cGMP-dependent cation channel of retinal rod outer segments

Light-modulated cytoplasmic cGMP simultaneously controls plasma membrane Na+ conductance in visual excitation and Ca2+ entry into rods by direct interaction with the cation channel. Cytoplasmic Ca2+ in turn may set operating points and contribute to the dynamics of several enzymes that regulate cGMP levels in the dark, recovery from excitation and receptor adaptation or down regulation. Similar channels may couple electrical activity to internal nucleotide metabolism in other tissues. We here report the identification, partial purification and behaviour after reconstitution of a protein of relative molecular mass 39,000 (Mr 39K) present in both disk and plasma membranes from bovine rod outer segments that mediates these cGMP-dependent cation fluxes. Its cGMP agonist specificity, kinetic cooperativity, ionic selectivity, membrane density and other features closely match the properties of the visual cGMP-dependent conductance inferred from electrophysiological measurements.     

Light-suppressible, cyclic GMP-sensitive conductance in the plasma membrane of a truncated rod outer segment

Recent experiments by Fesenko et al and ourselves have shown that excised membrane patches from retinal rod outer segments contain a cyclic GMP-sensitive conductance which has electrical properties similar to those of the light-sensitive conductance. This finding supports the notion that cGMP mediates phototransduction (see ref. 3) by directly modulating the light-sensitive conductance. However, some uncertainty remained about whether the patch experiments had discriminated completely between plasma and intracellular disk membranes; thus the cGMP response in an excised membrane could have resulted from contaminating disk membrane fragments, which are known to contain a cGMP-regulated conductance. Furthermore, the patch conductance has not yet been shown to be light-suppressible, an ultimate criterion for identity with the light-sensitive conductance. We now report experiments on a truncated rod outer segment preparation which resolved these issues. The results demonstrated that the cGMP-sensitive conductance was present in the plasma membrane of the outer segment, and that in the presence of GTP the conductance could be suppressed by a light flash. With added ATP, the effectiveness of the light flash was reduced and the suppression was more transient. The effects of both GTP and ATP were consistent with the known biochemistry. From the maximum current inducible by cGMP, we estimate that approximately 1% of the light-sensitive conductance is normally open in the dark; this would give an effective free cGMP concentration of a few micromolar in the intact outer segment in the dark.     

Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retinal rods.

In vertebrate photoreceptors, light reduces cyclic GMP concentration and closes cGMP-activated channels to induce a hyperpolarizing response. As Ca2+ can permeate the channels and the Na(+)-Ca2+ exchanger continuously extrudes Ca2+, closure of the channel results in a reduction of the inter-rod Ca2+ concentration. This is believed to be one of the mechanisms of light-adaptation produced by activation of guanylate cyclase. Effects of Ca2+ on the cGMP phosphodiesterase (PDE) have been reported, but their physiological significance has remained unclear. We have perfused the inside-out preparation of a frog rod outer segment (I/O ROS, originally termed truncated ROS, and find that Ca2+ in a physiological range regulates the light-activation of PDE. Therefore, PDE regulation by Ca2+ must be involved in light-adaptation in rods. The effect is mediated by a newly found protein which binds to disk membranes at high Ca2+ concentrations and prolongs PDE activation.     

Control of Ca2+ in rod outer segment disks by light and cyclic GMP

Photons absorbed in vertebrate rods and cones probably cause electrochemical changes at the photoreceptor plasma membrane by changing the cytoplasmic concentration of a diffusible transmitter substance, reducing the Na+ current flowing into the outer segment of the cell in the dark, to produce the observed membrane hyperpolarization that is the initial excitatory response. Cyclic GMP has been proposed as the transmitter because a light-activated cyclic GMP phosphodiesterase (PDE) has been found in rod disk membranes and because intracellularly injected cyclic GMP reduces rod membrane potentials. Free Ca2+ has also been proposed because increasing external [Ca2+] quickly and reversibly reduces the dark current and divalent cationophores increase the Ca2+ sensitivity. Ca2+ efflux from rod outer segments (ROS) of intact retinas occurs simultaneously with light responses. Vesicles prepared from ROS disk membranes become more permeable on illumination, releasing trapped ions or molecules, but intact outer segment disks have not previously been found to store sufficient Ca2+ in darkness and to release enough in light to meet the theoretical requirements for control of the dark current by varying cytoplasmic Ca2+ (refs 14-18). We now report experiments that show the required Ca2+ storage and release from rod disk membranes suspended in media containing high-energy phosphate esters and electrolytes approximating the cytoplasmic composition of live rod cells. Cyclic GMP stimulates Ca2+ uptake by ROS disks in such media.     

