Regulation of intracellular cyclic GMP concentration by light and calcium in electropermeabilized rod photoreceptors.

Abstract This study examines the regulation of cGMP by illumination and by calcium during signal transduction in vertebrate retinal photoreceptor cells. We employed an electropermeabilized rod outer segment (EP-ROS) preparation which permits perfusion of low molecular weight compounds into the cytosol while retaining many of the features of physiologically competent, intact rod outer segments (ROS). When nucleotide-depleted EP-ROS were incubated with MgGTP, time- and dose-dependent increases in intracellular cGMP levels were observed. The steady state cGMP concentration in EP-ROS (0.007 mol cGMP per mol rhodopsin) approached the cGMP concentration in intact ROS. Flash illumination of EP-ROS in a 250-nM free calcium medium resulted in a transient decrease in cGMP levels; this occurred in the absence of changes in calcium concentration. The kinetics of the cGMP response to flash illumination of EP-ROS were similar to that of intact ROS. To further examine the effects of calcium on cGMP metabolism, dark-adapted EP-ROS were incubated with MgGTP containing various concentrations of calcium. We observed a twofold increase in cGMP steady state levels as the free calcium was lowered from 1 μM to 20 nM; this increase was comparable to the behavior of intact ROS. Measurements of guanylate cyclase activity in EP-ROS showed a 3.5-fold increase in activity over this range of calcium concentrations, indicating a retention of calcium regulation of guanylate cyclase in EP-ROS preparations. Flash illumination of EP-ROS in either a 50- or 250-nM free calcium medium revealed a slowing of the recovery time course at the lower calcium concentration. This observation conflicts with any hypothesis whereby a reduction in free calcium concentration hastens the recovery of cytoplasmic cGMP levels, either by stimulating guanylate cyclase activity or by inhibiting phosphodiesterase activity. We conclude that changes in the intracellular calcium concentration during visual transduction may have more complex effects on the recovery of the photoresponse than can be accounted for solely by guanylate cyclase activation

Photoreceptor phosphodiesterase (PDE6): structure, regulatory mechanisms, and implications for treatment of retinal diseases

The photoreceptor phosphodiesterase (PDE6) is a member of large family of Class I phosphodiesterases responsible for hydrolyzing the second messengers cAMP and cGMP. PDE6 consists of two catalytic subunits and two inhibitory subunits that form a tetrameric protein. PDE6 is a peripheral membrane protein that is localized to the signaling-transducing compartment of rod and cone photoreceptors. As the central effector enzyme of the G-protein coupled visual transduction pathway, activation of PDE6 catalysis causes in a rapid decrease in cGMP levels that results in closure of cGMP-gated ion channels in the photoreceptor plasma membrane. Because of its importance in the phototransduction pathway, mutations in PDE6 genes result in various retinal diseases that currently lack therapeutic treatment strategies due to inadequate knowledge of the structure, function, and regulation of this enzyme. This review focuses on recent progress in understanding the structure of the regulatory and catalytic domains of the PDE6 holoenzyme, the central role of the multi-functional inhibitory γ-subunit, the mechanism of activation by the heterotrimeric G protein, transducin, and future directions for pharmacological interventions to treat retinal degenerative diseases arising from mutations in the PDE6 genes

Structural analysis of the regulatory GAF domains of cGMP phosphodiesterase elucidates the allosteric communication pathway

Regulation of photoreceptor phosphodiesterase (PDE6) activity is responsible for the speed, sensitivity, and recovery of the photoresponse during visual signaling in vertebrate photoreceptor cells. It is hypothesized that the physiological differences in the light responsiveness of rods and cones may result in part from differences in the structure and regulation of the distinct isoforms of rod and cone PDE6. Although rod and cone PDE6 catalytic subunits share a similar domain organization consisting of tandem GAF domains (GAFa and GAFb) and a catalytic domain, cone PDE6 is a homodimer whereas the rod PDE6 catalytic dimer is composed of two homologous catalytic subunits. Here we provide the x-ray crystal structure of cone GAFab regulatory domain solved at 3.3 Å resolution in conjunction with chemical cross-linking and mass spectrometric analysis of conformational changes to GAFab induced upon binding of cGMP and the PDE6 inhibitory γ-subunit (Pγ). Ligand-induced changes in cross-linked residues implicate the α4-helix of GAFa (close to the cGMP binding site) and the β1/β2 loop of GAFb as key motifs that have been previously proposed to communicate with the catalytic domains of PDE6. Molecular dynamics (MD) simulations of cone GAFab revealed asymmetry in the two GAFab subunits forming the homodimer and allosteric perturbations on cGMP binding. Cross-linking of Pγ to GAFab in conjunction with solution NMR spectroscopy of isotopically labeled Pγ identified the central polycationic region of Pγ interacting with the GAFb domain. These results provide a mechanistic basis for developing allosteric activators of PDE6 with therapeutic implications for halting the progression of certain retinal degenerative diseases

Cyclic Guanosine 5′-Monophosphate Binding to Regulatory GAF Domains of Photoreceptor Phosphodiesterase

