Abstract
Purpose: :
Garp2 (glutamic acid-rich protein 2) is a product of alternative splicing from the Cngb1 gene encoding the beta subunit of the cGMP-gated cation channel. Garp2 is expressed exclusively in rods and may function in maintaining rod outer segment structural integrity and have a role in disk morphogenesis. Garp2 was shown to bind directly to PDE6 thereby lowering the "dark noise" in rods by reducing basal PDE6 activity levels. In this study, we examined the role of Garp2 in rod photoreceptor structure and function by generating and analyzing a transgenic mouse over-expressing Garp2 (Garp2 tg).
Methods: :
Transgenic mice were generated by expressing mouse Garp2 driven by the 4.4kb mouse rhodopsin promoter. Quantitative Western blotting, immunocytochemistry, and light microscopy were performed using established protocols. Electroretinogram (ERG) analyses were performed on 1-month-old Garp2 tg and compared to age-matched wild-type controls. The leading edge of dark-adapted ERG a-wave responses were fitted to the rod phototransduction model of Hood and Birch [IOVS, 1994; 35(7)].
Results: :
In the transgenic retina, the overexpressed Garp2 protein is properly localized to the ROS. In the transgenic line used for analysis Garp2 levels were 3-fold greater than WT. Analysis of the Garp2 tg mice at PN 30 shows that ROS length is reduced 20% while ONL nuclei are unchanged. Average a-wave responses to dark-adapted flashes were decreased 33% in the Garp2 tg. However, the sensitivity parameter S derived from the fit to the rod model was increased more than 50%.
Conclusions: :
Reduction in a-wave responses in the Garp2 tg may be attributed to reduced ROS length. The increase in phototransduction gain indicated by the increased S value may be due to direct modulation of PDE6 activity by Garp2. This result suggests that Garp2 may have a role in regulating phototransduction.
Keywords: photoreceptors • transgenics/knock-outs • electrophysiology: non-clinical