Inactivation of cGMP-dependent conductance of rod outer segment plasma membrane induced by cGMP.

Integral cGMP-dependent currents as well as activity of single cGMP-activated channels in plasma membrane of rod outer segment (ROS) were recorded using the patch-clamp method. The dependence of integral currents on cGMP concentration is shown to be bell-shaped. Decrease in cGMP-dependent conductance at high cGMP concentration results from a decrease in channel opening probability. Thus, cGMP in high concentrations inactivates cGMP-dependent conductance.     

Cation channels in the plasma membrane of retinal rod outer segments activated by cGMP

Properties of cGMP-activated cation channels were investigated on isolated patches of the ROS plasma membrane using the "patch clamp" technique. The channels were shown to be characterized by ideal cation selectivity under physiological conditions and are nearly equally permeable for cations of alkaline metals. At the same time they are permeable for some bivalence cations (PNa approximately PCa). Other channel properties are described and their comparative analysis is given. It suggests that cGMP-activated cation channels represent a new type of cation channels.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Subunit stoichiometry of retinal rod cGMP phosphodiesterase

The cyclic GMP phosphodiesterase of the retinal rod is composed of three distinct types of polypeptides: alpha (90 kDa), beta (86 kDa), and gamma (10 kDa). The gamma subunit has been shown to inhibit phosphodiesterase activity associated with alpha and beta. To investigate the subunit stoichiometry of the retinal phosphodiesterase, we have developed a panel of monoclonal and peptide antibodies that recognize individual phosphodiesterase subunits. By quantitative and immunochemical analysis of the purified subunits, we have shown that each phosphodiesterase molecule contains one copy each of alpha and beta subunit and two copies of gamma subunit. Moreover, gamma can be chemically cross-linked to both alpha and beta, but not to itself, suggesting that alpha and beta may each bind one gamma. The phosphodiesterase is fully activated when both copies of gamma were removed by proteolysis with trypsin. Upon recombination of the purified gamma subunit with the trypsin-activated phosphodiesterase containing alpha beta, the alpha beta gamma 2 stoichiometry is once again restored, with concomitant total inhibition of activity. Our results suggest that at least two activated transducin molecules are required to fully activate one molecule of phosphodiesterase in retinal rods.     

Glycoproteins specific for the retinal rod outer segment plasma membrane

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of Mr 230,000 and 110,000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.     

The delta subunit of retinal rod cGMP phosphodiesterase regulates the membrane association of Ras and Rap GTPases.

Post-translational modifications of GTPases from the Ras superfamily enable them to associate with membrane compartments where they exert their biological activities. However, no protein acting like Rho and Rab dissociation inhibitor (GDI) that regulate the membrane association of Rho and Rab GTPases has been described for Ras and closely related proteins. We report here that the delta subunit of retinal rod phosphodiesterase (PDEdelta) is able to interact with prenylated Ras and Rap proteins, and to solubilize them from membranes, independently of their nucleotide-bound (GDP or GTP) state. We show that PDEdelta exhibits striking structural similarities with RhoGDI, namely conservation of the Ig-like fold and presence of a series of hydrophobic residues which could act as in RhoGDI to sequester the prenyl group of its target proteins, thereby providing structural support for the biochemical activity of PDEdelta. We observe that the overexpression of PDEdelta interferes with Ras trafficking and propose that it may play a role in the process that delivers prenylated proteins from endomembranes, once they have undergone proteolysis and carboxymethylation, to the structures that ensure trafficking to their respective resident compartments.     

Effects of fluoride on retinal rod outer segment cGMP phosphodiesterase and G-protein

The effects of fluoride on ROS phosphodiesterase and G-protein have been studied using membrane-free extracts. When G-protein was present NaF, at millimolar concentrations, stimulated PDE activity however, in a G-protein free extract, cGMP hydrolysis was inhibited by high fluoride concentrations. Fluoride was also found to profoundly inhibit the ability of G-protein to bind a GTP analogue, GTP gamma S, both in the presence and absence of rhodopsin. Aluminium greatly modified these effects of fluoride on PDE and G-protein. The possibility that fluoride activates PDE through its effect on G-protein is discussed.     

The effect of ATP, GTP and cAMP on the cGMP-dependent conductance of the fragments from frog rod plasma membrane

Using a 'patch-clamp' method in the 'inside-out' configuration, ATP, ADP, AMP-PCP and AMP-PNP have been shown to increase the cGMP-dependent component of the rod plasma membrane conductance 2-4-fold and GTP, GDP but not GMP or nonhydrolyzable GTP analogs GMP-PNP and GTP-gamma-S to abolish the ATP action. The ATP and GTP effects were observed at [EDTA] = 1 mM when magnesium and calcium ions were absent. In about half of the experiments the cGMP-dependent conductance was shown to be increased by cAMP in the micromolar concentration range by 10-50%, the cAMP action did not depend on the presence of nucleoside triphosphates. In vivo ATP, GTP and cAMP are assumed to modulate the sensitivity of the photoreceptor plasma membrane to cGMP.     

