Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

Similar to other members of this family, GCAPs have four EF hands, three of which actually bind divalent cations. Strictly speaking, GCAPs are Ca2+–Mg2+-binding proteins. The word activating in their name is a bit misleading: Mg2+- liganded GCAPs activate retGCs, whereas Ca2+-liganded forms actually inhibit cGMP synthesis. Thus, in darkadapted rods with high free-Ca2+ concentrations (estimates range from 250 to 600 nM in different species), GCAPs keep the retGC activity at low level. This makes perfect sense, because high Ca2+ indicates that there is enough free cGMP ( 2–5 mM) to keep the channels open. Light-induced decrease of intracellular Ca2+ (to 5–50 nM, based on different estimates) is the direct result of channel closure, reflecting reduced cGMP in need of replenishing. The loss of bound Ca2+ and its replacement by Mg2+ (which is always1 mM in the cytoplasm) switches GCAPs from the inhibitory to the activating mode exactly when rapid cGMP synthesis is necessary to restore its level. Increasing cGMP opens more channels, thereby gradually restoring Ca2+. Rising Ca2+ displaces Mg2+ on GCAPs, progressively reducing retGC activity, so that the cell returns to the initial state. After a dim flash, this process often overshoots, leading to a transient increase in the cGMP and Ca2+ concentration, likely because PDE is inactivated faster than retGC. The absence of GCAPs slows down cGMP resynthesis, so that light-induced PDE activity results in a more profound decrease of cGMP than in the normal rod. This results in closure of more channels and greatly increases the amplitude of single-photon response. This compromises temporal resolution, prolonging the rising and falling phase of the light response, and limits the working range of rods to lower light levels.

Interestingly, vertebrate photoreceptors express two isoforms of retGC, retGC1 and retGC2, and at least two GCAPs, GCAP1 and GCAP2. The presence of two isoforms of each protein in rods of all vertebrates, including fish, clearly indicates that the different isoforms have nonredundant functions. RetGCs are membrane proteins, suggesting that retGC1 and retGC2 may be localized to different membranes within the OS. RetGC1 was reliably detected in disks, and the possibility that a fraction may also be present in the plasma membrane remains open. The localization of RetGC2 was not studied with sufficient spatial resolution. Since retGC is a dimer, two isoforms of RetGC could give rise to three types of dimers, two homoand one heterodimer. The fact that each subunit interacts with either GCAP1 or GCAP2 further expands the number of combinatorial possibilities. Definitive experiments, such as knockouts of individual isoforms of either protein, singly and in different combinations, are needed to fully elucidate the biochemistry of the Ca2+ feedback mechanism. A recent study of GCAP2 knockout mice revealed that although each GCAP is responsible for about half of the total retGC activation, the functions of the two proteins are quite

distinct. Due to lower affinity for Ca2+, GCAP1 switches to the activation mode as soon as the concentration of Ca2+ begins to fall, whereas GCAP2 responds later, when Ca2+ levels drop further. Thus, together the two GCAPs ensure graded increase in retGC activity in a wider range of Ca2+ concentrations than either one could have covered alone.

Another issue in need of clarification is the physiological role of a remarkable buffering capacity of the OS cytoplasm for both second messengers. According to current estimates, total cGMP in the OS is as high as50 mM, with only 2–5 mM of it being free and the rest bound to the noncatalytic sites on PDE a- and b-subunits. The polycationic region of PDEg appears to stabilize the interaction of cGMP with these sites. Reciprocally, the presence of cGMP in noncatalytic sites enhances the interaction of the a- and b-subunits with PDEg. This mechanism implies that PDE activation by Td would release cGMP from noncatalytic sites. The role of this event in the photoresponse remains unclear. Similarly, free Ca2+ represents only a small fraction of the total Ca2+ in the OS cytoplasm, the rest being bound to several abundant proteins, such as recoverin, GCAPs, and calmodulin. Ca2+ binding by these proteins is a two-way street: on the one hand, it critically regulates their function, on the other hand, by soaking up Ca2+, they significantly change its concentration, modulating the input they respond to. Obviously, Ca2+ and cGMP buffering cannot be separated by purely experimental means from other functional modalities of the proteins involved. Therefore, rigorous experimentation must be supplemented with detailed biochemically realistic mathematical modeling to distinguish between the effects of binding on protein activity and on the concentration of free second messenger in the cytoplasm, which is necessary to elucidate the exact biological roles of both.

