Insights into cone function from the 'all-cone' neural retina leucine zipper null mouse

Cone photoreceptors mediate daylight and bright light vision and are thus critical to human sight. Rods outnumber cones in most mammalian retinas and this has hindered the study of cone function at the molecular and biochemical level. Cones comprise just 3% of the retina in mouse, the only mammalian system for the study of experimental gene modification within an intact system. A mouse null for the transcription factor neural retina leucine zipper protein (Nrl) generated by Alan Mears and Anand Swaroop showed promise as having a cone-enriched retina. However, the issue of whether these photoreceptors were cones or cone-rod intermediates was left unresolved. The first part of this thesis provides a thorough characterization of Nrl -/- mouse photoreceptors. Immunohistochemistry, electron microscopy, Western blot analysis, and electroretinography were used to characterize the protein expression, ultrastructure, morphology, and function of Nrl-/- mouse photoreceptors. The results of these experiments support the conclusion that Nrl -/- mouse photoreceptors are cones and not cone-rod intermediates.;Cone photoreceptors typically recover from light stimulation faster than rods. However, the molecular details of cone light response inactivation are not well understood. In the second part of this thesis the role of the G-protein coupled receptor kinase Grk1 in cone opsin phosphorylation and cone response recovery was investigated in suction pipette recordings from single Nrl-/--Grk1-/- and Nrl-/- mouse photoreceptors. Nrl-/- photoreceptor light responses recover more rapidly than wt rods. Like wt mouse cones, both short-wave (S-opsin, lambda max=360 nm) and mid wave (M-opsin, lambdamax∼510 nm) sensitive cone pigments are found to be functionally coexpressed in Nrl-/- mouse photoreceptors. Functional coexpression of S- and M-opsins with their distinct absorption maxima allowed the recording of dim flash responses of photoreceptors driven exclusively by either opsin. Grk1 was found to be essential for normal inactivation of both S- and M-opsins of mouse. However, the inactivation of the M-pigment response was much more delayed by the absence of Grk1, revealing a relatively effective, Grk1-independent inactivation pathway for S-opsin.