Na⁺/Ca²⁺, K⁺ Exchangers 4 And 2 Are Required for the Rapid Light Response Recovery and Normal Light Adaptation of Cones

Abstract

Purpose: Biochemical evidence suggests that Ca²⁺ is extruded by NCKX2 from cone outer segments. However, cone function is not severely compromised in Nckx2^-/- mice, indicating an additional Ca²⁺ extrusion mechanism in the cone outer segment. We sought to identify this mechanism and determine its role in cone physiology.

Methods: RNA microarray differential expression analysis in WT and cone-dominant Nrl^-/- mice was used to pinpoint a cone-specific exchanger candidate, NCKX4. In situ hybridization with WT and Nrl^-/- mice was used to establish the expression of NCKX4 in the cone photoreceptors. Cone-specific NCKX4 knock-out mice (^cNckx4^-/-) were generated by crossing Nckx4^f/f and HRGP-Cre mice that express Cre in cones. ^cNckx4^-/- and Nckx2^-/- mice were crossed to produce ^cNckx4^-/- Nckx2^-/- knockout mice (DKO). All mice were on Gnat1^-/- background to facilitate cone electrophysiology experiments. Cone light responses were recorded from isolated retinas perfused with Locke’s media (with DL-AP4, L-aspartate and barium) at 37 ^oC. Dark-adapted flash response properties and light adaptation were compared among ^cNckx4^-/-, DKO and HRGP-Cre control mice to determine the role of NCKX4 and NCKX2 in cone phototransduction.

Results: In situ hybridization revealed abundant NCKX4 expression in the photoreceptor layer of Nrl^-/- mouse retinas. In the WT retina the signal was observed only in sparse cells in the upper strata of the photoreceptor layer, a location where cones reside. Dark-adapted flash responses of ^cNckx4^-/- cones were slower than those of control cones but their response amplitudes were normal. The operating range of ^cNckx4^-/- cones was shifted to five-fold dimmer background light intensities but Weber-like adaptation was preserved. The cones from 3-month old mice lacking both NCKX4 and NCKX2 showed slower responses and about 10-fold smaller amplitudes as compared to control and ^cNckx4^-/- cones.

Conclusions: Our data suggest that NCKX4 is a novel cone-specific Ca²⁺ extrusion mechanism that, together with NCKX2, is required for the fast light response termination and normal light adaptation of mammalian cones. The use of two exchangers may contribute to the faster responses and more potent adaptation of cones compared to rods. Such redundancy may also protect cones from blinding diseases related to mutations in NCKX2 or NCKX4.