Light-Dependent Protein Translocation

and Rod Signaling

Arrestin localization to the OS in the light and to the IS in the dark was first described in 1985, before the role of this protein in signal termination was established. The subsequent discovery that Td also translocates in a light-dependent fashion, moving in the opposite direction, suggested an idea that translocation may underlie well-known adaptation of rods to different light levels. Preferential localization in the dark-adapted rod of a signal transducer to the rhodopsin-rich OS and a signal terminator to the IS could increase light sensitivity by slowing down shutoff. Conversely, the removal of Td from the OS and accumulation of arrestin in this compartment in the light could significantly reduce it by decreasing the number of Td molecules activated by

Rh* and speeding up rhodopsin inactivation. Subsequent studies showed that the fraction of recoverin, which presumably slows down rhodopsin phosphorylation by keeping RK away from Rh*, in the OS decreases dramatically from 12% in the dark to less than 2% in the light (the bulk of recoverin localizes to the IS in both conditions). The amplification constant in light-adapted rods was found to decrease 10-fold, in line with the reduction of Td concentration in the OS. While these results support the idea that Td translocation plays a role in rod adaptation, by fully explaining the changes in sensitivity by Td movement alone, they effectively rule out any significant contribution of the translocation of other proteins. The recent finding that phosducin, which interacts with free bg-dimer released upon Td activation and demonstrates robust translocation from the OS in the light, does not contribute to rod adaptation, supports this notion. The movement of Td, arrestin, phosducin, and recoverin in both directions is a relatively slow process that takes many minutes, which does not seem adequate to explain much faster photoreceptor adaptation. The translocation of arrestin in both directions is energy independent. It is driven in the dark by its low-affinity interactions with microtubules, particularly abundant in IS, and in the light by its binding to light-activated forms of rhodopsin. These findings suggest that the translocation of arrestin and other proteins is more likely to play a role in rod survival during daytime than in relatively fast light/dark adaptation. However, the translocation of different proteins may have distinct functions, which to a large extent remain to be elucidated.

Why Rods Do Not Have an Action Potential

In most neurons, extracellular Ca2+ enters presynaptic terminals during an action potential. A brief increase in its local concentration triggers transient exocytosis of neuro- trasmitter-containing vesicles. In contrast, vertebrate rod photoreceptors work backward. In the dark, rods are partially depolarized (OS membrane potential is about –35 mV) due to massive influx of Na+ and Ca2+ ions through cGMPgated channels. This results in continuous release of the neurotransmitter (L-glutamate) from ribbon synapses. By virtue of closing cGMP-gated channels, light of increasing intensity induces progressive hyperpolarization of rods up to –60 mV (a change of 25 mV). The activation of a single rhodopsin changes the membrane potential by as much as 1 mV. Thus, light intensity is encoded in the extent of hyperpolarization, which determines the magnitude of the decrease of neurotransmitter release. This mechanism makes the signaling graded, in contrast to the all-or-nothing type in neurons with a conventional action potential. It

directly couples the change in membrane potential with synaptic activity, so that both the closure of the cGMPgated channels upon light stimulation and their reopening upon signal termination described above immediately translate into corresponding changes in neurotransmitter release. The absence of thresholds ensures that the information is not lost in transmission, so that the brain can take full advantage of the single-photon sensitivity of the rod photoreceptors. In addition, this mechanism creates a natural ceiling: a full stop of neurotransmitter release is the maximum possible effect of the illumination of any intensity.

Conclusions

In many respects, rod photoreceptors are virtually perfect light sensors that combine single-photon sensitivity with a surprisingly wide dynamic range. Exquisitely timed and extremely efficient inactivation at every step of the signaling cascade between light absorption by rhodopsin and changes in the membrane potential plays an important role in their function. Not surprisingly, molecular errors in this complex multistep inactivation mechanism due to mutations in key proteins underlie a variety of congenital visual disorders in humans, ranging in severity from night blindness to retinal degeneration.

See also: Light-Driven Translocation of Signaling Proteins in Vertebrate Photoreceptors; Phototransduction: Adaptation in Cones; Phototransduction: Adaptation in Rods; Phototransduction: Inactivation in Cones; Phototransduction: Phototransduction in Cones; Phototransduction: Phototransduction in Rods; Phototransduction: Rhodopsin.

Further Reading

Burns, M. E. and Arshavsky, V. Y. (2005). Beyond counting photons: Trials and trends in vertebrate visual transduction. Neuron 48: 387–401.

Cote, R. H. (2006). Photoreceptor phosphodiesterase (PDE6):

A G-protein-activated PDE regulating visual excitation in rod and cone photoreceptor cells. In: Beavo, J. A., Francis, S. H., and Houslay, M. D. (eds.) Cyclic Nucleotide Phosphodiesterases in Health and Disease, pp. 165–193. Boca Raton, FL: CRC Press.

