Abstract
Purpose: :
We previously showed that mice lacking expression of the rod cGMP-gated cation channel β-subunit and its related GARP protein are also greatly reduced in channel α-subunit, yet still exhibit rod function at reduced sensitivity. We have now performed detailed EM analysis, quantitative Western analysis, and real time PCR to further define the phenotype in Cngb1 KO mice.
Methods: :
All mice used in the study were 18-60 days old. Knockout mice are deleted for expression of the cGMP-gated channel β-subunit and GARP proteins in the rod photoreceptors. Western analysis, real time PCR, SEM and TEM were done using established protocols.
Results: :
Our initial analysis of the Cngb1 knockout mouse revealed in addition to an absence of channel β-subunit and GARP proteins a dramatic reduction in α-subunit. Quantitative Western analysis indicates that the channel α-subunit is >90 % reduced in homozygous Cngb1 KO mice. Additionally, ABCA4 and GC1 appear to be about 80% reduced in the knockout mouse compared to WT levels. Rhodopsin levels were close to WT and peripherin, Rom-1 and PDE6 levels were only about 30% reduced which may reflect the observed slow progressing retinal degeneration. Real time PCR analysis of several transcripts encoded by photoreceptor-specific genes (Cngb1 β-subunit and GARP, RPGRIP1, CNGA1, NCKX1, Rom-1, Per-2, Rho) did not reveal any significant changes with the exception of the β-subunit and GARP that were undetectable and RPGRIP1 that was 30-40 % reduced compared to WT levels. Light microscopy of 30 do KO mice retina revealed normal stratification of retinal layers, slightly reduced ROS length and abnormal photoreceptor morphology. By TEM additional girth of rods is frequently observed on most cells appearing as ectopic disk-like material that extends along the confines of the plasma membrane, similar to that reported in RPGRIP1 KO mice and cytochalasin D treated animals. SEM reveals a non-uniform wavy appearing distribution of increased width of the outer segments consistent with the overextended disks observed by TEM.
Conclusions: :
While ROS disks can form normally in Cngb1 KO mice, the knockout does lead to a novel alteration of outer segment structure due to altered disk morphogenesis. Thus, GARP and/or the β-subunit may contribute to the structural integrity of the rod photoreceptor through a role in regulating disk morphogenesis. At least two integral membrane ROS proteins (ABCA4 and GC1) are reduced in abundance due to Cngb1 knockout that may be related to a proposed transport function for the β-subunit.
Keywords: photoreceptors • retinal degenerations: cell biology • ion channels