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.     

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.     

Photoreceptor light adaptation is mediated by cytoplasmic calcium concentration

The vertebrate visual system can operate over a large range of light intensities. This is possible in part because the sensitivity of photoreceptors decreases approximately in inverse proportion to the background light intensity. This process, called photoreceptor light adaptation, is known to be mediated by a diffusible intracellular messenger, but the identity of the messenger is still unclear. There has been considerable speculation that decreased cytoplasmic Ca2+ concentration (Cai2+) may play a role in light adaptation, and recent experiments in which Ca2+ buffer was incorporated into rod-cells have supported this notion. The extent of the contribution of calcium, however, remains unresolved. We now show that light-dependent changes in sensitivity in amphibian photoreceptors can be abolished by preventing movements of Ca2+ across the outer-segment plasma membrane. These experiments demonstrate that light adaptation in photoreceptors is mediated in cones primarily, and in rods perhaps exclusively, by changes in Cai2+.     

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.     

Cyclic GMP increases photocurrent and light sensitivity of retinal cones

Like retinal rods, cone photoreceptors contain cyclic GMP and light-activated phosphodiesterase. The cGMP phosphodiesterase cascade is thought to mediate phototransduction in rods. Biochemical assays of nucleotide content in cone-dominant retinas, however, have failed to demonstrate light-induced changes in cGMP. Changes in cyclic AMP following light exposure have been reported, leading to the suggestion that in cone phototransduction cAMP assumes a role analogous to that played by cGMP in rods. Cyclic GMP introduced from tight-seal pipettes into isolated cones of the larval tiger salamander, Ambystoma tigrinum, rapidly increases light-modulated membrane current more than 10-fold. In cones, as in rods, cGMP also causes an approximately 10-fold increase in photocurrent duration and a 5- to 10-fold increase in light-sensitivity. Cyclic AMP has no effect on cone photocurrents under the same conditions. Because cGMP has similar effects on photocurrent magnitude and kinetics in both rods and cones, we conclude that cGMP plays corresponding roles in transduction in both vertebrate photoreceptor classes.     

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.     

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.     

Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning

Signalling by intracellular second messengers such as cyclic nucleotides and Ca2+ is known to regulate attractive and repulsive guidance of axons by extracellular factors. However, the mechanism of interaction among these second messengers in determining the polarity of the guidance response is largely unknown. Here, we report that the ratio of cyclic AMP to cyclic GMP activities sets the polarity of netrin-1-induced axon guidance: high ratios favour attraction, whereas low ratios favour repulsion. Whole-cell recordings of Ca2+ currents at Xenopus spinal neuron growth cones indicate that cyclic nucleotide signalling directly modulates the activity of L-type Ca2+ channels (LCCs) in axonal growth cones. Furthermore, cGMP signalling activated by an arachidonate 12-lipoxygenase metabolite suppresses LCC activity triggered by netrin-1, and is required for growth-cone repulsion mediated by the DCC-UNC5 receptor complex. By linking cAMP and cGMP signalling and modulation of Ca2+ channel activity in growth cones, these findings delineate an early membrane-associated event responsible for signal transduction during bi-directional axon guidance.     

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.     

Electrogenic Na-Ca exchange in retinal rod outer segment

Previous work has suggested that a Na-Ca exchanger may have a key role in visual transduction in retinal rods. This exchanger is thought to maintain a low internal free Ca2+ concentration in darkness and to contribute to the rod's recovery after light by removing any internally released Ca2+. Little else is known about this transport mechanism in rods. We describe here an inward membrane current recorded from single isolated rods which appears to be associated with such external Na+-dependent Ca2+ efflux activity. External Na+, but not Li+, could generate this current; high external K+ inhibited it while small amounts of La3+ (10 microM) completely abolished it. The exchanger can also transport Sr2+, but not Ba2+ or other divalent cations. The exchange ratio was estimated to be 3Na+:1Ca2+. As well as demonstrating clearly the Na-Ca exchanger in the rod outer segment, our experiments also cast serious doubt on the commonly held view that light simply releases internal Ca2+ to bind to and block the light-sensitive conductance.     

Differences in the kinetics of rod and cone synaptic transmission

Photoreceptors of the vertebrate retina hyperpolarize in response to light. The hyperpolarization elicited by a brief flash is approximately ten times slower in rods than in cones of the same retina. We have examined the amplification and temporal properties of synaptic transfer of rod and cone signals to a common postsynaptic element, the horizontal cell. We find that the kinetics of signal transfer at these chemical synapses parallels the speed of the light-evoked signals themselves.     

