Introduction

Synthesis of cGMP is an essential part of the fundamental phototransduction process that defines the function of vertebrate rods and cones. Many excellent review articles describing the details of the phototransduction pathway have been published, to name a few, references [8, 9, 35, 52, 100, 106]. For that reason, we provide here only a brief overview of the signaling events that mediate absorption of a photon by visual pigment and the hyperpolarization of photoreceptor, the origin of the visual signaling (Fig. 1). Cyclic GMP plays a central role in phototransduction by regulating the permeability of the cyclic nucleotide-gated channels (CNG) in the plasma membrane of the photoreceptor’s outer segment. In the dark, retinal membrane guanylyl cyclase (RetGC) [25, 66, 125] produces enough cGMP to keep a small fraction of CNG channels in the open state, and that allows the channels to maintain an inward current of Na+ and Ca2+, partially depolarizing the photoreceptor membrane. Light activates cGMP hydrolysis by cGMP phosphodiesterase-6 (PDE6) through GDP/GTP exchange in the Gt protein, transducin, catalyzed by photoactivated rhodopsin or cone pigments. As GTP transducin activates PDE6, the rapid decline in cGMP concentration closes the CNG channels and stops the influx of Na+ and Ca2+ in the outer segment, thus causing hyperpolarization of rods and cones. The recovery of photoreceptors from excitation includes multiple steps, regulating all components of the phototransduction cascade. G-protein-coupled receptor kinases GRK1 and GRK7 phosphorylate photoactivated pigments to allow them to bind arrestins, which blocks activation of Gt; the intrinsic Gt GTPase activity accelerated by RGS9 protein and PDE6 rapidly deactivates Gt and thus returns its effector enzyme PDE6 to its inactive self-inhibited state, which halts cGMP decay. Quenching the rhodopsin-transducin-PDE6 cascade is a necessary, but not the only, step in photoreceptor recovery: timely re-opening of the CNG channels also requires acceleration of cGMP synthesis by RetGC via a negative Ca2+ feedback mechanism [52, 101].

Fig. 1
figure 1

The schematic of Ca2+ feedback and guanylyl cyclase regulation in photoreceptor. During excitation phase, photoactivated pigment (R*) activates the transduction cascade, R*➔Gt*➔PDE6*, which closes CNG channels in the outer segments and hyperpolarizes the photoreceptor membrane. Once the channels are closed, Ca2+ concentrations fall and thus allow Mg2+ to replace Ca2+ in GCAP. The Mg2+ GCAP stimulates cGMP production while the R* is being phosphorylated and blocked by arrestin and the transduction cascade is quenched by accelerated GTP hydrolysis in Gt, all resulting in reopening of the channels and restoring the inward cation current partially depolarizing photoreceptor; asterisks symbolize the activated state. Arr arrestin, GCAP guanylyl cyclase activating protein, Gt G protein transducin, CNG Ch cyclic nucleotide-gated channel, GRK G-protein receptor kinase, NCKX Na+/Ca2+, K+-exchanger, PDE6 cGMP phosphodiesterase 6, RGS9 regulator of G-protein signaling 9-1, RetGC retinal membrane guanylyl cyclase. See text for other details

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The main purpose of this short review is to discuss the molecular mechanisms of the Ca2+ feedback with an emphasis on its role in regulation of cGMP synthesis as a part of normal photoreceptor physiology and pathological processes underlying congenital blindness. In addition to that, we will review a recently emerged role of a non-Ca2+ sensor protein, retinal degeneration 3 (RD3), as a potent regulator of RetGC function in photoreceptors and its role in protecting rods and cones from degeneration. The authors do not intend to provide a comprehensive detailed review of Ca2+ and guanylyl cyclase regulation in photoreceptors, but choose instead to present their opinion on only some key aspects of such regulation in the physiology and disease of photoreceptors.

The origin of Ca2+ feedback in photoreceptors

Ca2+ feedback originates from fluctuations of intracellular Ca2+ in the outer segment between light and dark. Although the levels of free Ca2+ concentrations reported in the literature slightly vary between different measurements and different animal species, they all indicate a steep, up to tenfold, decline of free Ca2+ in response to light. Ca2+ continuously extruded from the outer segment via Na+/Ca2+, K+ exchangers, NCKX1 in rods, and NCKX2/NCKX4 in cones [116, 117], returns to the outer segment through the open CNG channels (Fig. 1), which keeps the free Ca2+ concentrations in dark-adapted photoreceptors in the range of ~250–560 nM [37, 121]. When the CNG channels close in light, the influx of Ca2+ into the outer segment stops and its free concentrations rapidly decline to the range of ~20–50 nM [37, 121]. Consequently, a number of processes regulated by Ca2+-binding proteins become affected, thus contributing to the sha** of photoresponses in rods and cones through negative Ca2+ feedback [35, 52, 100, 101]. Regulation of retinal membrane guanylyl (guanylate) cyclase (RetGC) by Ca2+ sensor proteins plays the major role in Ca2+ feedback [101].