Rhodopsin in the rod outer segment plasma membrane

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.     

A 48 kDa protein arrests cGMP phosphodiesterase activation in retinal rod disk membranes

Photolyzed rhodopsin (R) catalyzes GTP-binding to alpha-transducins (T alpha); T alpha X GTPs then activate cGMP phosphodiesterase (PDE). PDE activation is arrested by ATP in two ways: (i) initial velocity is suppressed, and (ii) PDE velocity rapidly returns to preactivation levels (turnoff). Arrestin (a 48 kDa protein) markedly enhances turnoff while not affecting initial velocity. Arrestin in the presence of ATP achieves rapid turnoff by directly inhibiting activated PDE, as indicated by its ability to inhibit the direct activation of PDE by T alpha X GMP--PNP (guanylyl-imidodiphosphate). Double reciprocal plots reveal a competition between arrestins and activated transducins for sites on PDE. Blocking R phosphorylation blocks initial velocity suppression but does not disturb rapid turnoff. Our data suggest a 2-fold mechanism for PDE deactivation: (i) formation of T alpha X GTPs is suppressed by R phosphorylation, while (ii) activation of PDE by T alpha X GTPs is competitively inhibited by arrestins when ATP is present.     

Binding stoichiometry of a fluorescent cGMP analogue to membranes of retinal rod outer segments

The high-affinity binding of the cGMP analogue 8-(5-thioacetamidofluorescein)-cGMP to rod outer segment membranes depleted of peripherally bound proteins has been defined by equilibrium dialysis (mean +/- SD): membranes contain about one cGMP binding site per 130 rhodopsin molecules; the concentration of free ligand for half saturation is 2.0 +/- 0.6 microM; the apparent Hill coefficient of the bound versus free ligand relationship is 1.7 +/- 0.5; half saturation of the binding sites is sufficient for 85% activation of calcium permeability. A gating mechanism is proposed.     

Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta

The photoreceptor cGMP phosphodiesterase (PDE6) plays a key role in vertebrate vision, but its enzymatic mechanism and the roles of metal ion co-factors have yet to be determined. We have determined the amount of endogenous Zn(2+) in rod PDE6 and established a requirement for tightly bound Zn(2+) in catalysis. Purified PDE6 contained 3-4-g atoms of zinc/mole, consistent with an initial content of two tightly bound Zn(2+)/catalytic subunit. PDE with only tightly bound Zn(2+) and no free metal ions was inactive, but activity was fully restored by Mg(2+), Mn(2+), Co(2+), or Zn(2+). Mn(2+), Co(2+), and Zn(2+) also induced aggregation and inactivation at higher concentrations and longer times. Removal of 93% of the tightly bound Zn(2+) by treatment with dipicolinic acid and EDTA at pH 6.0 resulted in almost complete loss of activity in the presence of Mg(2+). This activity loss was blocked almost completely by Zn(2+), less potently by Co(2+) and almost not at all by Mg(2+), Mn(2+), or Cu(2+). The lost activity was restored by the addition of Zn(2+), but Co(2+) restored only 13% as much activity, and other metals even less. Thus tightly bound Zn(2+) is required for catalysis but could also play a role in stabilizing the structure of PDE6, whereas distinct sites where Zn(2+) is rapidly exchanged are likely occupied by Mg(2+) under physiological conditions.     

Light changes the membrane potential and ion balances of retinal rod disks

Light stimulation of rod cells in vertebrate eyes may cause Ca2+ release from the intracellular disks. Radiolabelled tracers show that light causes a small hyperpolarization of intact disk stacks and redistribution of the ions Ca2+ and Cl-.     

Light-induced decreases in cGMP concentration precede changes in membrane permeability in frog rod photoreceptors

This study examines whether changes in cGMP concentration initiated by illumination of frog rod photoreceptors occur rapidly enough to implicate cGMP as an intermediate between rhodopsin activation in the disc membrane and permeability changes in the plasma membrane. Previous studies using whole retinas or isolated outer segments have provided conflicting evidence on the role of cGMP in the initial events of phototransduction. The rod photoreceptor preparation employed in this work consists of purified suspensions of outer segments still attached to the mitochondria-rich ellipsoid portion of the inner segment. These photoreceptors are known to retain normal electrophysiological responses to illumination and have cGMP levels comparable to those measured in the intact retina. When examined under several different conditions, changes in cGMP concentrations were found to occur as rapidly or more rapidly than the suppression of the membrane dark current. Subsecond changes in cGMP concentration were analyzed with a rapid quench apparatus and confirmed by comparison with a rapid freezing technique. In a 1 mM Ca2+ Ringer's solution, cGMP levels decrease to 65% of their final extent within 200 ms after bright illumination; changes in membrane dark current follow a similar time course. When the light intensity is decreased to 8000 rhodopsins bleached per rod per s, the light-induced cGMP decrease is completed within 50 ms, with 7 X 10(5) cGMP molecules hydrolyzed per rhodopsin bleached. During this time the dark current has not yet begun to change. Thus, under physiological conditions it is clear that changes in cGMP concentration precede permeability changes at the plasma membrane. The correlation of rapid changes in cGMP levels with changes in membrane current leave open the possibility that changes in cGMP concentration may be an obligatory step in the reaction sequence linking rhodopsin activation by light and the resultant decrease in sodium permeability of the plasma membrane.     