Cyclic nucleotides control a system which regulates Ca2+ sensitivity of platelet secretion

Cellular responses to extracellular signals are mediated by changes in the intracellular concentrations of one or more second messengers. In platelets, inhibitory agonists increase intracellular cyclic-3',5'-AMP [( cyclic AMP]i (refs 2, 3] whereas excitatory agonists increase [Ca2+]i and/or [1,2-diacylglycerol]i (refs 4-9), and in some cases decrease [cyclic AMP]i (refs 10, 11). Both activation and inhibition of platelet responses have been attributed to an increase in [cyclic-3',5'-GMP]i (refs 8, 12). The activity of protein kinase C, which is associated with the platelet secretory response, is increased by both 1,2-diacylglycerol and Ca2+ (refs 4, 7, 8). The role of cyclic AMP may involve either inhibition of Ca2+ mobilization to the cytosol or stimulation of intracellular Ca2+ uptake, and in addition inhibition of 1,2-diacylglycerol formation. The relationship between cyclic-3',5'-GMP (cyclic GMP) and other second messengers in platelet activation has not been defined. Using platelets made permeable by exposure to an intense electric field, we demonstrate here modulation of the Ca2+ sensitivity of platelet secretion by thrombin, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleyl-2- acetylglycerol ( OAG ), both potent activators of protein kinase C. The effect of thrombin is selectively modified by cyclic GMP and cyclic AMP. The response to OAG and TPA is also modulated by cyclic AMP but to a much lesser extent.     

cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin-induced Ca2+ current increase in snail neurones

Protein phosphorylation catalysed by cyclic AMP-dependent, Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases is important both in the modulation of synaptic transmission and in the regulation of neuronal membrane permeability (for reviews see refs 5-7). However, there has previously been no evidence for the involvement of cyclic GMP-dependent protein kinase (cGMP-PK) in the regulation of neuronal function. Serotonin induces an increase of Ca2+ current in a group of identified ventral neurones of the snail Helix aspersa. This effect is probably mediated by cGMP because it is mimicked by the intracellular injection of cGMP or the application of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase. We have now found that the effect of either serotonin or zaprinast on the Ca2+ current is potentiated by the intracellular injection of cGMP-PK. Moreover, the intracellular injection of activated cGMP-PK (cGMP-PK + 1 microM cGMP) greatly enhances the Ca2+ current of the identified ventral neurones seen in the absence of serotonin. These results indicate that cGMP-PK has a physiological role in the control of the membrane permeability of these neurones.     

Signal clipping by the rod output synapse

The properties of synapses between retinal neurons make an essential contribution to early visual processing. Light produces a graded hyperpolarization in photoreceptors, up to 25 mV in amplitude, and it is conventionally assumed that all of this response range is available for coding visual information. We report here, however, that the rod output synapse rectifies strongly, so that only potential changes within 5 mV of the rod dark potential are transmitted effectively to postsynaptic horizontal cells. This finding is consistent with the voltage-dependence of the calcium current presumed to control neurotransmitter release from rods. It suggests functional roles for the strong electrical coupling of adjacent rods and the weak electrical coupling of adjacent rods and cones. The existence of photoreceptor coupling resolves the apparent paradox that rods have a 25 mV response range, while signals greater than 5 mV in amplitude are clipped during synaptic transmission. We predict that the strengths of rod-rod and rod-cone coupling are quantitatively linked to the relationship between the rod response range and the synapse operating range.     

Role of visual pigment properties in rod and cone phototransduction

Retinal rods and cones share a phototransduction pathway involving cyclic GMP. Cones are typically 100 times less photosensitive than rods and their response kinetics are several times faster, but the underlying mechanisms remain largely unknown. Almost all proteins involved in phototransduction have distinct rod and cone variants. Differences in properties between rod and cone pigments have been described, such as a 10-fold shorter lifetime of the meta-II state (active conformation) of cone pigment and its higher rate of spontaneous isomerization, but their contributions to the functional differences between rods and cones remain speculative. We have addressed this question by expressing human or salamander red cone pigment in Xenopus rods, and human rod pigment in Xenopus cones. Here we show that rod and cone pigments when present in the same cell produce light responses with identical amplification and kinetics, thereby ruling out any difference in their signalling properties. However, red cone pigment isomerizes spontaneously 10,000 times more frequently than rod pigment. This high spontaneous activity adapts the native cones even in darkness, making them less sensitive and kinetically faster than rods. Nevertheless, additional factors are probably involved in these differences.     

Augmentation of cardiac calcium current by flash photolysis of intracellular caged-Ca2+ molecules

The entry of calcium ions into cells through voltage-activated Ca2+ channels in the plasma membrane triggers many important cellular processes. The activity of these channels is regulated by several hormones and neurotransmitters, as well as intracellular messengers such as Ca2+ itself (for examples, see refs 1-9). In cardiac muscle, myoplasmic Ca2+ has been proposed to potentiate Ca2+ influx, although a direct effect of Ca2+ on these channels has not yet been demonstrated. Photosensitive 'caged-Ca2+' molecules such as nitr-5, however, provide powerful tools for investigating possible regulatory roles of Ca2+ on the functioning of Ca2+ channels. Because its affinity for Ca2+ is reduced by irradiation, nitr-5 can be loaded into cells and induced to release Ca2+ with a flash of light. By using this technique we found that the elevation of intracellular Ca2+ concentration directly augmented Ca2+-channel currents in isolated cardiac muscle cells from both frog and guinea pig. The time course of the current potentiation was similar to that seen with beta-adrenergic stimulation. Thus Ca2+ may work through a similar pathway, involving phosphorylation of a regulatory Ca2+-channel protein. This mechanism is probably important for the accumulation of Ca2+ and the amplification of the contractile response in cardiac muscle, and may have a role in other excitable cells.     