Dizhoor, A. M., Olshevskaya, E. V., and Peshenko, I. V. (2006). Calcium sensitivity of photoreceptor guanylyl cyclase (RetGC) and congenital photoreceptor degeneration: Modeling in vitro and in vivo. In:

Philippov, P. P. and Koch, K.-W. (eds.) Neuronal Calcium Sensor Proteins, pp. 203–219. New York: Nova Science Publishers, Inc.

Gurevich, V. V. and Gurevich, E. V. (2004). The molecular acrobatics of arrestin activation. Trends in Pharmacological Science 25: 105–111.

Gurevich, V. V., Hanson, S. M., Gurevich, E. V., and Vishnivetskiy, S. A. (2007). How rod arrestin achieved perfection: Regulation of its availability and binding selectivity. In: Kisselev, O. and Fliesler, S. J. (eds.) Signal Transduction in the Retina, Methods in Signal Transduction Series, pp. 55–88. Boca Raton, FL: CRC Press.

Glossary

Confocal fluorescence microscopy – An optical microscopy method that uses a focused laser spot to measure fluorescence within an extremely small volume.

Cytosol – The liquid portion of a cell’s contents, or cytoplasm.

Depolarization – A positive-going change in the membrane potential of a cell.

Diacylglycerol (DAG) – A glyceride consisting of two fatty acid chains covalently bound to a glycerol molecule through ester linkages.

d-myo-Inositol (1,4,5) trisphosphate (IP3) – An inositol derivative having three phosphate groups covalently bound to the inositol ring.

Guanine-nucleotide-binding protein (G protein) –

A family of signaling proteins that are activated by the exchange of guanine triphosphate for guanine diphosphate bound to the protein.

On-cell patch clamp – An electrophysiological recording technique that uses a polished glass micropipette to measure currents flowing through a small patch of cellular plasma membrane containing ion channels.

Phosphoinositide – A phospholipid containing a polar inositol headgroup.

Smooth endoplasmic reticulum (SER) – A network of membrane sacs found in animal cells that, among other things, functions as a store of calcium ions.

Transient receptor potential (TRP) channels –

A widespread family of ion channels. The lightactivated channels of Drosophila are founding members of the family.

Arrangement of Eyes in Limulus

American horseshoe crabs (Limulus polyphemus) have 10 eyes. They have two large lateral compound eyes, each containing about 1000 clusters of photoreceptors or ommatidia. A small lens within each ommatidium focuses light from a small patch of visual space onto each photoreceptor cluster, which transmits information about local changes in light intensity to the brain through a nerve fiber. There are five additional eyes on the top side of its first (anterior) major

body section, two median eyes, one endoparietal eye, and two rudimentary lateral eyes. Two ventral eyes are located on the underside of the animal above the mouth (Figure 1). Photoreceptors located on the telson (tail) constitute the 10th eye. Of these eyes, the lateral compound, median ocellar and ventral eyes have been extensively studied. All have microvillar photoreceptors, in which the visual pigment, rhodopsin, is embedded in the membrane of fingerlike projections of the plasma membrane called microvilli (Figure 2). Each eye has allowed for a study of different aspects of invertebrate vision. The large compound eyes have been favorite preparations for the study of image processing by compound eyes, the biochemistry of visual transduction, and the circadian control of visual sensitivity. The medial eye has been studied for its sensitivity to ultraviolet (UV) light which has allowed the elucidation of the physiological consequences of the reversible photoisomerization of invertebrate rhodopsin. Lastly, the ventral eyes have played a role in the understanding of phototransduction in invertebrate photoreceptors.

The Microvillus is the Cellular Structure Mediating Visual Transduction

Central to the performance of each photoreceptor cell is the rhabdomere – an array of photoreceptive microvilli positioned so as to maximally absorb light entering the eye. Each microvillus is a cylindrical outgrowth of the plasma membrane, 50–80 nm in diameter and 0.5–2 mm in length (Figure 2). Electron micrographs often show an axial filament within each microvillus. The axial filament contains a bundle of actin filaments with their þ ends directed toward the tip of the microvillus. The actin filaments appear to extend through the bottom of the microvillus into the cytoplasm, through fenestrations in submicrovillar cisternae (SMC) of smooth endoplasmic reticulum (SER). The actin cytoskeleton is responsible for the presence of an unconventional motor protein, myosin III, in the microvilli of Limulus lateral eye photoreceptors. The membrane of a typical microvillus contains 1000 or more particles, which are presumed to be mostly molecules of the visual pigment, rhodopsin, which absorbs light and initiates the physiological response of the photoreceptor. A photoreceptor might possess 105 microvilli, resulting in a total rhodopsin content of 108 molecules, comparable to that of vertebrate retinal photoreceptors.

