Fig. 1. The cyclic guanosine monophosphate (cGMP) signaling pathway for visual excitation in vertebrate rod photoreceptors. Top: In dark-adapted rod photoreceptors, cytoplasmic cGMP (small gray circle) and calcium concentrations are high, and some of the cGMP-gated cation channels in the plasma membrane are fully liganded with cGMP and in their open state. This permits entry of Na+ and Ca2+ through the pore. Rhodopsin, transducin, and phosphodiesterase 6 (PDE6) are in their nonactivated states. Bottom: On absorption of a photon by rhodopsin, isomerization of the 11-cis retinal chromophore causes receptor activation (R*). This leads to binding of transducin to R*, guanine nucleotide exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP), and formation of the activated transducin α-subunit with bound GTP (Tα*). The Tα* species then binds PDE6 holoenzyme, causing deinhibition by the γ-subunit (Tα*-P*) and a large acceleration of catalysis of cGMP to 5’-GMP at the active site. The light-induced drop in cGMP concentration induces the ligand-gated ion channel to close, causing membrane hyperpolarization. Ongoing extrusion of calcium by the Na+/Ca2+-K+ exchanger in the absence of calcium influx through the channel also causes intracellular calcium concentration to decline (which is vital for the recovery process).

second-messenger concentration (i.e., cGMP) leads to dissociation of cGMP bound to the CNG ion channel, closure of the ion channel, and membrane hyperpolarization.

Central Components of the cGMP Signaling Pathway

The first step in vertebrate vision is the photoisomerization of the retinal chromophore (11-cis retinal) of the visual pigment on the outer segment membrane (Fig. 1).

Rhodopsin is a member of the G protein-coupled receptor superfamily, in which 11-cis retinal locks rhodopsin into its inactive conformation in the dark. Photoactivation causes isomerization of 11-cis retinal to all-trans retinal. This causes movement of the transmembrane α-helices that surround the chromophore, producing metarhodopsin II, the activated form of the receptor [48, 49].

Conformational changes in the C-terminal tail and cytoplasmic loops of metarhodopsin II allow rhodopsin to bind with high affinity to the heterotrimeric G protein, transducin [50]. This interaction catalyzes the exchange of bound GDP for GTP on the transducin α-subunit, causing dissociation of the α-subunit (with bound GTP) from the βγ dimer [51]. Activated transducin then binds to its effector in this signaling cascade, PDE6, displacing the inhibitory PDE6 γ-subunit and accelerating the catalysis of cGMP (see the section “Transducin Activation of Rod PDE6 During Visual Excitation” for details). Because of the significant lifetime of metarhodopsin II, 20–100 transducin molecules (and hence PDE6 molecules) can be activated per photoisomerization event [52, 53]; this represents the first stage of amplification of the signaling cascade.

A second stage in signal amplification follows stoichiometric activation of PDE6 by transducin: Each activated PDE6 can break down many thousands of cGMP molecules per second (see the section “PDE6 Has Evolved to Meet the Special Demands of the Central Effector of Visual Transduction”). The overall gain of this amplified excitation pathway is well over 100,000 cGMP molecules hydrolyzed per activated rhodopsin [51].

The very rapid drop in cytoplasmic cGMP levels on light activation ensures that cGMP dissociates quickly from binding sites on the CNG ion channel in the plasma membrane. This sequence leading from photoisomerization of visual pigment to channel closure and membrane hyperpolarization constitutes the set of reactions defined as visual excitation.

Termination and Adaptation of the Light Response

The recovery of the dark-adapted state following illumination occurs in a precisely controlled manner that optimizes both the temporal resolution of visual stimuli as well as the ability of photoreceptors to light adapt over the 1012 range of photic stimuli on earth. Each step in the visual excitation pathway must be deactivated to restore cGMP levels and to return the components of the excitation pathway to their inactive states. The recovery of the photoresponse depends on inactivation of the metarhodopsin II state of rhodopsin as well as deactivation of GTP-bound form of the transducin α-subunit. Because the kinetics of the recovery phase of the light response are highly stereotypical, it has been appreciated for some time that a single deactivation step must be rate limiting [43]. Using a transgenic approach, it has been shown that the rate of GTP hydrolysis by transducin α-subunit represents the rate-limiting step of rod photoresponse recovery [53]. By regulating the guanosine triphosphatase (GTPase) rate of activated transducin, the lifetime of activated PDE6 is thereby precisely controlled (see section “Deactivation of Transducin”).