Calcium signalling in the guidance of nerve growth by netrin-1

Pathfinding by growing axons in the developing nervous system is guided by diffusible or bound factors that attract or repel the axonal growth cone. The cytoplasmic signalling mechanisms that trigger the responses of the growth cone to guidance factors are mostly unknown. Previous studies have shown that the level and temporal patterns of cytoplasmic Ca2+ can regulate the rate of growth-cone extension in vitro and in vivo. Here we report that Ca2+ also mediates the turning behaviour of the growth cones of cultured Xenopus neurons that are induced by an extracellular gradient of netrin-1, an established diffusible guidance factor in vivo. The netrin-1-induced turning response depends on Ca2+ influx through plasma membrane Ca2+ channels, as well as Ca2+-induced Ca2+ release from cytoplasmic stores. Reduction of Ca2+ signals by blocking either of these two Ca2+ sources converted the netrin-1-induced response from attraction to repulsion. Activation of Ca2+-induced Ca2+ release from internal stores with a gradient of ryanodine in the absence of netrin-1 was sufficient to trigger either attractive or repulsive responses, depending on the ryanodine concentration used. These results support the model that cytoplasmic Ca2+ signals mediate growth-cone guidance by netrin-1, and different patterns of Ca2+ elevation trigger attractive and repulsive turning responses.     

Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes

Hydrolysis of membrane-associated phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)-P2) to water soluble inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is a common response by many different kinds of cells to a wide variety of external stimuli (see refs 1 and 2 for review). Ins (1,4,5)P3 is a putative second messenger which increases intracellular Ca2+ by mobilizing internal Ca2+ stores, a hypothesis which has been substantiated by studies with chemically permeabilized cells and with isolated microsomal membrane fractions. But the possibility that Ins(1,4,5)P3 could induce in intact cells an influx of external Ca2+ through transmembrane channels, originally hypothesized by Michell in 1975, has never been directly tested. We report here single-channel recordings of an Ins(1,4,5)P3-activated conductance in excised patches of T-lymphocyte plasma membrane. The Ins(1,4,5)P3-activated transmembrane channel appears to be identical to the recently described mitogen-regulated, voltage-insensitive Ca2+ permeable channel involved in T-cell activation. We suggest that Ins(1,4,5)P3 acts as the second messenger mediating transmembrane Ca2+ influx through specific Ca2+-permeable channels in mitogen-stimulated T-cell activation.     

Retinal light adaptation--evidence for a feedback mechanism

Light adaptation is the adjustment of retinal response properties to variations in ambient illumination. It enables the encoding of visual information over a millionfold intensity range, from moonlight to broad daylight, despite the relatively small dynamic range of response of visual neurones. We have studied the effects of light adaptation on the dynamics and sensitivity of visual responses of neurones in the turtle retina, by measuring the responses of horizontal cells in the retina to light which was modulated with a sinusoidal time course around various mean levels. As a quantitative measure of the transduction from light to neural signals, we calculated the gain of response at each frequency. Gain is defined as the amplitude of the modulated response component divided by the amplitude of light modulation. We report here that the gain (mV photon-1) at low temporal frequencies decreased as the mean light level increased. Over a 2 log-unit range of mean light levels, low-frequency gain was inversely proportional to the mean light level, as in Weber's law. However, at high temporal frequencies, the gain was almost independent of mean light level. Our results are reminiscent of Kelly's results on human temporal-frequency sensitivity in various states of light adaptation. We found that a family of horizontal-cell temporal frequency responses, measured at various mean light levels, could be accounted for by a negative feedback model in which the feedback strength is proportional to mean light level.     

Suppression by glutamate of cGMP-activated conductance in retinal bipolar cells.

Depolarizing bipolar cells (DBCs) of the retina are the only neurons in the vertebrate central nervous system known to be hyperpolarized by the neurotransmitter glutamate. Both glutamate and its analogue L-2-amino-4-phosphonobutyrate (APB) hyperpolarize DBCs by decreasing membrane conductance. Furthermore, glutamate responses in DBCs slowly decrease during whole-cell recording, suggesting that the response involves a second messenger system. Here we report that intracellular cyclic GMP or GTP activates a membrane conductance that is suppressed by APB, resulting in an enhanced APB response. In the presence of GTP-gamma-S, APB causes an irreversible suppression of the conductance. Inhibitors of G-protein activation or phosphodiesterase activity decrease the APB response. Thus, the DBC glutamate receptor seems to close ion channels by increasing the rate of cGMP hydrolysis by a G protein-mediated process that is strikingly similar to light transduction in photoreceptors.