0.5 μm

Figure 2 Electron micrograph of a section through the R-lobe of a ventral photoreceptor, showing the microvilli (MV) and submicrovillar cisternae of smooth endoplasmic reticulum (SER; arrow). Adapted from Dabdoub, A., Payne, R., and Jinks,

R. N. (2002). Journal of Comparative Neurology 442: 217–225. Copyright 2009 Wiley-Liss, Inc.

Studies of Visual Transduction Using

Limulus Ventral Photoreceptors

The Limulus ventral photoreceptor (Figure 3) is a highly polarized cell divided into two lobes, analogous to the inner and outer segments of vertebrate retinal photoreceptors. The rhabdomeral (R) lobe bears microvilli on its plasma membrane and is therefore light sensitive. The light-insensitive arhabdomeral (A) lobe contains the cell’s

nucleus. An axon projects from the A lobe toward the animal’s central nervous system. Ventral photoreceptors were originally chosen as a model for invertebrate phototransduction because of their large size (>200 mm). This facilitates insertion of multiple electrodes and makes it possible to clamp the membrane potential of the cells, measure electrical current flow across the plasma membrane, and inject compounds of interest into the cytoplasm (Figure 4).

The essential electrical response to illumination is the activation of a very large flow of current into the cell, carried mostly by sodium ions (Figure 4). The result is a depolarization (positive-going change) of the cell membrane which is graded with light intensity, of up to 60 mV. The reversal potential of the light-sensitive current is between þ10 mV and þ20 mV and its dependence on extracellular ion concentrations indicates that this conductance is sodiumand potassium-, but not Ca2+-per- meable. This is in contrast to the light-activated transient receptor potential (TRP) channel conductance in the photoreceptors of the fruit fly, Drosophila, which is highly Ca2+ permeable.

The study of ventral photoreceptors has revealed that they have remarkable performance characteristics, most notably the very large amplification of the transduction process. Amplification refers to the amount of charge that is carried across the plasma membrane as a result of excitation by a single photon. In ventral photoreceptors, this gain can be directly measured because the single-photon response, termed a quantum bump, is easily recorded using glass micropipettes. In response to very dim illumination,

quantum bumps over 10 mV in amplitude occur randomly as individual photons are effectively absorbed by rhodopsin molecules. Under voltage clamp, the peak current across the membrane generated by an effectively absorbed photon can exceed 1 nA and appears to be generated over several square microns of membrane surface, containing hundreds of microvilli. The comparatively large currents flowing indicate that quantum bumps are caused by the passage across the plasma membrane of hundreds of millions of cations through ion channels. By contrast, in the smaller Drosophila photoreceptors or amphibian rods, a singlephoton event involves a maximum current of less than 10 pA. Limulus photoreceptors achieve this large amplification in only 100–200 ms, faster than amphibian rods. A further remarkable feature of Limulus photoreceptors is their broad dynamic range. Whereas most vertebrate rods

work over about a 4-log-unit range of light intensity before saturating, Limulus photoreceptors work over 8. They achieve this range through a strong adaptation process that reduces amplification at high light intensity. The large dynamic range of these cells obviates the need for the dual system of photoreceptors (rods and cones) used to achieve a large dynamic range in the vertebrate eye.

The Light-Sensitive Conductance

Consists of the Summed Effect of

Conventional Ion Channels

The glial cells that surround individual ventral photoreceptors can be removed, exposing the plasma membrane surface. This preparation allows On-cell patch-clamp

recording of single ion channels in the microvillar membrane. As expected, channels whose opening was triggered by light were recorded by this method, with singlechannel conductance ranging from 18 to 50 pS. Identification of these channels as the light-activated conductance requires establishing a close correspondence between the properties of individual channel currents and those of photocurrents recorded from the whole cell. Depolarizing potentials applied in the dark did not open the channels; therefore the light-activated opening was not just a secondary consequence of the depolarizing receptor potential. Light-evoked single-channel activity was graded with light intensity, reduced by light adaptation, and the singlechannel currents reversed in the same membrane potential range as the macroscopic photocurrent. While these characteristics are consistent with the recorded channels being those that carry the light-activated current, definitive molecular or pharmacological proof is still needed.

