Investigating the Ca2+-dependent and Ca2+-independent mechanisms for mammalian cone light adaptation.

Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods. In addition, cones can function over a wide range of light conditions whereas rods saturate in moderately bright light. Calcium plays an important role in regulating phototransduction and light adaptation of rods and cones. Notably, the two dominant Ca2+-feedbacks in rods and cones are driven by the identical calcium-binding proteins: guanylyl cyclase activating proteins 1 and 2 (GCAPs), which upregulate the production of cGMP; and recoverin, which regulates the inactivation of visual pigment. Thus, the mechanisms producing the difference in adaptation capacity between rods and cones have remained poorly understood. Using GCAPs/recoverin-deficient mice, we show that mammalian cones possess another Ca2+-dependent mechanism promoting light adaptation. Surprisingly, we also find that, unlike in mouse rods, a unique Ca2+-independent mechanism contributes to cone light adaptation. Our findings point to two novel adaptation mechanisms in mouse cones that likely contribute to the great adaptation capacity of cones over rods.

Molecular determinants of Guanylate Cyclase Activating Protein subcellular distribution in photoreceptor cells of the retina.

Retinal guanylate cyclase (RetGC) and guanylate cyclase activating proteins (GCAPs) play an important role during the light response in photoreceptor cells. Mutations in these proteins are linked to distinct forms of blindness. RetGC and GCAPs exert their role at the ciliary outer segment where phototransduction takes place. We investigated the mechanisms governing GCAP1 and GCAP2 distribution to rod outer segments by expressing selected GCAP1 and GCAP2 mutants as transient transgenes in the rods of GCAP1/2 double knockout mice. We show that precluding GCAP1 direct binding to RetGC (K23D/GCAP1) prevented its distribution to rod outer segments, while preventing GCAP1 activation of RetGC post-binding (W94A/GCAP1) did not. We infer that GCAP1 translocation to the outer segment strongly depends on GCAP1 binding affinity for RetGC, which points to GCAP1 requirement to bind to RetGC to be transported. We gain further insight into the distinctive regulatory steps of GCAP2 distribution, by showing that a phosphomimic at position 201 is sufficient to retain GCAP2 at proximal compartments; and that the bovine equivalent to blindness-causative mutation G157R/GCAP2 results in enhanced phosphorylation in vitro and significant retention at the inner segment in vivo, as likely contributing factors to the pathophysiology.

Enzymatic relay mechanism stimulates cyclic GMP synthesis in rod photoresponse: biochemical and physiological study in guanylyl cyclase activating protein 1 knockout mice.

Regulation of cGMP synthesis by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) in rod and cone photoreceptors by calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) is one of the key molecular mechanisms affecting the response to light and is involved in congenital retinal diseases. The objective of this study was to identify the physiological sequence of events underlying RetGC activation in vivo, by studying the electrophysiological and biochemical properties of mouse rods in a new genetic model lacking GCAP1. The GCAP1(-/-) retinas expressed normal levels of RetGC isozymes and other phototransduction proteins, with the exception of GCAP2, whose expression was elevated in a compensatory fashion. RetGC activity in GCAP1(-/-) retinas became more sensitive to Ca(2+) and slightly increased. The bright flash response in electroretinogram (ERG) recordings recovered quickly in GCAP1(-/-), as well as in RetGC1(-/-)GCAP1(-/-), and RetGC2(-/-)GCAP1(-/-) hybrid rods, indicating that GCAP2 activates both RetGC isozymes in vivo. Individual GCAP1(-/-) rod responses varied in size and shape, likely reflecting variable endogenous GCAP2 levels between different cells, but single-photon response (SPR) amplitude and time-to-peak were typically increased, while recovery kinetics remained faster than in wild type. Recovery from bright flashes in GCAP1(-/-) was prominently biphasic, because rare, aberrant SPRs producing the slower tail component were magnified. These data provide strong physiological evidence that rod photoresponse recovery is shaped by the sequential recruitment of RetGC isozyme activation by GCAPs according to the different GCAP sensitivities for Ca(2+) and specificities toward RetGC isozymes. GCAP1 is the 'first-response' sensor protein that stimulates RetGC1 early in the response and thus limits the SPR amplitude, followed by activation of GCAP2 that adds stimulation of both RetGC1 and RetGC2 to speed-up photoreceptor recovery.