Ca2+ sensors in photoreceptors are recoverin-like EF-hand proteins

Proteins that mediate Ca2+ feedback belong to a distinct group of recoverin-like proteins in the EF-hand protein superfamily [13, 14]. The recoverin-like proteins harbor some notable features separating them from other groups of the EF-hand superfamily. To date, several photoreceptor-specific Ca2+-sensor proteins have been identified: recoverin (or S-modulin in lower vertebrate species), visinin, and guanylyl cyclase activating proteins (GCAPs) [13, 14, 57]. The latter subgroup includes several homologs, GCAP1 through GCAP8, in different vertebrate species [26, 43, 44, 81]. However, only two—GCAP1 and GCAP2—are ubiquitous among the animal species, and in some species, they are the only two GCAP isoforms present in photoreceptors [126]. A subpopulation of S-cones in humans also expresses GCAP3 [43]. Another EF-hand protein, GCIP [59], has been suggested to also play a physiological role in Ca2+ feedback, but later found not to be involved in sha** photoresponses [15, 72]. The recoverin-like Ca2+ sensor proteins have a number of common features: they have similar size (~21–25 kDa), are fatty acylated at the N-terminus, and have four helix-loop-helix EF-hand motifs, from EF-hand 1 (proximal to the N-terminus in primary structure) to EF-hand 4 (proximal to the C-terminus) [13, 14, 57] (Figs. 2, 3). The characteristic shape of the recoverin-like protein, distinct from other families of EF-hand proteins, presents two semi-globular lobes, each formed by a pair of EF-hand structures, connected via a “hinge” Gly residue [13, 57, 62]. In contrast to calmodulin-like proteins, the recoverin-like proteins do not undergo a major rearrangement of their core structures in response to binding and release of Ca2+ [62] (Figs. 2, 3).

Fig. 2
figure 2

Ribbon diagrams of four Ca2+-liganded sensor proteins of the recoverin family. a Myristoylated recoverin [3, 33], b myristoylated GCAP1 [111], c non-myristoylated GCAP2 [6], and d non-myristoylated GCAP3 [110]. EF1 through EF4–EF-hands; Myr fatty acyl moiety; yellow spheres indicate the positions of metal ions in the EF-hand loops. Hereafter, the images were created using Shrödinger PyMol software by OpenGL version 2.0

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Fig. 3
figure 3

Ca2+-dependent conformational changes in recoverin and GCAP1. a Calcium-myristoyl switch in recoverin. In the apo form, the fatty acyl residue is buried inside the semiglobule comprised of EF1 and EF2. Upon transition from the apo form to the Ca2+-liganded state, the fatty acylated N-terminus becomes expelled from the protein structure [3]. b Calcium-myristoyl tug [91] in GCAP1, NMR structures of Mg2+ (green)—and Ca2+ (blue)—liganded states; NMR model by Lim et al. [63]. The myristoyl fatty chain remains constrained in the fold of the N-terminal lobe of the protein in the both physiological metal-liganded states, but changes its orientation as a result of the push-pull action of the exiting helix of EF-hand 4 transmitted via Leu-176 by partial unwinding/rewinding of the alpha-helix 10 [61, 63, 91]. See detailed explanations in the text

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The N-fatty acyl residue plays different structural roles in the function of photoreceptor Ca2+-sensor proteins. The N-fatty acyl groups in recoverin and GCAPs expressed in photoreceptors include saturated and non-saturated C12 and C14 residues, among which the saturated C14:0 myristoyl chain represents a relatively minor component [23, 81]. Despite that, the N-fatty acylation is commonly referred to as “myristoylation” for simplicity. In recoverin, the myristoyl group changes its position in protein structure: in a metal-free state of recoverin, the myristoyl residue is held inside the N-terminal lobe of the protein, but swings out when EF-hand 2 binds Ca2+ [4, 5, 24, 33, 128] (Fig. 3a). This change, commonly referred to as “calcium-myristoyl switch” [128, 130], has been characterized by detailed structural studies [5, 33]. The extrusion of the fatty acyl moiety increases recoverin’s affinity for the membrane [24], evidently by utilizing the extruded fatty chain as an anchor. Although the structures of visinin and S-modulin were not studied as comprehensively as that of recoverin, their primary structures are very closely homologous to that of recoverin [48], so both proteins are likely to share common three-dimensional characteristics with recoverin.