The Response of the Ventral Photoreceptor is Mediated by the Phosphoinositide Cascade

A large body of evidence now demonstrates that the phosphoinositide (PI) pathway links the absorption of light to the activation of ion channels in the plasma membrane of invertebrate microvillar photoreceptors (Figure 5). The PI pathway is a mechanism for releasing intracellular messengers upon the activation of a receptor

protein, using inositol phospholipids as a substrate. In microvillar photoreceptor cells, the receptor protein is rhodopsin and the cascade is localized to the membrane of the microvilli that cover the plasma membrane of the light-sensitive R-lobe. Activated rhodopsin catalyzes the exchange of guanine triphosphate (GTP) for guanine diphosphate (GDP) bound to the alpha subunit of a heterotrimeric GTP-binding protein of the Gq subfamily (Gqa), which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5 bisphosphate (PIP2), a minor membrane phospholipid, into a lipid messenger, diacylglycerol (DAG), and the water-soluble messenger, d-myo-inositol 1,4,5 trisphosphate (IP3). In support of this biochemical pathway, light-activated PIP2 hydrolysis and/ or IP3 production has been reported in ventral photoreceptors of Limulus. Gqa has been amplified and sequenced from Limulus ventral eye tissue and immunolocalized to the rhabdomeral microvilli. Pharmacological agents that inhibit PLC, neomycin and U-73122, dramatically desensitize the light response of Limulus photoreceptors.

The PI Cascade Generates at Least Two

Intracellular Messenger Molecules

The PI cascade generates two messenger molecules with very different properties (Figure 5) DAG is essentially confined to the plasma membrane, while IP3 can diffuse into the surrounding cytoplasm. In addition, the decline of the precursor, PIP2 may act as an additional signal within

ER

TRPC?

Cytoplasm

the membrane. The difficult task of assessing the relative roles of these messengers in activating the light-sensitive ion channels has dominated research on the invertebrate phototransduction cascade.

Roles of IP3 and Intracellular Ca2+ Ions in

Excitation of Limulus Ventral

Photoreceptors

There is no evidence that IP3 can itself activate the lightsensitive channels of ventral photoreceptors. The bestcharacterized alternative target of IP3 is a Ca2+ channel, the IP3 receptor protein (IP3R), located in the membrane of endoplasmic reticulum (ER). IP3 therefore releases Ca2+ from intracellular stores within the ER. In Limulus photoreceptors, suitable calcium stores are located close to the base of the rhodopsin-containing microvilli – the subrhabdomeral cisternae (SMC) of SER. Extensions of the SMC are juxtaposed less than 100 nm from the bases of the microvilli. The cytoplasm of both the R lobe containing the SER and, to a lesser extent, the A lobe are immunoreactive when probed with an anti-IP3R antibody.

In darkness, ventral photoreceptors, like most cells, maintain a low free cytosolic Ca2+ concentration ([Ca2+]i) of 200–600 nM. Release of calcium from the SMC results in a very large elevation of [Ca2+]i during the first few hundred milliseconds of the light response. Confocal fluorescent light microscopy has enabled the measurement of the light-induced elevation of [Ca2+]i in ventral photoreceptors at spots within 4 mm of the microvillar membrane in the R lobe (Figures 6 and 7).

Following a very bright flash, [Ca2+]i begins to rise after a latent period of approximately 20 ms. Thereafter, [Ca2+]i rises at an initial rate of 1–2 mM s–1 to reach a peak of 100–200 mM within 200 ms. For less-intense flashes, the peak elevation of [Ca2+]i is graded with flash intensity, increasing from 2 mM to more than 140 mM as light intensity increases from 10 effective photons to 10 000 effective photons. For the dimmest flashes so far investigated, these concentrations represent 600 free Ca2+ ions per effective photon generated within the confocal measurement volume.

Very high levels of [Ca2+]i are reached only transiently upon illumination. Following a dim or moderate-intensity light flash, [Ca2+]i falls close to its resting value within 1 s, although a small lingering elevation may persist for up to a minute afterward. Even during sustained intense illumination, [Ca2+]i falls within 5 s to a sustained plateau elevation of less than 20 mM. As expected, if calcium release from stores occurs, the light-induced rise in [Ca2+]i is unaltered by removal and chelation of extracellular Ca2+, but is severely reduced by pharmacological agents expected to deplete Ca2+ stores.

