Abstract
Purpose:
Ca2+ homeostasis in rod and cone outer segments is important for visual adaptation and the health of photoreceptors. Rods and cones are thought to use different isoforms of Na+/Ca2+, K+ exchangers (NCKX) to extrude Ca2+ from their outer segments. We sought to determine the physiological importance of Nckx1 in mammalian rods by generating Nckx1 knockout mice and characterizing the functional, morphological, and molecular properties of its rods.
Methods:
To remove Nckx1, most of the first exon of Slc24a1 gene was deleted and replaced by neomycin cassette. Standard methods were used to produce Nckx1+/- mice which were crossed to produce Nckx1-/- and WT control mice. We used transretinal ERG to study photoreceptor function from intact isolated retinas perfused with Ringers’ solution supplemented with L-15 (0.72 g/L), 40 μM DL-AP4 to block the b-wave, and 100 μM BaCl2 to remove glial component. Contrast sensitivity as a function of mean luminance was determined with the OptoMotry system.
Results:
The light responses from Nckx1-/- rods were 100-fold smaller and with slower shut-off kinetics compared to those of WT controls. However, fractional sensitivity of Nckx1-/- rods was over 2-fold larger than in the control rods. Weber-like background adaptation of Nckx1-/- rods was preserved. In behavior experiments, optimal contrast sensitivity of Nkcx1-/- mice required more light than in WT mice but still less than in Gnat1-/- controls. Cone response amplitudes were comparable to those of Gnat1-/- mice and photopic visual acuity and contrast sensitivity were normal in Nckx1-/- mice. Retinal structure and rod morphology in Nckx1-/- mice were slightly degenerated and expression analysis revealed a significant downregulation of CNG channels in the Nckx1 KO mouse retinas.
Conclusions:
Nckx1 is required for normal rod function but not for Weber adaptation. The removal of Nckx1 had a subtle effect on rod morphology. We find a novel mechanism of regulation by Nckx1 of CNG channels expression in mouse rods that likely preserves a normal steady state Ca2+ and prevents large scale rod degeneration in Nckx1-/- mice. Channel regulation by Nckx1 expression levels could be a therapeutic target for preventing degeneration in rod channelopathies.
Keywords: 648 photoreceptors •
439 calcium •
688 retina