In contrast to recoverin, the myristoyl fatty chain in GCAPs remains buried inside the N-terminal semi-globule of the protein (Figs. 2, 3), regardless of its cation-liganded state [39, 60, 77, 111]. Instead of engaging in a calcium-myristoyl switch, the structural role of the myristoyl residue in GCAP1 is to link the N-terminal portion of the protein with its C-terminal lobe internally, via a structural link called the “calcium-myristoyl tug” (Fig. 3b) that plays an important role in fine-tuning GCAPs’ affinity for both Ca2+ and the target enzyme [91] as will be reviewed later in more detail.

Another common feature shared by the photoreceptor Ca2+ sensor proteins is that not all four EF-hand motifs in their primary structure are the actual metal binding domains [62]. The N-terminal EF-hand 1 in all proteins of the recoverin family lacks some of the essential oxygen-containing side chains required for coordination of Ca2+ [13, 14, 62] (Fig. 2). Recoverin has only two functional Ca2+-binding EF-hands (EF-hand 2 and-3), whereas three EF-hands (-2, -3 and -4) in GCAPs bind Ca2+ or Mg2+ [83] and play a major role in the GCAP-dependent regulation of the photoreceptor guanylyl cyclase, as described in the following sections.

Metal binding properties of the photoreceptor-specific EF-hand sensor proteins

A possible function of recoverin is to help rhodopsin, by extending the life of its active state, to more effectively propagate activation of the phototransduction cascade in dim light, before the receptor is quenched by phosphorylation and arrestin binding [1, 7, 16, 47, 48]. GRK1 is considered to be the primary target for Ca2+ recoverin [1, 129] although a more direct effect on PDE6 activity was also recently proposed [75] (Fig. 1). In dark-adapted photoreceptors, recoverin can more effectively stick to the disk membranes as a result of its anchoring via calcium-myristoyl switch [17, 24, 129, 130]. Recoverin could also be the intracellular Ca2+ buffer in photoreceptors, although its contribution to the overall Ca2+-buffering capacity of photoreceptors has been estimated to be relatively modest [67].

Although the details of the functional role of GCAPs in regulation of the photoresponse require further study, it has been generally well established. GCAPs control the activity of RetGC in a Ca2+-sensitive manner by activating RetGC in the light, when Ca2+ concentrations fall, and decelerating the cyclase in the dark, when Ca2+ influx through the open CNG channels elevates free Ca2+ levels in the outer segment [35, 52, 100, 101, 106] (Fig. 1). GCAP-dependent regulation of the cyclase dominates the Ca2+ feedback, contributing to the feedback much stronger than other regulatory components of the photoresponse affected by Ca2+ [101]. Therefore, the metal-binding properties of GCAPs are especially important for their role in photoreceptors. GCAPs regulate RetGC within the physiological submicromolar range of free Ca2+ [82]. The affinities of EF-hands for Ca2+ in GCAPs are also very close to the physiological levels of free Ca2+ in the outer segment [83, 84]. However, the reported estimated Kd values of the EF-hands for Ca2+ vary rather widely depending on the methods used for their evaluation. Below, we examine in more detail how the metal binding properties of individual EF-hands in GCAP1 relate to the sensor function of GCAPs as the RetGC regulators. We will also address possible reasons for the variability between different estimated EF-hand affinities for Ca2+ in GCAP1.

GCAPs have three active metal binding EF-hands: EF2, EF3, and EF4 (Figs. 2, 4). Ca2+ binding in different EF-hands has been studied by selectively inactivating other EF-hands using point mutations introduced in 12-residue EF-hand metal binding loops. Subsequent assessment of the individual EF-hand affinities for Ca2+ using the intrinsic GCAP1 tryptophan fluorescence spectra and titration using Ca2+ indicator dyes [83] gives a range of their KdCa between ~26 and ~120 nM, being the lowest in EF2 (the highest-affinity site) and the highest in EF4 (the lower-affinity site). Another important observation from studying the Ca2+ sensitivity of individual EF-hands was that although binding of Ca2+ in EF2 does not require Ca2+ to occupy EF-hands 3 or 4, binding of the metal in EF4 strictly requires EF3 being liganded by Ca2+ [84, 87]. In other words, there are two Ca2+-sensor parts of GCAP1: one operates through EF2 and the other through a pair of EF-hands 3 and 4 acting in concert [84]. The high-resolution crystal structure of myristoylated GCAP1 in its Ca2+-bound state established by Stephen et al. [111] also helps to weigh the relevance of the apparent KdCa values for Ca2+-binding in EF-hands to the structural organization of GCAP1 (Fig. 4). Overall, the Ca2+ binding affinities derived from the tryptophan fluorescence spectroscopy and titration using  Ca2+ indicator dyes [83] agrees quite well with the three-dimensional structure of GCAP1 EF-hands in a Ca2+-liganded state (Fig. 4). The three EF-hands in GCAP1 hold Ca2+ using a typical for EF-hands seven-dentate coordination in a 12-residue loop. Similarly to some EF-hands of this type [13, 14], a water molecule provides one of the essential hydrogen bonds coordinating Ca2+ in the loop. All three metal binding EF-hands in GCAP1 use water to hold Ca2+ in the loop, much like a “stopper” plugging the exit channel for the metal ion (Fig. 4). These water molecules are clearly visible in the crystal structure established by Stephen et al. [111]. The three-dimensional EF-hand structure reveals that the Ca2+:H2O pairs are better embedded into the EF2 than in the other two metal-binding EF-hands. Conversely, in EF4, the water molecule mediating coordination of Ca2+ is exposed near the very surface, indicating that the metal ion is held in the EF4 loop more flexibly than in EF2 (Fig. 4b).