IP3 Can Release Ca2+ from the SER

Microinjections of IP3, or photolysis of caged IP3, rapidly release Ca2+ from the R lobe in darkness (Figure 8). Photolysis of caged IP3 by UV light delivered to a spot beneath the microvillar membrane results in local elevations of Ca2+ that are comparable in magnitude and rate

Beam splitter

100 μM

1 s

Intense light

Figure 7 (a) Receptor potential (solid line) and reconstructed elevation of [Ca2+]i (red symbols) recorded following a flash from an attenuated laser beam that delivered 50 effective photons to a photoreceptor. The bar beneath the trace indicates the timing of the flash. (b) The responses to the dim flash used in (b) are shown on an expanded timescale on the right. On the left is shown the rising edge of the responses to a much brighter step of light produced by the unattenuated laser beam, delivering 108 effective photons per second. The bars below the traces indicate the onset and duration of the stimuli. Adapted from Payne, R. and Demas, J. (2000). Journal of General Physiology 115: 735–748.

of rise to those elicited by bright visible light. However, the latency of the Ca2+ signal that follows illumination by visible light is 30 ms longer than that of the response to the release of caged IP3 (compare Figures 8(a) with 8(b)), the difference being presumably the time required for light to activate the PI pathway. There is therefore convincing evidence that the light-induced release of Ca2+ from internal stores is mediated by the PI pathway acting on IP3Rs in SER that are closely juxtaposed to the microvillar membrane.

Released Ca2+ Ions can Activate an Inward Current

Pulsed pressure injections of solutions containing 1–2 mM Ca2+ into the R lobe of ventral photoreceptors activates a current in the plasma membrane of up to 20 nA, with a similar reversal potential, sodium dependence, and outward rectification to that activated by light. Release of Ca2+

F/Fo

15 mV

0.5

20 ms

488 nm

(a)

Caged InsP3 released

488 nm

(b)UV

Figure 8 Timing of Ca2+ release by caged InsP3. Photoreceptors were loaded with caged IP3 and the Ca2+ indicator dye fluo-3; 488 nm and UV laser beams were focused onto the edge of the R-lobe. As an indicator of [Ca2+]i, uncalibrated dye fluorescence is shown, expressed as a fraction of the background fluorescence during the latent period of the response (F/F0). (a) Membrane potential (solid line) and fluo-3 fluorescence (red dots) recorded during illumination by the 488-nm laser alone, stimulating Ca2+ release and depolarization through the photoisomerization of rhodopsin, that is, through the natural phototransduction pathway. Laser stimulation began at the beginning of the fluorescence trace. (b) Effect of superimposing a 20-ms duration UV flash and so releasing caged InsP3 into the cytoplasm of the cell, stimulating an earlier release of Ca2+ directly from the SER. Adapted from Ukhanov, K. and Payne, R. (1997). Journal of Neuroscience 17: 1701–1709.

ions through IP3 activates the same conductance, indicating that the [Ca2+]i generated through the endogenous Ca2+ release pathway is sufficient. The current, typically 5–20 nA in amplitude following a pulse of 100 mM IP3, generates a transient depolarization of the photoreceptor lasting for less than 1 s. The coupling between the elevation of [Ca2+]i and the depolarization of the photoreceptor is rapid. Depolarization follows caged IP3-induced Ca2+ release after 2.5 3.3 ms (Figure 8), while photolysis of caged Ca2+ (O-nitrophenyl ethylene glycol tetraacetic acid (EGTA)) at the edge of the R lobe activates current within 1.8 0.7 ms.

Light-Induced Ca2+ Release can be Detected before the Electrical Response

The above experiments indicate that micromolar [Ca2+]i, released from internal stores by IP3, can activate an

inward current through the plasma membrane within a few milliseconds. It follows that if light-induced Ca2+ release is to act similarly, then a component of the photocurrent must be initiated a few milliseconds after Ca2+ is released. Certainly, the onset of the two signals is highly correlated if measured confocally beneath the microvillar membrane. The time for [Ca2+]i to exceed 2 mM is approximately equal to that for the receptor potential to exceed 8 mV (mean difference: 2.2 6.4 ms). However, the question of which event occurs first is difficult to address, since two signals with different noise levels are compared (Figure 7). The detection of the Ca2+ signal lead the electrical response by up to 5 ms within about one-third of cells examined. The lag in other cells could indicate the presence of an early Ca2+-independent component of the response present in some cells, but it is difficult to be sure of this because the placement of the confocal measuring spot relative to the microvillar membrane is critical. However, in any case, given the rapidity with which Ca2+ can elicit an inward current (1–3 ms; see above) and the fact that it takes the photocurrent 50 ms to rise to peak, this timing appears to be sufficient for released Ca2+ ions to contribute to the activation of the photocurrent during the rising edge of the response to light.

How Does IP3-Induced Ca2+ Release Activate Inward Current and is

this Current Flowing through the Light-Sensitive Conductance?