Bleaching of mouse rods: microspectrophotometry and suction-electrode recording.

When a substantial fraction of rhodopsin in a rod photoreceptor is exposed to bright light, the rod is desensitized by a process known as bleaching adaptation. Experiments on isolated photoreceptors in amphibians have revealed many of the features of bleaching adaptation, but such experiments have not so far been possible in mammals. We now describe a method for making microspectrophotometric measurements of pigment concentration and suction-electrode recording of electrical responses over a wide range of bleaching exposures from isolated mouse rods or pieces of mouse retina. We show that if pigment is bleached at a low rate in the presence of bovine serum albumin (BSA), and intermediate photoproducts are allowed to decay, mouse rods are stably desensitized; subsequent treatment with exogenous 11-cis retinal results in pigment regeneration and substantial recovery of sensitivity to the dark-adapted value. Stably bleached wild-type (WT) rods show a decrease in circulating current and acceleration of the time course of decay, much as in steady background light; similar effects are seen in guanylyl cyclase-activating protein knockout (GCAPs(-/-)) rods, indicating that regulation of guanylyl cyclase is not necessary for at least a part of the adaptation produced by bleaching. Our experiments demonstrate that in mammalian rods, as in amphibian rods, steady-state desensitization after bleaching is produced by two components: (1) a reduction in the probability of photon absorption produced by a decrease in rhodopsin concentration; and (2) an equivalent background light whose intensity is proportional to the fraction of bleached pigment, and which adapts the rod like real background light. These two mechanisms together fully account for the 'log-linear' relationship in mammalian retina between sensitivity and per cent bleach, which can be measured in the steady state following exposure to bright light. Our methods will now make possible an examination of bleaching adaptation and pigment regeneration in mouse animal lines with mutations or other alterations in the proteins of transduction.

Enzymatic properties and regulation of the native isozymes of retinal membrane guanylyl cyclase (RetGC) from mouse photoreceptors.

Mouse photoreceptor function and survival critically depend on Ca(2+)-regulated retinal membrane guanylyl cyclase (RetGC), comprised of two isozymes, RetGC1 and RetGC2. We characterized the content, catalytic constants, and regulation of native RetGC1 and RetGC2 isozymes using mice lacking guanylyl cyclase activating proteins GCAP1 and GCAP2 and deficient for either GUCY2F or GUCY2E genes, respectively. We found that the characteristics of both native RetGC isozymes were considerably different from other reported estimates made for mammalian RetGCs: the content of RetGC1 per mouse rod outer segments (ROS) was at least 3-fold lower, the molar ratio (RetGC2:RetGC1) 6-fold higher, and the catalytic constants of both GCAP-activated isozymes between 12- and 19-fold higher than previously measured in bovine ROS. The native RetGC isozymes had different basal activity and were accelerated 5-28-fold at physiological concentrations of GCAPs. RetGC2 alone was capable of contributing as much as 135-165 µM cGMP s(-1) or almost 23-28% to the maximal cGMP synthesis rate in mouse ROS. At the maximal level of activation by GCAP, this isozyme alone could provide a significantly high rate of cGMP synthesis compared to what is expected for normal recovery of a mouse rod, and this can help explain some of the unresolved paradoxes of rod physiology. GCAP-activated native RetGC1 and RetGC2 were less sensitive to inhibition by Ca(2+) in the presence of GCAP1 (EC(50Ca) ∼132-139 nM) than GCAP2 (EC(50Ca) ∼50-59 nM), thus arguing that Ca(2+) sensor properties of GCAP in a functional RetGC/GCAP complex are defined not by a particular target isozyme but the intrinsic properties of GCAPs themselves.