Fig. 4
figure 4

a Three-dimensional ribbon diagram of GCAP1 indicates the positions of Ca2+ (yellow) and H2O (light cyan) coordinated in three EF-hand loops; the calcium-myristoyl tug structure is highlighted in blue and the hinge Gly-86 between the two semi-globules is shown in pink. b Space-filled/mesh close-up diagram of the Ca2+:H2O position in EF-hand structures; red space-filled side chains in the loop, yellow Ca2+, cyan H2O (the models utilize the molecular coordinates of the GCAP1 crystal structure reported by Stephen et al. [111]

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Notably, the affinities of the individual EF-hands for metal ions estimated using isothermal titration calorimetry (ITC) [2, 60, 96] rank their relative affinities for Ca2+ that are different from those derived from the tryptophan fluorescence spectroscopy and are not consistent with the crystal structure of GCAP1 (Fig. 4). In addition, contrary to the apparent Kd determined by the fluorescence spectroscopy, some of the affinities estimated by ITC do not fit well with the physiological range of free Ca2+ in the outer segment. A likely reason for that is that the heat release for GCAPs in response to the binding metal does not present a simply exothermic or endothermic pattern, but includes a combination of both [60, 96]. When a complex kinetics of the heat release is simplified by formally applying one- or two-center binding models, it evidently skews the accuracy of measuring the individual EF-hand affinities for the metal ligand. In contrast, the binding isotherms derived from the tryptophan fluorescence directly reflect conformational changes in the protein that occur as a result of metal binding and are not affected by a complex pattern of the heat release and absorption. So even though ITC microcalorimetry can be very useful in some aspects of structural studies in GCAPs [2, 96], its usefulness for measuring specific parameters of metal binding should be evaluated with a great deal of caution.

As an important characteristic of the Ca2+-sensitive regulation of RetGC by GCAPs, the effect of Ca2+ is highly cooperative (the Hill coefficient ≥ 2) [53, 68, 69, 82, 89]. The isotherm for Ca2+ binding to a purified GCAP1 shows that the binding occurs within the same physiological range of Ca2+ as the regulation of RetGC, but surprisingly does not show cooperativity (the Hill coefficient of 1) [83]. It is therefore possible that in GCAPs bound to its target enzyme, the interaction between different EF-hands change to bind the metal in a cooperative manner.

Another important aspect of GCAP-mediated Ca2+-sensitive regulation of retinal guanylyl cyclase is that it is also highly sensitive to Mg2+[29, 82], because all metal binding EF-hands in GCAP also bind Mg2+ with submillimolar affinities [83, 84]. Although GCAPs, like other Ca2+ binding proteins, undergo conformational changes between different functional states, these conformational changes are rather subtle when compared to calmodulin or even to recoverin [62]. These conformational changes in GCAPs are not robust because GCAPs are liganded by divalent cations in both functional states [60, 62]. In the “activator” state of GCAP, Mg2+ almost fully substitutes Ca2+ in at least two and probably all three metal binding EF-hands [83, 84]. The affinity of GCAP1 for Mg2+ is ~1000-fold lower than for Ca2+ [82, 83], but the physiological concentrations of Mg2+ in the outer segment, near 1 mM [20], vastly exceed the ~20–50 nM Ca2+ concentration in illuminated photoreceptors [37, 121]. Therefore, when the free Ca2+ concentrations in the outer segment fall after illumination, Mg2+ can easily displace Ca2+ from EF-hands 3 and 4 (and even from EF2 in a substantial fraction of GCAP1) [82–84]. Preservation of the cation-bound state both in the dark and in the light is essential for the regulatory function of GCAP, because the apo-form of GCAP1 is a completely inactive protein that fails to bind with RetGC [84, 87]. To preserve the GCAP ability to bind to the target enzyme, a divalent cation, either Mg2+ or Ca2+, must occupy both EF-hands 2 and -3 [84, 87]. Hence, the replacement of Ca2+ by Mg2+ in the light enables GCAPs to stay in complex with RetGC even after the CNG channels become closed and the concentration of intracellular free Ca2+ in light-adapted photoreceptors falls. Mg2+ binding also plays a crucial role in adjusting the Ca2+ sensitivity of GCAPs to the physiological range of free Ca2+ in the outer segment. The exact concentrations of Mg2+ in mammalian photoreceptors have never been directly measured, but the Ca2+ sensitivity of RetGC controlled by GCAP1 and GCAP2 in mouse photoreceptors best fits into the physiological range of free Ca2+ at the free Mg2+ concentrations near 1 mM [83], close to the physiological levels of Mg2+ measured in rods of lower vertebrates [20]. If GCAP EF-hands were unable to bind Mg2+, it would effectively undercut the dynamic range of RetGC regulation: when Mg2+ concentrations are reduced, the inhibition of the RetGC/GCAP complex by Ca2+ becomes too sensitive, and the cyclase activity decelerates well before the free Ca2+ reaches its normal dark-adapted level. Conversely, increase of GCAP affinity for Mg2+ versus Ca2+ would require much higher concentrations of Ca2+ to decelerate the cyclase. As we will further discuss in this article, such a low sensitivity of the cyclase to the inhibition by Ca2+ abnormally elevates Ca2+ influx through CNG channels and provokes photoreceptor death [105]. Evidently, the structures of EF-hands in GCAPs evolved to achieve the optimal balance between their affinities for Mg2+ versus Ca2+ and thus to optimize the dynamic range of the cyclase regulation within the physiological ranges of the two cations in photoreceptors [29].