Metarhodopsin I/metarhodopsin II transition triggers light-induced change in calcium binding at rod disk membranes

The hypothesis of Yoshikami and Hagins that calcium ions act as diffusible transmitter molecules between the photochemistry of rhodopsin and the subsequent electrical events at the outer plasma membrane of rods initiated many investigations on light-stimulated calcium release in vertebrate photoreceptor cells (see refs 2, 3). Although it not seems firmly established that light has some effect on the redistribution of calcium in various disk preparations, reconstituted systems and intact rod outer segments, the physiological significance remained unclear. We previously reported a rapid, light-triggered calcium release from binding sites at the disk membrane in the presence of calcium ionophore A23187 (refs 3, 8). However, there is no evidence for rapid calcium release into the cytosol in the absence of ionophore. On fragmentation of intact rod outer segments, calcium release due to a light-requlated change of calcium binding appeared almost completely abolished. We describe here experiments with sonicated rod outer segments in which the previously observed loss of the calcium release capacity has been prevented. Calcium release in sonicated disks in the presence of A23187 kinetically follows the metarhodopsin I/metarhodopsin II transition (tau 1/2 = 10 ms, activation energy EA = 34 kcal mol-1), suggesting that calcium release is triggered by this photochemical transition.     

Effects on the photoresponse of calcium buffers and cyclic GMP incorporated into the cytoplasm of retinal rods

It is generally accepted that the light response in retinal rods involves a reduction of ionic permeability (predominantly to Na+) in the plasma membrane of the outer segment and that this is mediated by an internal messenger which diffuses between the disk and plasma membranes. There is controversy, however, over the identity of the diffusible substance; two alternative schemes have received widespread support (for review see refs 1,2). According to the 'calcium hypothesis', light stimulates the release into the cytoplasm of calcium, leading to the blockage of channels which are normally open in darkness, whereas based on the 'cyclic nucleotide hypothesis', cyclic GMP causes the opening of channels in the dark, but is hydrolysed by a light-activated phosphodiesterase. We report here effects of introducing calcium buffers and cyclic GMP into the rod cytoplasm by means of a patch pipette, which seem to be inconsistent with the calcium hypothesis.     

Cyclic GMP-sensitive conductance of retinal rods consists of aqueous pores

The surface membrane of retinal rod and cone outer segments contains a cation-selective conductance which is activated by 3',5'-cyclic guanosine monophosphate (cGMP). Reduction of this conductance by a light-induced decrease in the cytoplasmic concentration of cGMP appears to generate the electrical response to light, but little is known about the molecular nature of the conductance. The estimated unitary conductance is so small that ion transport might occur via either a carrier or a pore mechanism. Here we report recordings of cGMP-activated single-channel currents from excised rod outer segment patches bathed in solutions low in divalent cations. Two elementary conductances, of approximately 24 and 8 pS, were observed. These conductances are too large to be accounted for by carrier transport, indicating that the cGMP-activated conductance consists of aqueous pores. The dependence of the channel activation on the concentration of cGMP suggests that opening of the pore is triggered by cooperative binding of at least three cGMP molecules.     

Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors.

