small pieces in Ringer’s buffer, and the EGFP fluorescence in living photoreceptor cells was imaged using a confocal fluorescence microscope LSM510 (Zeiss). Immunofluorescence For dark adaptation, tadpoles were kept in the dark overnight. For light adaptation, dark-adapted tadpoles were exposed to room light (500 lux) for at least 60 min. Detached tadpole eyeballs were fixed in 4% formaldehyde in phosphate-buffered saline for 2 h at 22 C. After fixation, the eyeballs were submersed in a 30% sucrose answer in phosphate-buffered saline for 5 KLF1 h at 4 C and then embedded in tissue freezing medium (Tris-buffered saline) and frozen on dry ice. Radial sectioning (10 m) of the retina was performed using a cryomicrotome Microm HM 505E. Retinal cryosections were air-dried.