Whereas holding Mg2+ or Ca2+ in EF2 and EF3 allows GCAPs to always bind its target enzyme [58, 87], binding and release of Ca2+ in EF4 are most critical for switching GCAPs between its RetGC-activator and RetGC-inhibitor states. Inactivation of the cation binding in EF4 locks GCAPs in a perpetual activator state, accelerating RetGC activity even at such free Ca2+ concentrations that by far exceed the normal physiological levels in the dark [22, 83, 87]. Mg2+ binding in EF4 is not essential for the ability of GCAPs to activate RetGC, but helps to adjust its Ca2+ sensitivity to the physiological range of Ca2+ [82]. Structural changes in EF4 have a major regulatory effect even though the main part of the RetGC-binding interface in GCAP1 is located in the lobe comprised by the N-terminal EF-hands 1 and 2 [92]. This happens because the relatively subtle movement of the exiting helix in EF4 affects the GCAP1 fold on the other side of the molecule [63] (Fig. 3) and dimerization state of GCAP1 [12, 64]. Extensive site-directed mutagenesis of GCAP2 [32, 78] and especially GCAP1 [92] identified the cyclase binding interface of GCAP as a compact patch of the residues located on one side of the molecule and occupying large portions of EF1, -2, and a part of the entering helix of EF3 [92] (Fig. 5). Notably, the loop motif in GCAP EF-hand 1 lacks some side chains required for effective coordination of Ca2+ [13, 14, 57, 61]. Evidently, in the course of the GCAPs’ evolution, their N-terminal EF-hand traded its original function of binding metal for a new function: binding the target enzyme [32, 42, 92].

Fig. 5
figure 5

RetGC-binding interface in GCAP1. The surface-exposed residues critical for the regulatory binding to RetGC are highlighted in red, and the non-essential residues are highlighted in blue [92]. The main part of the binding interface on GCAP1 occupies EF-hand 1 and 2 in semi-globule I, whereas the most critical Ca2+ sensor function belongs to the semi-globule II containing EF-hands 3 and 4

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In GCAP1, a structural link between the C-proximal EF4 and the N-terminal lobe occurs through a “calcium-myristoyl tug” [91], which directly links the two parts of the molecule: one responsible for recognition of the target enzyme and the other acting as the Ca2+ sensor for the change of its functional state (Fig. 3). The main portion of the GCAP1 molecule that creates the cyclase-binding interface also contains the N-myristoyl residue embedded in the semi-globule I, between EF-hands 1 and -2 (Figs. 3, 4). The molecular dynamic of this part of the molecule also indicates that these residues are most likely to allow GCAP1 to change its interactions with the cyclase in response to the change of the metal-liganded state [103]. As the exiting helix of EF4 continues to the C-terminal helix 10 extending to the opposite side of GCAP1, Leu-176 directly contacts the fatty acyl moiety (Figs. 3, 4) [111]. This link enables the tug action between EF4, the main Ca2+ sensor part in the cyclase regulation, and the cyclase-binding interface [91]. The pull-push action of the tug [70, 71, 90, 91] tunes EF4 affinity for Ca2+, adjusting the Ca2+ sensitivity of the GCAP1 activator-to-inhibitor conversion and its affinity for RetGC at the same time [90, 91]. Artificial changes in the length and/or composition of the tug can further increase GCAP1 affinity for the cyclase, but only at the expense of reducing its affinity for Ca2+, and vice versa [91]. Apparently, the calcium-myristoyl tug has evolved as yet another fine adjustment of the GCAP1 structure to its role as a Ca2+ sensor in regulation of RetGC in physiological conditions optimal for the photoreceptor function.

