Abstract
Rod and cone photoreceptor neurons utilize discrete PDE6 enzymes that are crucial for phototransduction. Rod PDE6 is composed of heterodimeric catalytic subunits (αβ), while the catalytic core of cone PDE6 (α') is a homodimer. It is not known if variations between PDE6 subunits preclude rod PDE6 catalytic subunits from coupling to the cone phototransduction pathway. To study this issue, we generated a cone-dominated mouse model lacking cone PDE6 (Nrl(-/-) cpfl1). In this animal model, using several independent experimental approaches, we demonstrated the expression of rod PDE6 (αβ) and the absence of cone PDE6 (α') catalytic subunits. The rod PDE6 enzyme expressed in cone cells is active and contributes to the hydrolysis of cGMP in response to light. In addition, rod PDE6 expressed in cone cells couples to the light signaling pathway to produce S-cone responses. However, S-cone responses and light-dependent cGMP hydrolysis were eliminated when the β-subunit of rod PDE6 was removed (Nrl(-/-) cpfl1 rd). We conclude that either rod or cone PDE6 can effectively couple to the cone phototransduction pathway to mediate visual signaling. Interestingly, we also found that functional PDE6 is required for trafficking of M-opsin to cone outer segments.
Highlights
The variation in cGMP binding affinity is thought to affect the transducin-dependent activation of PDE6 and subsequent removal of inhibition by ␥- subunits of PDE6 [13, 14]. It is not known if the changes in properties of GAF domains between rod and cone PDE6 dictate their interaction with cellspecific transducin and inhibitory subunits (PDE6 ␥) and affect the ability of rod PDE6 to function in cone photoreceptor cells
Light-dependent PDE6 activation in cones lacking cone PDE6 was abrogated when rod PDE6- subunit was removed in NrlϪ/Ϫ cpfl1 rd mice
We demonstrated the presence of rod PDE6 in cone photoreceptor cells lacking NRL using multiple lines of evidence as listed below; 1) RT-polymerase chain reaction (PCR) with subunit specific primers showing the expression of pde6a and pde6b message, 2) Western blotting with subunit specific antibodies establishing the presence of rod PDE6 protein subunits and absence of cone PDE6 catalytic subunit, 3) mass spectrometry confirming the presence of rod PDE6 subunits with 100% confidence
Summary
Cone Photoreceptor Cells in NrlϪ/Ϫ cpfl1 Mice Respond to Light—To assess retinal function in the all-cone mouse model lacking cone PDE6, we performed ERGs. ERG measurements under dark and light-adapted conditions reflect rod and cone photoreceptor cells activities, respectively. To verify the expression of rod PDE6 protein in retina obtained from NrlϪ/Ϫ cpfl1/ϩ and NrlϪ/Ϫ cpfl1 mice, we performed immunoblotting of retinal extracts using catalytic subunit-specific PDE6 antibodies. In agreement with our immunoblotting results, cone PDE6 was absent in retinal sections from NrlϪ/Ϫ cpfl1 mice (Fig. 3D, top row).
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