The cGMP Signaling Pathway in Retinal Photoreceptors and the Central Role of Photoreceptor Phosphodiesterase (PDE6)

Regulation of Intracellular cGMP Levels in Photoreceptor Cells

Guanylate cyclase (GC) catalyzes the synthesis of cGMP from guanosine triphosphate (GTP). Vertebrates have two major families of GC, soluble and membrane associated. The two membrane-associated GCs found in photoreceptor cells (GC-2E and GC-2F; also abbreviated as ROS-GCs or Ret-GCs in the literature) consist of an extracellular domain (of unknown function), a single-pass transmembrane segment, a kinase-homology domain, and a catalytic domain [4, 5]. The photoreceptor GCs are not regulated by binding of ligands to the extracellular domain as is the case for several other membrane-associated GCs [5]. Instead, photoreceptor GCs are regulated in a calcium-dependent manner by three distinct GC-activating proteins (GCAPs; GCAP1, GCAP2, and GCAP3 [4, 6, 7]).

The breakdown of cyclic nucleotides in cells is catalyzed by cyclic nucleotide PDEs. In vertebrates, there are 11 families of PDEs that share a conserved catalytic domain but differ in their substrate specificity (cAMP-, cGMP-, or dual-specific), regulatory mechanisms, and pharmacological sensitivity [8]. Rods and cones express a photoreceptor-specific PDE named PDE6 that has a very high catalytic efficiency when activated by light, is regulated by its inhibitory γ-subunit, and shares structural and pharmacological sensitivity with PDE5, a PDE abundant in vascular smooth muscle and other tissues [9].

In addition to metabolic regulation of cGMP by GC and PDE activities, the free cytoplasmic cGMP concentration can also be regulated by cGMP transport out of the cell or by cGMP sequestration (i.e., binding to specific binding sites) within the cell. Transport systems have been identified that selectively pump cGMP out of various cell types [3, 10, 11], but to date evidence for cGMP efflux from photoreceptors is lacking. In contrast, high-affinity cGMP-binding proteins (e.g., PDE6 itself) are present in photoreceptor cells and are likely to contribute substantially to reducing the free cGMP concentration [2]. Sequestration of cGMP is indeed a major factor in determining the cytoplasmic free cGMP concentration of 2–4 µM (inferred from electrophysiological studies; [12, 13]) since the total cGMP concentration in the rod outer segment is tenfold higher [14].

Downstream Targets of cGMP Action in Photoreceptor Cells

All of the above-mentioned downstream targets of cGMP are present in photoreceptor cells: the PKG, the CNG ion channels, and the cGMP-binding PDE6.

cGMP-Dependent Protein Kinase

There is not much known about the abundance of PKG in photoreceptor cells, potential protein substrates for reversible phosphorylation, or the relevance of PKG for the phototransduction pathway. Some evidence indicates that cAMP-dependent protein kinase (PKA) is more prevalent than PKG [15, 16], and other studies do not unequivocally distinguish PKG from PKA [17, 18]. Considering the relatively high cGMP levels in photoreceptor cells, the potential for cGMP to bind to and activate PKA (i.e., “cross-talk”) cannot be ruled out.

The observation that several PKG/PKA substrates undergo light-dependent phosphorylation/ dephosphorylation supports the idea that cyclic nucleotide-dependent kinases may be involved in some aspects of visual signaling. For example, light-dependent dephosphorylation of components I and II (in amphibian photoreceptors) and phosducin (in mammalian and fish photoreceptors) is consistent with the idea that the light-induced drop in free cGMP levels results in PKA/PKG inactivation and thereby causes dephosphorylation of

the above-mentioned phosphoproteins [19–25]. The abilities of these dephosphorylated proteins to preferentially bind to transducin βγ dimers [25–27] and to assist in lightdependent protein translocation within the photoreceptor cell [28] are consistent with a role for PKG/PKA in long-term light adaptation.

Cyclic Nucleotide-Gated Ion Channels

The CNG ion channels in rod and cone cells belong to a large superfamily of ion channels that share a similar structure, including six transmembrane segments that selfassociate to constitute the pore of the ion channel [29]. The rod and cone CNG channels are heterotetramers (rod, 3 CNGA1 and 1 CNGB1; cone, 2 CNGA3, 2 CNGB3 [30, 31]). Activation (opening) of the CNG ion channel results from a highly cooperative binding of four cyclic nucleotide molecules to the C-terminal cyclic nucleotide-binding domain in the channel [32]. Channel closure in the plasma membrane resulting from the drop in cGMP levels induces hyperpolarization of the cell membrane and the generation of the receptor potential.