The roles of GCAPs in sha** photoresponse

Ca2+-sensitive regulation of RetGC is the predominant component of the Ca2+ feedback in photoreceptors [101]. The kinetics of rod recovery is limited primarily by the rate of rhodopsin-transducin-PDE6 cascade deactivation, rather than by the replenishment of cGMP by guanylyl cyclase [38, 55]. The cyclase activity not only does not rate-limit the recovery of normal rods, but even when overexpression of RGS9 stimulates deactivation of transduction and makes rod responses recover more quickly, the activity of RetGC apparently remains high enough not to limit the accelerated responses [55]. However, the high activity of RetGC during the photoreceptor recovery only becomes possible after its robust activation by GCAPs via negative Ca2+ feedback. In the absence of GCAPs, rods and cones retain their general ability to hyperpolarize in the light, but they recover from excitation much more slowly than normal [15, 72].

GCAP1 and GCAP2 are the two ubiquitous isoforms of GCAPs present in all vertebrate species tested to date. They are also the only two isoforms encoded by the mouse genome [72]. When the tail-to-tail adjacent Guca1a and Guca1b mouse genes, coding respectively for GCAP1 and GCAP2, are simultaneously disrupted by a gene knockout construct, rod dim flash responses grow very large and recover very slowly [15, 72]. So RetGC, even without its activator proteins, can restore the permeability of the CNG channels, but the rods operating only on the basal cyclase activity fail to restrict the amplitude of a single-photon response and to recover in a timely fashion, thus losing the temporal resolution of their responses and becoming hypersensitive to light. Cones lacking GCAP1 and GCAP2 also prolong their recovery phase, albeit less dramatically than rods [104]. The responses in GCAP-deficient cones still remain much faster than in rods, possibly because cones have higher RetGC content than rods [11, 25, 123].

Rods express both GCAP1 and GCAP2, whereas cones almost exclusively express GCAP1 [68, 69, 123]. Rods need both GCAP1 and GCAP2 to properly shape their responses to a dim flash. Although very similar in their main structural characteristics, the two Ca2+ sensors have slightly different operational ranges: GCAP1 is less and GCAP2 is more sensitive to Ca2+ [41, 89]. Under physiological conditions, RetGC activity stimulated by GCAP1 requires higher Ca2+ concentrations to decelerate it (IC50 ~130 nM) [68, 69, 89]. Conversely, deceleration of the cyclase stimulated by GCAP2 (IC50 ~50–60 nM) occurs at free Ca2+ concentrations lower than in GCAP1 [68, 69, 89]. Individual GCAP gene knockouts [68, 69] indicate that the two GCAPs activate RetGC in response to light by acting in a sequential mode (Fig. 6). GCAP1 is the first-response sensor that starts to activate RetGC as soon as the intracellular Ca2+ begins to drop following the activation of the phototransduction cascade. RetGC activity stimulated by GCAP1 restricts the amplitude of the dim flash response, by not allowing rods to close an excessive number of channels after absorbing a small number of photons, which helps to extend the dynamic range of rod responses. The free Ca2+ levels have to fall deeper in mid-phase of the response, before GCAP2-stimulated RetGC activity can kick in and accelerate the recovery. The amplitudes of dim-flash rod responses in GCAP1-/- mice, where upregulated GCAP2 takes over the RetGC activation, grow larger than normal, albeit not as large as in GCAP1/GCAP2 double-knockouts, and reach the peak later than normal, yet their recovery from the excitation occurs with a near-normal kinetics [69]. Conversely, in GCAP2-/- mice, the amplitude and the time to peak of rod responses remain similar to normal, but they recover more slowly [68].

Fig. 6
figure 6

Sequential activation of RetGC isozymes by GCAPs in rods via Ca2+ feedback. GCAP1 has lower affinity for Ca2+ and therefore activates RetGC1 soon after the high dark Ca2+ levels in rods start to decline at the beginning of the photoresponse. The early activation of RetGC1 restricts the amplitude of a photon response. GCAP2 has higher affinity for Ca2+ and converts to the Mg2+-liganded state only after the free Ca2+ levels further decline in mid-phase of the response; it then activates the ancillary RetGC2 (and possibly a fraction of RetGC1 unoccupied by GCAP1) [68, 69]

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It is also worth mentioning that the term “relay” that has been used to describe the sequential activation of the cyclase by GCAPs [51, 69] is somewhat inaccurate, because the activation of the cyclase by GCAP1 continues even after Ca2+ falls to the levels sufficient for activation by GCAP2. Contrary to the “relay” fashion, the two GCAPs do not replace each other; rather, the fraction of the cyclase activity stimulated by GCAP2 in mid-phase of the response adds to the activity of the cyclase pool stimulated by GCAP1 in the earlier phase of the response (Fig. 6).

