Expression of an N-terminal GARP region truncation of CNG channel β-subunit on a KO background partially rescues structure/function and alters calcium feedback

Abstract

Purpose : The rod cGMP-gated cation channel β-subunit and two associated GARP proteins encoded by the Cngb1 locus are required for normal phototransduction, disk morphogenesis, and rod structural integrity. Deletion of the promoter region and first coding exon in mice (X1 KO) eliminates expression of these proteins and leads to structural/ functional photoreceptor deficits and retinal degeneration. The aim of this study is to determine if a truncated β-subunit (Tβ) can substitute for full length β in the X1 KO mouse retina.

Methods : The murine β subunit is composed of 1326 aa, of which 550 correspond to GARP1 and 326 to GARP2. We introduced a 285 aa N-terminal truncation product (Tβ) into WT mice (WT Tβ) and crossed the transgene allele onto the X1 KO background (X1 Tβ). Mice were assessed by OCT, histology, EM Western, ERG, and rod single cell recordings.

Results : At 1 month, OCT and light histology in X1 Tβ mice was normal, but by four months EM revealed overgrowth of rod outer segments (OS). By 8 months, OS were widely spaced and discs not as tightly packed. In WT Tβ mice the transgene localized exclusively in the rod photoreceptor in both inner and outer segments, with levels 3-fold above WT levels. At 1 month, functional recovery assessed by ERG was normal. Rod single cell suction electrode recordings also showed that the X1 Tβ rods were functioning similarly to WT, with similar dark currents and collecting areas. However, X1 Tβ rods displayed greater dark calcium levels; indicated by a 2-fold larger Na⁺/Ca⁺⁺, K⁺ exchange current that led to larger than normal single photon responses, smaller I_o, and slower kinetics.

Conclusions : Tβ expression partially restores structure/function in the X1 KO mouse. This partial recovery does not persist for the life of the animal, indicating that the N-terminus of the β-subunit, and possibly GARPs are required for sustained normal structure/function. The truncated β-subunit is sufficient to at least partially establish a plasma/disk membrane connection and significantly improve the photoresponse compared to X1 KO mice. Lastly, higher than normal calcium levels may be indicative of abnormal calcium feedback in the X1 Tβ mouse that may normally be mediated by the low affinity, high capacity Ca⁺⁺-binding GARP region.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.