Abstract
Purpose: :
The rod cGMP-gated cation channel β-subunit and associated soluble GARP proteins are required for normal phototransduction, disk morphogenesis and rod cell structural integrity, yet the function of these proteins in these processes is not fully understood. We have generated several mouse models designed to dissect the function of these proteins in the rod cell. Here we report on the rescue of structure and function in a Cngb1 knockout mouse model expressing a truncated β-subunit.
Methods: :
Transgenic mice were generated on WT and photoreceptor null Cngb1-X1 knockout backgrounds expressing opsin promoter-driven truncated β-subunit 2-3 fold above WT levels. Immunohistochemistry, Western, functional analysis by ERG, and OCT were performed using established protocols.
Results: :
Two lines of mice were generated that contain the truncated β-subunit transgene that expresses residues 370-1326 of the full-length β-subunit. The transgene is devoid of any GARP2 sequence (1-291), and contains only 160 aa of GARP1-related sequence. In WT mice, transgene expression was observed in several layers of the retina and endogenous β-subunit levels were reduced, however a- and b-wave ERG responses were comparable to WT mice without the transgene. In 2 month old Cngb1-X1 knockout mice truncated beta subunit expression restored the structural loss observed in the knockout mice and exhibited near normal retina function by ERG analysis. At 7.5 months when knockout mice rods have degenerated, truncated transgene mice exhibit some cell loss but near normal rod structure and function.
Conclusions: :
Truncated β-subunit can partially substitute for full-length beta subunit and GARP proteins in Cngb1-X1 knockout mice indicating that soluble GARPs are not essential for rod structure and function until later ages. In WT mice expressing the truncated β-subunit, transport is impaired or altered indicating that sequence in the truncated N-terminal region may be necessary for proper transport of the β-subunit to the outer segment.
Keywords: ion channels • retinal degenerations: cell biology • transgenics/knock-outs