Protein kinase C-mediated phosphorylation of retinal rod outer segment membrane proteins

We have previously reported that the purified GDP-bound alpha-subunit of the GTP-binding protein transducin (TD), present in outer segments of retinal rod cells (ROS), serves as a high affinity substrate (Km = 1 microM) for protein kinase C (PKC) [Zick et al. (1986) Proc. natn. Acad. Sci., U.S.A. 83, 9294-9297]. In the present study we demonstrate that TD-alpha undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-gamma-S which activates TD, suggesting that it is only the inactive conformation of TD-alpha that serves as a substrate for PKC. Indeed, both vanadate and AlF4, that confer an active conformation on TD-alpha-GDP, inhibit PKC-mediated phosphorylation of purified TD-alpha-GDP. We demonstrate that the purified beta subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-alpha and beta form high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the alpha and beta subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.     

Changes in cGMP concentration correlate with some, but not all, aspects of the light-regulated conductance of frog rod photoreceptors

Cyclic GMP has been implicated in controlling the light-regulated conductance of rod photoreceptors of the vertebrate retina. However, there is little direct evidence correlating changes in cGMP concentration with the light-regulated permeability mechanism in living cells. A preparation of intact frog rod outer segments suspended in a Ringer's medium containing low Ca2+ has been used to demonstrate that initial changes in total cellular cGMP concentration parallel changes in the light-regulated membrane current over a wide range of light intensities. At light intensities bleaching from 160 to 5.6 X 10(6) rhodopsin molecules/rod/s, decreases in the response latency for the cGMP kinetics parallel decreases in the latent period of the electrical response. Further, changes in the rate of the cGMP decrease parallel the rate of membrane current suppression as the light intensity is varied. Up to 10(5) cGMP molecules are hydrolyzed per photolyzed rhodopsin, consistent with in vitro studies showing that each bleached rhodopsin can activate over 100 phosphodiesterase molecules. Addition of the Ca2+ ionophore, A23187, does not affect the initial kinetics of the cGMP decrease or of the electrical response, excluding a direct role for Ca2+ in the initial events of phototransduction. These results are consistent with cGMP being the intracellular messenger that links rhodopsin isomerization with changes in membrane permeability upon illumination. It is unlikely, however, that light-induced changes in total cGMP concentration are the sole regulators of membrane current. This is suggested by several observations: at bright light intensities, the subsecond light-induced cGMP decrease is essentially complete prior to complete suppression of membrane current; maximal light-induced decreases in cGMP concentration occur at all light intensities tested, whereas the extent of membrane current suppression varies over the same range of light intensities; changing the external Ca2+ concentration from 1 mM to 10 nM in the dark causes an increase in membrane current that is significantly more rapid than corresponding changes in cGMP concentration. Thus, light-induced changes in total cellular cGMP concentration correlate with some, but not all, aspects of the visual excitation process in vertebrate photoreceptors.     

Functional regions of the inhibitory subunit of retinal rod cGMP phosphodiesterase identified by site-specific mutagenesis and fluorescence spectroscopy.

In the dark, the activity of the cGMP phosphodiesterase (PDE) of retinal rod outer segments is held in check by its two inhibitory gamma subunits. Following illumination, gamma is rapidly removed from its inhibitory site by transducin, the G-protein of the visual system. In order to probe the functional roles of specific regions in the PDE gamma primary sequence, 10 variants of PDE gamma have been produced by site-specific mutagenesis and expression in bacteria and their properties compared to those of protein containing the wild-type bovine PDE gamma amino acid sequence. Three questions were asked about each mutant: What is its affinity for the alpha beta catalytic subunit of PDE? Does it inhibit catalytic activity? If so, can transducin relieve this inhibition? Binding to PDE alpha beta was determined directly using fluorescein-labeled gamma by measuring the increase in emission anisotropy that occurs when gamma binds to alpha beta. Inhibition of PDE alpha beta was measured by reconstitution of the gamma variants with gamma-free PDE generated by limited digestion with trypsin or endoproteinase Arg-C. Unlike trypsin, the latter enzyme did not remove PDE's ability to bind membranes and be activated by transducin, so that transducin activation of PDE containing specific gamma variants could be assayed directly. The results indicate that mutations in many regions of gamma affect its binding to alpha beta. A mutant missing the last five carboxy-terminal residues (83-87) was totally lacking in inhibitory activity. However, it still bound to PDE alpha beta tightly, although with a 100-fold lower dissociation constant (approximately 5 nM) than that of wild-type gamma (approximately 50 pM).(ABSTRACT TRUNCATED AT 250 WORDS)