In cones, the need for sequential activation of RetGC by GCAPs is less evident, because GCAP1 heavily dominates regulation of RetGC by Ca2+ [123]. However, GCAP2 can accelerate the recovery in cones lacking GCAP1, making it faster than in cones lacking both GCAP1 and GCAP2 altogether [118].

Regulatory properties of retinal guanylyl cyclase

Retinal membrane guanylyl cyclase (RetGC) regulated in photoreceptors by GCAPs exists as two isozymes, RetGC1 and RetGC2 [25, 66, 125]. In mouse and rat genomes, Gucy2e and Gucy2f genes code for the respective orthologs, GC-E and GC-F, of human RetGC1 and RetGC2 [126]. The suggested elsewhere use of the name GC-E for human RetGC1, in our opinion, creates unnecessary confusion, because RetGC1 in humans is coded by GUCY2D gene, not by GUCY2E, which in primates codes for a pseudogene homologous to the olfactory guanylyl cyclase (conversely, in rodents the Gucy2d gene codes for an olfactory cyclase). This is why we hereafter prefer, for the sake of clarity, to keep the original designations for the two isozymes, RetGC1 and RetGC2 [25, 66] for all species, regardless of the names of their respective coding genes.

The joined activity of RetGC1 and RetGC2 presents the only source of cGMP in phototransduction. Ca2+-sensitive cGMP production is undetectable in mice lacking both isozymes, and their retinas do not respond to light [11, 89]. However, RetGC1 and RetGC2 do not contribute equally to cGMP synthesis in photoreceptors, and they also show different distributions between rods and cones. RetGC1 is the main isozyme of the cyclase, present in both rods and cones [11, 89, 123]. In rods, RetGC1 is the predominant form of the cyclase, responsible for the bulk (≥70%) of the cGMP production in outer segment, and RetGC2 isozyme acts as the ancillary component, producing < 30% of the total cGMP [89]. RetGC2 is virtually undetectable in cones [123], whereas RetGC1 is expressed in cones better than in rods [11, 25, 123]. Consequently, rod mass responses (as detected by scotopic electroretinography (ERG)) to dim flashes strongly diminish in mice lacking RetGC1 [95]. The cause of the degeneration is a nonsense mutation that truncates half of a 23-kDa RD3 polypeptide [34]. In a similar manner, mutations that truncate a human RD3 cause a recessive blindness from birth, Leber’s congenital amaurosis type 12 (LCA12) [34]. A long frame-shift in RD3 sequence also produces a canine retinal dysplasia [56].

RD3 has a dual function in photoreceptor physiology. Firstly, photoreceptors require RD3 in order to properly accumulate RetGC in the outer segment. There are indications that RD3 can be directly involved in the process of RetGC relocation from the photoreceptor inner segment to the outer segment [10, 131]. The content of RetGC1 and RetGC2 in rd3 mice declines at least tenfold [10, 95, 99]. The much lower than normal levels of RetGC activity can well explain the very early photoreceptor dysfunction in rd3 mice, resulting in a dramatic reduction of rod and cone ERG responses [95]. Gene therapy using an adenoviral vector to deliver the normal RD3 cDNA rescues rd3 photoreceptors from degeneration and dysfunction [73].

However, the lower guanylyl cyclase activity in rd3 photoreceptors per se is not the actual reason why they undergo rapid degeneration. Despite being significantly reduced, RetGC activity in rd3 retinas remains detectable and retains regulation by Ca2+ and GCAPs [95]. Furthermore, rods and cones in young rd3 mice still respond to light, albeit with much lower amplitudes of the ERG a-wave [31, 95]. Evidently, rd3 photoreceptors still produce enough cGMP to support at least their rudimentary physiological responses. In a sharp contrast to that, in mice devoid of all RetGC activity by deletion of both RetGC1 and RetGC2 genes, rods and cones are completely unable to produce cGMP for phototransduction [11, 89] and fail to respond to even brightest light stimuli [11, 95]. Yet, the photoreceptors in rd3 mice degenerate much faster than in the RetGC-deficient mice [95]. Importantly, the different degeneration patterns cannot be attributed to the differences in strain backgrounds, because both rd3 and RetGC knockout mice in those studies were congenic with the C57B6 strain [95].

