Abstract
Purpose :
Cpfl3 (cone photoreceptor function loss 3) is a mouse model commonly used as a functional null of cones due to a naturally occurring mutation in the alpha subunit of cone transducin (Gnat2). However, we have found that Cpfl3 cones have a normal dark current and can respond to light though with decreased sensitivity. Here we investigate the mechanisms underlying this altered physiology.
Methods :
Western blots were performed on retinas isolated from Cpfl3, rod transducin knockout (Gnat1), Cpfl3/Gnat1 double knockouts, and control C57 age-matched mice at 4, 9, and 14 weeks of age. Relative levels of GNAT2 protein were compared between strains. In addition, retinas isolated from Cpfl3, Cpfl3/Gnat1, and control C57 mice were mounted and stained with antibodies against GNAT2 and cone arrestin (ARR3) as well as peanut agglutinin. Physiological recordings were made from identified cones with whole-cell patch clamp in retinal slices.
Results :
Cpfl3 and Cpfl3/Gnat1 mice have 2 to 3-fold less GNAT2 protein compared to C57 at 4 weeks. There was a statistically significant difference in the number of cones between the three strains at both 4 and 14 weeks, and a significant difference in number of cones in C57 versus Cpfl3 and Cpfl3/Gnat1 mice. At 4 weeks, Cpfl3 and Cpfl3/Gnat1 had a higher average number of cones than C57, but by 14 weeks, Cpfl3 and Cpfl3/Gnat1 had fewer cones than C57. There was a significant decrease in the number of cones at 4 weeks versus 14 week-old Cpfl3 and Cpfl3/Gnat1 mice, but not C57. Cones from animals 2 – 3 months of age had normal dark currents but greatly reduced sensitivity and activation constants. Responses decayed more slowly than in WT, indicating an altered mechanism of inactivation of least one step in transduction. At dim intensities rod responses could be recorded from cones, indicating intact rod/cone gap junctions.
Conclusions :
A reduction in GNAT2 protein level in Cpfl3 mice is at least in part responsible for the decreased sensitivity and activation rate of cones. The number of cones is also reduced after 14 weeks in Cpfl3 mice; abnormal response decay could be a harbinger of eventual degeneration. Our results show that Cplf3 cones can respond to light with currents of normal amplitude and cannot be assumed to be a Gnat2 null.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.