Rod and cone CNG channels are optimized to instantaneously sense and respond to changes in cGMP concentration induced by activation of the visual excitation pathway. Rapid responsiveness to fluctuations in cGMP levels reflects the relatively low affinity of cGMP binding along with fast dissociation and association rates for the cGMP-binding sites. The cooperativity (Hill coefficient ~3) with which four cGMP molecules bind amplifies small changes in cGMP concentration [33]. An important aspect of the ionic permeability of the CNG channel is its relatively high calcium permeability; this generates a calcium feedback signal (in concert with a Na+/K+-Ca2+ exchanger) when the channels close during visual excitation, thus allowing for calcium-dependent reactions involved in recovery and adaptation to occur [29, 33].

Calcium regulatory proteins (calmodulin in rods, an uncharacterized calcium-binding protein in cones) bind to and regulate the photoreceptor CNG channel by reducing its cGMP sensitivity. Desensitization of CNG channels by reducing cGMP-binding affinity is also reported to be affected by tyrosine or serine/threonine phosphorylation, diacylglycerol or related metabolites, and retinoids [29, 32, 34–36], but the physiological significance of these regulatory mechanisms is uncertain.

PDE6 Is a High-Affinity cGMP-Binding Protein

In addition to having a catalytic domain responsible for lowering cGMP levels during visual excitation, PDE6 contains a regulatory domain consisting of two tandemly arrayed domains that bind cGMP with high affinity. These cGMP-binding sites serve to sequester a majority of the total cGMP in the photoreceptor cell as well as having regulatory properties, both of which are discussed in detail in this chapter.

THE CELLULAR CONTEXT OF cGMP SIGNALING IN VERTEBRATE RETINAL PHOTORECEPTORS

Compartmentation of cGMP Signaling in Photoreceptor Outer Segments

Vertebrate rod and cone photoreceptor cells are specialized neurons consisting of several functionally and structurally distinct cellular compartments: (1) The phototransducing outer segment portion of the cell contains densely packed membranes optimized for

photon capture, signal transduction, and the initial membrane hyperpolarization. (2) The nonmotile “connecting cilium” region connects the outer segment to the inner segment and actively regulates the transport of proteins and metabolites between these two compartments. (3) The inner segment is comprised of the metabolic machinery and is itself compartmentalized: Mitochondria are concentrated in the “ellipsoid” region nearest the connecting cilium, whereas organelles dedicated to protein biosynthesis are located between the mitochondria-rich ellipsoid region and the cell nucleus. (4) The synaptic terminals of rods and cones tonically release neurotransmitters in the dark and respond to membrane hyperpolarization by suppressing synaptic vesicle release in the light. The photoreceptor synapse communicates with second-order bipolar and horizontal cells to propagate the photoresponse. (For reviews, see [37, 38].)

Physiology of the Photoreceptor Response to Light

In the dark, a circulating “dark current” is maintained by entry of sodium and calcium through open CNG ion channels in the outer segment plasma membrane; this is concurrent with the extrusion of sodium by a Na+/K+-ATPase (adenosine triphosphatase) and the efflux of potassium by K+ channels (both localized to the inner segment). A Na+/Ca2+-K+ exchanger (in the outer segment) and other ion channels in the inner segment further regulate ion conduction and transport in photoreceptors. On illumination, this dark current is interrupted, and the change in membrane potential that results from channel closure is passively propagated from the outer segment through the inner segment to the synaptic terminal [39–41].

Remarkably, rod photoreceptors can detect individual photons and can generate discrete, reproducible photoresponses from these single-photon events [42]. In contrast, cone photoreceptors are less light sensitive than rods; their photoresponses are smaller and faster than for rods, but they operate over an enormous range of ambient light intensities.

The rising phase of the photoresponse (termed visual excitation) is dominated by the kinetics of activation of the cGMP signaling pathway (discussed in detail in the next section). However, to rapidly respond to changes in light stimuli, the visual excitation process must be rapidly terminated. Photoresponse recovery is tightly coordinated with the components of the cGMP excitation pathway but also depends on another second messenger, calcium, to control the kinetics of the recovery to the dark-adapted state. Furthermore, photoreceptors also undergo “light adaptation” in the presence of background illumination, a process that allows rods and cones to increase the range of light intensities over which visual transduction can operate. On exposure to background illumination, the sensitivity of rods and cones to flash stimulation is decreased, resulting in smaller photoresponses that have faster recovery kinetics. Calcium plays a central role in the underlying mechanisms of light adaptation, acting through calcium regulatory proteins to modulate several steps in the cGMP signaling pathway. (For reviews, see [40, 43–47].)

Biochemical Cascade of Visual Excitation

The phototransduction cascade is a prototypical heterotrimeric G protein-coupled signaling pathway (Fig. 1). The excitation process is triggered by absorption of a photon by the visual receptor rhodopsin, which activates a photoreceptor-specific G protein, transducin, that then activates the effector enzyme PDE6. The resultant drop in