Given the detailed evidence above, it seems reasonable to propose that IP3-induced Ca2+ release can activate the light-activated conductance in ventral photoreceptors (Figure 5). However, the molecular nature of the channel and the site of calcium’s action are not yet known. An ideal preparation for electrophysiology, ventral photoreceptors do not have the advantage of molecular genetic approaches that have allowed the identification of the light-sensitive channels in Drosophila as members of the TRP family. Still, two hypotheses have been developed for the nature of the light-activated ion channels based on physiological and molecular evidence.

The first hypothesis is that the channels are not Ca2+- gated, but are cyclic guanosine monophosphate (cGMP) gated. This seems a remote possibility at first, given the evidence that IP3-induced Ca2+ release can rapidly activate a plasma membrane conductance in intact cells. However, the proposal that a further messenger exists downstream from Ca2+ is driven by the experimental inability of Ca2+ to directly activate ion channels when applied to the inside of patches of plasma membrane excised from the rhabdomeral lobe. Instead, application of cGMP activated channels in a minority of excised patches. The channel events activated during application

of cGMP had a similar conductance to light-activated channels, similar reversal potential when bathed in media mimicking intracellular and extracellular ion concentrations and a similar increase in open probability upon membrane depolarization. Since intracellular injections of cGMP or its analogs depolarize the photoreceptor, it was proposed that cGMP might be a terminal messenger in the visual cascade in ventral photoreceptors, as well as in vertebrate rods and cones. A putative cGMPgated channel has been sequenced from ventral photoreceptors and localized to the microvillar photoreceptors. However, to reconcile this hypothesis with the large body of evidence for the initiation of the light response by the PI pathway, some coupling mechanism must be found to link IP3-induced Ca2+ release to the production of cGMP. There is some pharmacological evidence that a Ca2+- activated guanylate cyclase (GC) might provide this link (Figure 5), but no biochemical or molecular evidence has so far been obtained for this hypothesis.

The second hypothesis is that Limulus channels are members of the TRP family which, unlike Drosophila TRP and TRPL, do not display a high Ca2+- permeability (Figure 5). TRP channel homologs have been cloned from ventral photoreceptor messenger RNA (mRNA), and an established activator of some TRP-family channels, the synthetic lipid, 1-oleoyl-2-acetyl-sn-glycerol (OAG), activates a conductance with reversal potential similar to that activated by light. Activation of this conductance by OAG apparently requires the presence of free Ca2+ ions in the injection pipette, which may explain why extracellularly applied OAG has no effect and why there is a complete dependence of the light response on light-induced Ca2+ release from intracellular stores. The hypothesis of a Limulus homolog of the TRP channel provides a testable alternative to the proposed role of a cyclic nucleotide channel and may resolve the differences in phototransduction between Limulus and Drosophila photoreceptors.

Adaptation, a Decrease in the Sensitivity of the Visual Cascade, is Mediated by Small, Lingering Elevations of Ca2+

The onset of prolonged illumination of ventral photoreceptors, or the huge elevations of [Ca2+]i that occur following flashes of light are not sustained but fall back to the micromolar range within seconds. Concurrently, the photocurrent also falls from tens or hundreds of nA to a few nA. These declines are the result of a decrease in the photoreceptor’s sensitivity that prevents saturation of the photoreceptors in bright light and so extends its dynamic range. The initial light-induced elevation of [Ca2+]i therefore appears to function as a feedback signal that subsequently reduces the sensitivity of the visual cascade.

In support of this concept, slow injection of Ca2+ ions into the cytosol of ventral photoreceptors diminishes the sensitivity and latency of the light response, mimicking the effect of an adapting light. Injection of Ca2+ chelators, such as EGTA or 1,2-bis(o-aminophenoxy)ethane-N,N, N0,N0-tetraacetic acid (BAPTA), not only slows down and diminishes the initial photocurrent, but also blocks the decline of the light-induced current during prolonged illumination. The site of this feedback inhibition of the light response by Ca2+ could be at several points in the phototransduction cascade. For example, IP3-induced Ca2+ release is known to be inhibited by lingering elevations of [Ca2+]i in ventral photoreceptors. In addition, pharmacological activation of protein kinase C greatly reduces the sensitivity of the light-induced current, apparently acting upstream of IP3-induced Ca2+ release.