The assembly of signalling molecules into macromolecular complexes (transducisomes) provides specificity, sensitivity and speed in intracellular signalling pathways. Rod photoreceptors in the eye contain an unusual set of glutamic-acid-rich proteins (GARPs) of unknown function. GARPs exist as two soluble forms, GARP1 and GARP2, and as a large cytoplasmic domain (GARP' part) of the beta-subunit of the cyclic GMP-gated channel. Here we identify GARPs as multivalent proteins that interact with the key players of cGMP signalling, phosphodiesterase and guanylate cyclase, and with a retina-specific ATP-binding cassette transporter (ABCR), through four, short, repetitive sequences. In electron micrographs, GARPs are restricted to the rim region and incisures of discs in close proximity to the guanylate cyclase and ABCR, whereas the phosphodiesterase is randomly distributed. GARP2, the most abundant splice form, associates more strongly with light-activated than with inactive phosphodiesterase, and GARP2 potently inhibits phosphodiesterase activity. Thus, the GARPs organize a dynamic protein complex near the disc rim that may control cGMP turnover and possibly other light-dependent processes. Because there are no similar GARPs in cones, we propose that GARPs may prevent unnecessary cGMP turnover during daylight, when rods are held in saturation by the relatively high light levels.     

Circadian rhythm and light regulate opsin mRNA in rod photoreceptors

Disk membranes in the outer segment of rod photoreceptors are continuously renewed, being assembled at the outer segment base, displaced outward by new disks and eventually shed at the tip. In lower vertebrates, disk assembly occurs with a diurnal rhythm with 2-4% of the outer segment length produced daily. We have discovered that in toad and fish retinas the level of mRNA for opsin, the most abundant protein in rod disks, fluctuates with a daily rhythm and is regulated both by light and by a circadian oscillator. The mRNA level rises before light onset, remains high during the light phase of a diurnal cycle and decreases four to tenfold during the dark phase. In constant darkness, mRNA elevation occurs during subjective daytime. At night, rod opsin mRNA can be elevated by exposure to light.     

Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue

Specialized oxygen-sensing cells in the nervous system generate rapid behavioural responses to oxygen. We show here that the nematode Caenorhabditis elegans exhibits a strong behavioural preference for 5-12% oxygen, avoiding higher and lower oxygen levels. 3',5'-cyclic guanosine monophosphate (cGMP) is a common second messenger in sensory transduction and is implicated in oxygen sensation. Avoidance of high oxygen levels by C. elegans requires the sensory cGMP-gated channel tax-2/tax-4 and a specific soluble guanylate cyclase homologue, gcy-35. The GCY-35 haem domain binds molecular oxygen, unlike the haem domains of classical nitric-oxide-regulated guanylate cyclases. GCY-35 and TAX-4 mediate oxygen sensation in four sensory neurons that control a naturally polymorphic social feeding behaviour in C. elegans. Social feeding and related behaviours occur only when oxygen exceeds C. elegans' preferred level, and require gcy-35 activity. Our results suggest that GCY-35 is regulated by molecular oxygen, and that social feeding can be a behavioural strategy for responding to hyperoxic environments.     

Cyclic GMP-sensitive conductance in outer segment membrane of catfish cones

A cyclic GMP-sensitive conductance has recently been observed with patch-clamp recording in excised inside-out patches of plasma membrane from frog and toad rod outer segments. This conductance has properties suggesting that it is probably the light-sensitive conductance involved in visual transduction. We now report a similar conductance in the outer segment membrane of catfish cones. Cyclic GMP showed positive cooperativity in opening this conductance, with a Hill coefficient of 1.6-3.0 and a half-saturating cGMP concentration of 35-70 microM. Cyclic AMP at 1 mM, or changing Ca concentration (in the presence of Mg), had little effect on the conductance. In physiological solutions the cGMP-induced current had a reversal potential near +10 mV; the current amplitude increased roughly exponentially with membrane potential in both depolarizing and hyperpolarizing directions. Our results suggest that cGMP is also the internal transmitter for phototransduction in cones.     

A cyclic nucleotide-gated conductance in olfactory receptor cilia

Olfactory transduction is thought to be initiated by the binding of odorants to specific receptor proteins in the cilia of olfactory receptor cells. The mechanism by which odorant binding could initiate membrane depolarization is unknown, but the recent discovery of an odorant-stimulated adenylate cyclase in purified olfactory cilia suggests that cyclic AMP may serve as an intracellular messenger for olfactory transduction. If so, then there might be a conductance in the ciliary plasma membrane which is controlled by cAMP. Here we report that excised patches of ciliary plasma membrane, obtained from dissociated receptor cells, contain a conductance which is gated directly by cAMP. This conductance resembles the cyclic GMP-gated conductance that mediates phototransduction in rod and cone outer segments, but differs in that it is activated by both cAMP and cGMP. Our data provide a mechanistic basis by which an odorant-stimulated increase in cyclic nucleotide concentration could lead to an increase in membrane conductance and therefore, to membrane depolarization. These data suggest a remarkable similarity between the mechanisms of olfactory and visual transduction and indicate considerable conservation of sensory transduction mechanisms.     