Light adaptation extends the operating range of the photoresponse of rods and cones by dampening the response amplitude in response to an incremental change in light intensity as well as accelerating the kinetics of the photoresponse. Whereas visual excitation requires consideration only of one second messenger (i.e., cGMP), calcium plays a central role in many aspects of photoreceptor adaptation. The cytoplasmic concentration of calcium

in dark-adapted photoreceptor outer segments (400–600nM) rapidly decreases on light exposure to 10–50nM as a consequence of channel closure concomitant with continued extrusion of calcium by the Na+/Ca2+-K+ exchanger.

Calcium regulates light adaptation primarily through three mechanisms: regulation of GC activity, regulation of the rate of inactivation of rhodopsin by rhodopsin kinasemediated phosphorylation, and modulation of the affinity cGMP-gated ion channel for cGMP. Each process is regulated by distinct calcium-binding proteins: GC-activating proteins (GCAPs) for GC, recoverin/S-modulin for rhodopsin kinase, and calmodulin for the ion channel. Of these three calcium-dependent control steps, the dominant one for light adaptation is the regulation of GC activity [44–46, 54].

Deactivation of Rhodopsin

Metarhodopsin II inactivation involves phosphorylation by a specific G protein-coupled receptor kinase (GRK1 in rods, GRK7 in human cones) with activity that is regulated by the calcium-binding protein recoverin/S-modulin [55–57]. Calcium-recoverin binds to rhodopsin kinase and inhibits its ability to phosphorylate activated rhodopsin, thereby prolonging the activated state of the receptor [58]. Once phosphorylated, arrestin binds to phosphorylated rhodopsin to complete the inactivation process [59]. Pigment regeneration of the photobleached chromophore requires enzymatic and transport reactions, termed the retinoid cycle [60].

Deactivation of Transducin

The inactivation of the α-subunit of transducin requires hydrolysis of bound GTP. The intrinsic GTPase rate of transducin is slow but can be accelerated when complexed with the regulator of G protein signaling 9 (RGS9 [61]), the type 5 G-protein β-subunit [62], and the RGS9 anchor protein (R9AP [63]).

The PDE6 γ-subunit plays an elegant negative-feedback role by binding to RGS9 and enhancing the affinity of the RGS9 protein complex for α*-GTP (α-subunit with GTP bound) [64–66], thereby accelerating its intrinsically slow GTPase activity. This role of the γ-subunit to bind RGS9 serves to turn off PDE6 activation in a precisely timed manner that is critical to the kinetics of the recovery process. Importantly, this GTPase accelerating role of the γ-subunit does not interfere with the ability of α*-GTP to efficiently activate PDE6 during the initial stage of visual excitation [51].

Deactivation of PDE6

PDE6 inactivation occurs when the PDE6 γ-subunit is released from its binding site on the deactivated α-subunit of transducin and reinhibits the enzyme’s active site. The strength of the interaction of the γ-subunit for transducin versus PDE6 is modulated by the state of occupancy of cGMP at the PDE6 regulatory GAF domains. This feedback regulation mechanism is discussed in detail in the section “Functions of the Regulatory cGMP-Binding GAF Domains of PDE6”.

A nonenzymatic mechanism for restoring cGMP levels by utilizing cGMP bound to high-affinity sites on the PDE6 GAF domains [67] is theoretically possible [2] but has not been experimentally supported [68], in large part because the catalytic power of activated PDE6 will hydrolyze cGMP as it dissociates from its binding sites.