Drosophila and Limulus Photoreceptors Operate Differently and Illustrate Two General Mechanisms Coupling the PI Cascade to an Electrical Response

The two most extensively studied microvillar photoreceptors, those of Limulus and Drosophila, have many aspects of their phototransduction mechanism in common. Both are thought to utilize the PI cascade to open non-selective cation channels and both exhibit large elevations of [Ca2+]i, which are necessary for signal amplification and speed, as well as light adaptation. However, there are also clear differences: Limulus photoreceptors utilize IP3-induced Ca2+ release to elevate intracellular free Ca2+ ion concentrations, while Drosophila photoreceptors are thought to utilize DAG or derived products to open Ca2+-permeable TRP channels that allow Ca2+ entry from the extracellular space. The photoreceptors of the two species are therefore specific examples of two general mechanisms in cells for coupling the PI pathway to [Ca2+]i elevation. Why is there this difference? One explanation is the need in Limulus for the extra amplification provided by IP3-induced Ca2+ release. One IP3 molecule can release hundreds of Ca2+ ions from the ER through the IP3R channel. These Ca2+ ions can then diffuse along the inner surface of the plasma membrane to activate downstream targets, such as the hundreds of channels required to open in order to produce a significant quantal event in these giant photoreceptors. The trade-off for this amplification is slower speed. The extra amplification step (Ca2+ release) required in the visual cascade may explain why Limulus photoreceptors are slower than fly photoreceptors to respond to light. This speed difference is entirely reasonable, given the animals’ differing demands on their visual systems. Horseshoe crabs do not fly; rather they use their lateral eyes to find mates near moonlit beaches.

Extension of Phototransduction Mechanisms to other Microvillar Photoreceptor Types

Detailed knowledge of the mechanisms of both Limulus and Drosophila photoreceptors may outline general approaches used by other cell types to the problem of tailoring the outcome of the PI cascade to the need for either speed or amplification. An outstanding question is the extent to which other photoreceptor types utilize these mechanisms. It is apparent that light-induced Ca2+ release from intracellular stores occurs in photoreceptors of the file clam, leech, and honeybee. Furthermore, the light-sensitive channels that mediate the immediate electrical response in the file clam are relatively impermeable to Ca2+ ions, like those of Limulus, but not Drosophila. Barnacle photoreceptors, on the other hand, like those of Drosophila, display prominent lightinduced Ca2+ influx. Thus, elements of the two mechanisms for coupling phototransduction to an elevation of Ca2+ seem to be scattered across the invertebrate phyla. Whether there is a functional or evolutionary rationale for these differences remains to be seen. Indeed, it is not known yet whether photoreceptors in the other eight eyes of Limulus function similarly to the much-studied ventral eye.

See also: Circadian Rhythms in the Fly’s Visual System; Evolution of Opsins; Genetic Dissection of Invertebrate Phototransduction; Limulus Eyes and Their Circadian Regulation; Microvillar and Ciliary Photoreceptors in Molluskan Eyes; The Photoresponse in Squid.

Further Reading

Bandyopadhyay, B. C. and Payne, R. (2004). Variants of TRP ion channel mRNA present in horseshoe crab ventral eye and brain.

Journal of Neurochemistry 91: 825–835.

Battelle, B. A. (2006). The eyes of Limulus polyphemus (Xiphosura, Chelicerata) and their afferent and efferent projections. Arthropod Structure and Development 35: 261–274.

Brown, J. E. and Blinks, J. R. (1974). Changes in intracellular free calcium concentration during illumination of invertebrate photoreceptors. Journal of General Physiology 64: 643–665.

Brown, J. E., Rubin, L. J., Ghalayini, A. J., et al. (1984). myo-Inositol polyphosphate may be a messenger for visual excitation in Limulus photoreceptors. Nature 311: 160–162.

Chen, F. H., Baumann, A., Payne, R., and Lisman, J. E. (2001).

A cGMP-gated channel subunit in Limulus photoreceptors. Visual Neuroscience 18: 517–526.

Fein, A., Payne, R., Corson, D. W., Berridge, M. J., and Irvine, R. F. (1984). Photoreceptor excitation and adaptation by inositol 1,4,5 trisphosphate. Nature 311: 157–160.

Hardie, R. C. and Minke, B. (1992). The trp gene is essential for a lightactivated Ca2+ channel in Drosophila photoreceptors. Neuron 8: 643.

Nasi, E., Gomez, M., and Payne, R. (2000). Phototransduction mechanisms in microvillar and ciliary photoreceptors of invertebrates. In: Hoff, A. J., Stavenga, D. G., de Grip, W. J., and Pugh, E. N. (eds.) Molecular Mechanisms in Visual Transduction – Handbook of Biological Physics, vol. 3, pp. 389–448. Amsterdam: Elsevier Science.