In addition to the support of the normal content of RetGC in photoreceptor outer segment, RD3 also has no less important second role in the physiology of photoreceptors, which is protecting them from degeneration. This is directly related to another main property of RD3, which is inhibition of RetGC activity [88]. RD3 strongly suppresses catalytic activity of RetGC1 and RetGC2 and also blocks their stimulation by Mg2+ GCAPs [88, 95]. RD3 demonstrates nearly 1000-fold higher apparent affinity for RetGC than GCAPs in vitro, such that RD3 at nanomolar concentrations outcompetes GCAP1 and GCAP2 present at the micromolar concentrations required to effectively stimulate RetGC [88, 95]. Importantly, the RD3-dependent inhibition does not change the Ca2+ sensitivity of the RetGC:GCAP complex, but rather inactivates the complex entirely [88]. Recent in vivo studies in transgenic mice strongly indicate that the inhibitory function of RD3 plays the principal role in protecting photoreceptors from rd3 degeneration. After deletion of GCAP1 and GCAP2, the cyclase activity in rd3 retinas declines even further, because now the cyclase is no longer activated by GCAPs at low Ca2+. Consequently, their ERG photoresponses are further suppressed as well [31]. Yet, quite surprisingly, the vast majority of GCAP-deficient rd3 photoreceptors remain preserved at the ages when photoreceptors in rd3 mice expressing GCAPs have already died out [31, 99]. These results argue that photoreceptors need RD3 to block RetGC activation by GCAPs that may occur in the wrong place and/or at the wrong time. The intracellular localization of RD3—predominantly in the inner segment [31, 99]—further supports the hypothesis that the most likely function of RD3 is to suppresses aberrant activation of RetGC in the inner segment, before the cyclase reaches its proper destination in the outer segment and becomes a part of the Ca2+ feedback regulating phototransduction in the outer segment (Fig. 8).

Fig. 8
figure 8

A proposed biological role of RD3 in photoreceptors [31, 74, 95]. RD3 stimulates the delivery of RetGC to the outer segment while blocking its activation by GCAPs in the inner segment. In RD3-deficient photoreceptors, the content of RetGC in the outer segment declines and the photoresponses become compromised due to insufficient production of cGMP; aberrant activation of the unprotected RetGC occurs in the inner segment and triggers degeneration of the photoreceptor cell

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Three-dimensional NMR structure of RD3 [97] shows a rather unique fold of this protein, with an elongated bundle of four alpha-helices as its central core adjacent to non-structured regions at the N- and C-termini (Fig. 9). Site-directed mutagenesis probing the entire surface of the molecule by point mutations [85, 95] has identified two main clusters of the surface-exposed side chains that enable the high-affinity binding of RD3 to RetGC: one in the loop connecting helices α1 and α2, and the other on the surface of the helix α3 (Fig. 9) [85]. It is much less clear where the interface for binding RD3 is located on RetGC. The removal of a short C-terminal fragment from RetGC1 disrupts binding of RD3 [10, 98] but not of GCAPs [93, 94, 98]. Conversely, substitutions in the cyclase dimerization domain can block the binding of GCAPs but not of RD3 [93]. On the other hand, some point mutations in the RetGC1 kinase homology domain disrupt binding of both RD3 and GCAP [93, 98]. Therefore, it is possible that while competing with each other for the cyclase, RD3 and GCAP use different binding interfaces on RetGC that either partially overlap within the quaternary structure of the complex or the two different binding sites can affect each other indirectly by rearranging the quaternary structure of the cyclase. This and many other outstanding questions about the molecular dynamics of RetGC in its complexes with RD3 and GCAP remain to be addressed in future studies.

Fig. 9
figure 9

Ribbon diagram of the three-dimensional RD3 structure [97]. The two clusters of surface-exposed residues critical for the high-affinity inhibitory binding of RD3 to RetGC [85] are shown as sticks highlighted in red

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Does RD3 contribute to the photoreceptor degenerations caused by mutations in RetGC1 and GCAP1?

Recent studies also indicate that a CORD6-related substitution in the cyclase dimerization domain, Arg838Ser, not only increases RetGC1 affinity for Mg2+ GCAP and reduces its sensitivity to deceleration by Ca2+, but also makes the Mg2+ GCAP1: RetGC1 complex more resistant to inhibition by RD3 [30]. The most probable reason why higher concentrations of RD3 are required in that case is the increased affinity of the mutant RetGC1 for GCAP, which makes it more difficult for RD3 to displace GCAP from the complex with the cyclase. Likewise, a substitution in GCAP1 that also causes a dominant retinopathy, Gly86Arg, not only shifts the Ca2+ sensitivity of RetGC deceleration to a higher Ca2+ range, but also makes RetGC better resist the inhibition by RD3, again, most likely because of the increased affinity of the Gly86Arg GCAP1 for RetGC1 [96]. If the main role of RD3 is protecting photoreceptors against an aberrant activation of RetGC by GCAPs in the inner segment, then in the cases with both Arg838Ser RetGC1 and Gly86Arg GCAP1, the protective function of RD3 can be weakened by the higher stability of the GCAP:RetGC complex. This could present another factor contributing to the severity of retinal degeneration. Further studies should evaluate this possibility.