Icariin on relaxation effect of corpus cavernosum smooth muscle

NO-cGMP pathway in penile corpus cavernosal smooth muscle plays an important role in penile erection. The level of cGMP is regulated by a balance between the rate of synthesis by guanylate cyclase and the rate of hydrolytic breakdown to guanosine 5′ monophosphate (GMP) by phosphodiesterase 5(PDE5). Icariin is isolated from natural drug Epimedii herba, it is shown to have the relaxation effect on corpus cavernosal smooth muscle of rabbit (IC50: 4×10⁻⁴ mol/L), and the mechanism of the relaxation effect of Icariin on corpus cavernosum believed to have the inhibiting effect on PDE5 and activation of NO-cGMP pathway to enhancing penile erection.     

Light-induced reduction of cytoplasmic free calcium in retinal rod outer segment

The response of retinal rod photoreceptors to light consists of a membrane hyperpolarization resulting from the decrease of a light-sensitive conductance in the outer segment. According to the calcium hypothesis, this conductance is blocked by a rise in intracellular free Ca triggered by light, a notion supported by the findings that an induced rise in internal Ca leads to blockage of the light-sensitive conductance and that light triggers a net Ca efflux from the outer segment via a Na-Ca exchanger, suggesting a rise in internal free Ca in the light. We have now measured both Ca influx and efflux through the outer segment plasma membrane and find that, contrary to the calcium hypothesis, light seems to decrease rather than increase the free Ca concentration in the rod outer segment. This result implies that Ca does not mediate visual excitation but it probably has a role in light adaptation.     

A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor

Atrial natriuretic peptide (ANP) is a polypeptide hormone whose effects include the induction of diuresis, natriuresis and vasorelaxation. One of the earliest events following binding of ANP to receptors on target cells is an increase in cyclic GMP concentration, indicating that this nucleotide might act as a mediator of the physiological effects of the hormone. Guanylate cyclase exists in at least two different molecular forms: a soluble haem-containing enzyme consisting of two subunits and a non-haem-containing transmembrane protein having a single subunit. It is the membrane form of guanylate cyclase that is activated following binding of ANP to target cells. We report here the isolation, sequence and expression of a complementary DNA clone encoding the membrane form of guanylate cyclase from rat brain. Transfection of this cDNA into cultured mammalian cells results in expression of guanylate cyclase activity and ANP-binding activity. The ANP receptor/guanylate cyclase represents a new class of mammalian cell-surface receptors which contain an extracellular ligand-binding domain and an intracellular guanylate cyclase catalytic domain.     

Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain

In the vascular system, endothelium-derived relaxing factor (EDRF) is the name of the local hormone released from endothelial cells in response to vasodilators such as acetylcholine, bradykinin and histamine. It diffuses into underlying smooth muscle where it causes relaxation by activating guanylate cyclase, so producing a rise in cyclic GMP levels. It has been known for many years that in the central nervous system (CNS) the excitatory neurotransmitter glutamate can elicit large increases in cGMP levels, particularly in the cerebellum where the turnover rate of cGMP is low. Recent evidence indicates that cell-cell interactions are involved in this response. We report here that by acting on NMDA (N-methyl-D-aspartate) receptors on cerebellar cells, glutamate induces the release of a diffusible messenger with strikingly similar properties to EDRF. This messenger is released in a Ca2+-dependent manner and its activity accounts for the cGMP responses that take place following NMDA receptor activation. In the CNS, EDRF may link activation of postsynaptic NMDA receptors to functional modifications in neighbouring presynaptic terminals and glial cells.     

Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases

Alpha atrial natriuretic peptide (alpha-ANP) and brain natriuretic peptide are homologous polypeptide hormones involved in the regulation of fluid and electrolyte homeostasis. These two natriuretic peptides apparently share common receptors and stimulate the intracellular production of cyclic GMP as a second messenger. Molecular cloning has defined two types of natriuretic peptide receptors: the ANP-C receptor of relative molecular mass (Mr) 60-70,000 (60-70 K), which is not coupled to cGMP production and may function in the clearance of ANP and the ANP-A receptor of Mr 120-140 K, which is a membrane form of guanylate cyclase in which ligand binding to the extracellular domain activates the cytoplasmic domain of the enzyme. Here we report the cloning and expression of a second human natriuretic peptide-receptor guanylate cyclase, the ANP-B receptor. The ANP-B receptor is preferentially activated by porcine brain natriuretic peptide rather than human alpha-ANP, whereas the ANP-A receptor responds similarly to both natriuretic peptides. These observations may have important implications for our understanding of the central and peripheral control of cardiovascular homeostasis.     

Single cyclic GMP-activated channel activity in excised patches of rod outer segment membrane

The plasma membrane of retinal rod outer segments contains a cyclic GMP-activated conductance which appears to be the light-sensitive conductance involved in phototransduction. Recently, it has been found that this conductance is partially blocked by Mg2+ and Ca2+ at physiological concentrations, thus possibly accounting for the absence of observable single-channel activity in excised membrane patches and for the unusually small apparent unit conductance deduced from noise measurements on intact cells. We now report that, as expected from this idea, single cGMP-activated channel activity can be detected from an excised rod membrane patch in the absence of divalent cations. The most prominent unitary current had a mean conductance of approximately 25 pS. Both individual channel openings (mean open time approximately 1 ms) and short bursts of openings (mean burst duration of about a few milliseconds) were observed. In addition, there were smaller events which probably represented other states of the conductance. The mean current increased with the third power of cGMP concentration, suggesting that there are at least three cGMP-binding sites on the channel molecule. With 0.2 mM Mg2+ in the cGMP-containing solution, a flickering block of the open channel was observed; the effect of Ca2+ was similar. The results resolve a puzzle about the light-sensitive conductance by demonstrating that it is an aqueous pore rather than a carrier.     

Cyclic GMP is involved in the excitation of invertebrate photoreceptors

The hyperpolarizing receptor potential in vertebrate rod photoreceptors appears to be mediated by the second messenger, cyclic GMP. Injection of cGMP into rods or application of cGMP to inside-out membrane patches activates a conductance resembling that produced by light. Light produces a rapid reduction of cGMP in living rods, leading to closure of sodium channels and membrane hyperpolarization. In most invertebrate photoreceptors the response to light is depolarizing. We have investigated whether cGMP is involved in controlling the increase in sodium conductance that underlies this depolarization. We show here that injection of cGMP into Limulus photoreceptors produces a depolarization that mimics the receptor potential. We also show that the cGMP concentration of the squid retina increases rapidly during exposure to light. These results support the hypothesis that cGMP mediates the light-induced depolarization in invertebrate photoreceptors and suggests that vertebrate and invertebrate phototransduction may be more similar than previously thought.     

Membrane guanylate cyclase is a cell-surface receptor with homology to protein kinases

Guanylate cyclase has been strongly implicated as a cell-surface receptor on spermatozoa for a chemotactic peptide, and on various other cells as a receptor for atrial natriuretic peptides. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), the chemotactic peptide released by sea urchin Arbacia punctulata eggs, is specifically crosslinked to A. punctulata spermatozoan guanylate cyclase. After the binding of the peptide the state of guanylate cyclase phosphorylation modulates enzyme activity. We report here that the deduced amino-acid sequence of the spermatozoan membrane form of guanylate cyclase predicts an intrinsic membrane protein of 986 amino acids with an amino-terminal signal sequence. A single transmembrane domain separates the protein into putative extracellular and cytoplasmic-catalytic domains. The cytoplasmic carboxyl-terminal 95 amino acids contain 20% serine, the likely regulatory sites for phosphorylation. Unexpectedly, the enzyme is homologous to the protein kinase family.