Rod Photoreceptors Are Functional in Rod Channel ß–Subunit Knockout Mice: A Role for GARP in the Maintenance of Outer Segment Structural Integrity

Abstract

Purpose: : Mice lacking expression of the rod cGMP–gated cation channel ß–subunit and its related GARP proteins show reduced expression of channel α–subunit, yet the b–wave photoresponse, while reduced in sensitivity at least 30–fold, shows a maximum amplitude that is near wild type. Rods also show disorganized disks and malformed outer segments. We have analyzed the single cell response to light and have begun to characterize components of the phototransduction cascade to determine the basis for the photoresponse and observed structural abnormalities.

Methods: : All mice used in the study were 18–30 days old. Knockout mice are deleted for the predicted promoter region and the first two exons of the Cngb1 locus precluding expression of the ß–subunit and GARP proteins in rods. Immunocytochemistry was done with standard protocols and antibodies against α and ß channel subunits and peripherin–2. Rod recordings and [Ca²⁺] measurements were made by drawing individual OS into the lumen of a suction electrode with established techniques. Outer segment [Ca²⁺] measurements were made with fluorescent dye fluo–5F). Freeze fracture replicas were prepared and viewed by TEM as previously described.

Results: : Labeling with a ß–subunit antibody did not show any signal in the knockout retina confirming the absence of full–length ß–subunit protein. An α–subunit antibody showed sparse labeling in rod outer segments throughout the knockout retina. Analysis of signal intensity indicates a greater than 85 % reduction in α–subunit levels. Peripherin–2 antibodies labeled outer segments prominently and were not significantly different from labeling observed in wild type mice. Single cell responses of rod photoreceptors, which had outer segments only 1/4 to 1/3 the length of wild type rods, showed an 8–30 fold lower sensitivity and a significant reduction in maximum response amplitude. [Ca²⁺] was moderately lower than in wild type rod outer segments. Structural analysis of wild type retinas revealed a network of disk/disk and plasma membrane/disk membrane connections that may be affected in the knockout mouse.

Conclusions: : Rod photoreceptors in the knockout mice are functional despite the absence of ß–subunit and significant reduction of α–subunit. Both GARP and the ß–subunit may contribute to the structural integrity of the rod photoreceptor.

Keywords: photoreceptors • retinal degenerations: cell biology • ion channels