Of the 11 families of mammalian cyclic nucleotide phosphodiesterases (PDEs), 5 contain regulatory domains capable of binding cyclic guanosine 5′-monophosphate (cGMP). The best understood of the GAF-containing PDEs is the family of rod (PDE6R) and cone (PDE6C) photoreceptor PDEs. Binding of cGMP to the rod PDE6 catalytic dimer (αβ) allosterically regulates the affinity of the inhibitory subunits of PDE6 (γ) for the enzyme. Two nonidentical, highaffinity cGMP-binding sites exist on the nonactivated mammalian PDE6R holoenzyme (αβγγ). One of the sites does not readily exchange with free cGMP when the catalytic dimer is complexed with Pγ. On dissociation of γ from the catalytic dimer, one of the two cGMP-binding sites undergoes a transition from high to low affinity. This chapter describes techniques to quantify cGMP binding to PDE6 in order to study the regulatory significance of the GAF domains. For high-affinity cGMP binding sites on PDE6, membrane filtration is the method of choice because of its speed, simplicity, and sensitivity. However, lower-affinity cGMP-binding sites require a method that does not perturb the equilibrium between bound and free ligand. The use of ammonium sulfate solutions during filtration extends to lower-binding affinities the useful range of membrane filtration. However, a centrifugal separation technique that minimizes perturbation of the cGMP-binding equilibrium is also presented for measuring lower-affinity cGMP-binding sites. These methods are applicable to understanding the regulatory mechanisms regulating other GAF-containing PDEs as well

Intracellular cGMP concentration in rod photoreceptors is regulated by binding to high and moderate affinity cGMP binding sites.

Abstract cGMP is the second messenger for visual excitation in vertebrate rod photoreceptors. However, no direct correlation has been observed between the measured total cGMP concentration in the rod outer segment and the electrical response of these cells to photic stimulation. To address this discrepancy, we have quantitated the number and affinities of cGMP binding sites in the rod outer segment to determine the cytoplasmic free cGMP concentration that is involved in visual transduction. We identified two distinct classes of cGMP binding sites in amphibian rod outer segments: 1) high affinity binding sites with a KDI = 60 nM and a site density of 30 μM, and 2) moderate affinity binding sites with a KD2 = 6.6 μM and a site density of 78 μM. These two classes of binding sites are calculated to bind 94% of the total cellular cGMP, thereby lowering the cytoplasmic cGMP concentration to 3.5 μM in dark-adapted rod outer segments. This value is consistent with predictions of the cytoplasmic cGMP concentration based on activation of the cGMP-gated ion channel of rod photoreceptors. The kinetics of cGMP dissociation from high affinity binding sites indicate that this class of sites would dissociate its bound cGMP too slowly to participate in visual excitation and recovery to flash illumination. This binding of cGMP to intracellular binding sites provides a non-enzymatic mechanism by which photoreceptor cells regulate the concentration and restrict the diffusion of this second messenger during visual transduction

Photoreceptor Phosphodiesterase (PDE6): Activation and Inactivation Mechanisms During Visual Transduction in Rods and Cones

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes recent advances in the structural characterization of the rod and cone PDE6 catalytic and regulatory subunits in the context of previous biochemical studies of the enzymological properties and allosteric regulation of PDE6. Emphasis is given to recent advances in understanding the structural and conformational changes underlying the mechanism by which the activated transducin α-subunit binds to—and relieves inhibition of—PDE6 catalysis that is controlled by its intrinsically disordered, inhibitory γ-subunit. The role of the regulator of G-protein signaling 9-1 (RGS9-1) in regulating the lifetime of the transducin-PDE6 is also briefly covered. The therapeutic potential of pharmacological compounds acting as inhibitors or activators targeting PDE6 is discussed in the context of inherited retinal diseases resulting from mutations in rod and cone PDE6 genes as well as other inherited defects that arise from excessive cGMP accumulation in retinal photoreceptor cells

Characteristics of Photoreceptor PDE (PDE6): similarities and differences to PDE5

Phosphodiesterase 6 (PDE6) is highly concentrated in the retina. It is most abundant in the internal membranes of retinal photoreceptors, where it reduces cytoplasmic levels of cyclic guanosine monophosphate ( cGMP) in rod and cone outer segments in response to light. The rod PDE6 holoenzyme comprises alpha and beta catalytic subunits and two identical inhibitory gamma subunits. Each catalytic subunit contains three distinct globular domains corresponding to the catalytic domain and two GAF domains ( responsible for allosteric cGMP binding). The PDE6 catalytic subunits resemble PDE5 in amino-acid sequence as well as in three-dimensional structure of the catalytic dimer; preference for cGMP over cyclic adenosine monophosphate ( cAMP) as a substrate; and the ability to bind cGMP at the regulatory GAF domains. Most PDE5 inhibitors inhibit PDE6 with similar potency, and electroretinogram studies show modest effects of PDE5 inhibitors on visual function - an observation potentially important in designing PDE5-specific therapeutic agents