Neuron,
Vol. 8, 995-1002,
June, 1992, Copyright
0 1992 by Cell Press
Signal Flow in Visual Transduction
Leon Lagnado and Denis Baylor The Department of Neurobiology Sherman Fairchild Science Center Stanford University School of Medicine Stanford, California 94305
Over the past decade our understanding of visual transduction has advanced at a rapid pace, revealing molecular strategies for intracellular signaling that are exploited in a wide range of other cells, including olfactory receptor neurons, pinealocytes, retinal bipolar cells, and hepatocytes. Retinal photoreceptors have proved especially valuable in attacking signal transduction mechanisms because of their experimental advantages. The outer segment, the specialized organelle in which phototransduction occurs, can readily be obtained in quantity. The natural stimulus, light, is easily controlled. In photoreceptors, cGMP has been identified as the ligand controlling the ion channels that generate the light-evoked membrane hyperpolarization (Fesenko et al., 1985). Ca*+, once a candidate ligand for the channels, is now known to be important in regulating the sensitivity of the photoreceptor. The mechanism of transduction appears similar in the rods, which mediate vision at low light levels, and in the cones, which operate at higher light levels. Key questions now concern the molecular mechanics of individual components of the transduction apparatus and the way in which thecomponents function in concert to produce the light response. Our review focuses on the latter aspect and attempts to present recent advances as well as current problems. Visual transduction is the subject of a number of recent reviews (Liebman et al., 1987; Stryer, 1986,199l; Lamb and Pugh, 1990; Hurley, 1987; Signal
McNaughton, Flow
from
1991). Visual
Pigment
to Ion
Channels
Figure 1 diagrams the pathway bywhich signals originating in the visual pigment modulate the membrane channels that generate the rod’s electrical response. Absorption of light isomerizes rhodopsin’s retinal chromophore from the II-cis to the all-trans configuration, causing the protein to become enzymatically active. Active rhodopsin (Rh*) catalyzes the replacement of GDP by CTP on the G protein transducin (T). T-GTP activates cCMP phosphodiesterase (PDE), which hydrolyzes cGMP. In darkness cGMP binds to the ion channels and holds them open. Light-triggered hydrolysis of cGMP allows the channels to close, interrupting an inward current of Na+, CaZ+, and Mg 2+. The resulting membrane hyperpolarization lowers the rate of transmitter release from the synaptic terminal, triggering responses in second-order cells.
Review
Psychophysical experiments by Hecht et al. (1942) showed that human rods register single photon absorptions, which change only 1 of the 108rhodopsin molecules in the cell, and that a coincidence of single quanta1 absorptions in 5 rods reaches consciousness. The electrical response to absorption of a single photon is highly amplified (see upper trace in Figure 2A). At the peak of the response, 500 channels close; this is 3% of the number open in darkness. The response blocks the entry of several hundred thousand cations. The steps shown in Figure 1 account for this amplification. One photoisomerized rhodopsin activates hundreds of copies of transducin within a fraction of a second. Additional gain results from the powerful hydrolytic activity of PDE and the sizeable ion fluxes through individual open channels. A quantitative treatment, based on the known properties of the individual steps shown in Figure 1, accounts for the rising phase of the flash response over awide range of intensity (Lamb and Pugh, 1992), and models of the entire response have also been advanced (Forti et al., 1989; Sneyd and Tranchina, 1989). The single photon response of cones is too small to measure directly, but the dim flash response (lower trace in Figure 2A) is a scaled up replica of the single photon effect. This response is briefer than the rod’s and is diphasic; the oscillation is characteristic of a system with a negative feedback that operates after a delay. If the the large gain of the tranduction mechanism were constant, a steady background light of moderate intensity would close all the channels in the outer segment, preventing further changes in light intensity from being encoded. Instead, again control automatically reduces the sensitivity of transduction in background light so that some channels remain open and available for modulation by changes in light intensity. The intracellular Ca2+ concentration tracks the number of open channels and controls the gain of transduction. The Ca2+ level in the cell is determined by the balance of Ca2+ entry through the channels in the surface membrane and extrusion through the Na+: Ca2+,K+ exchanger (Hodgkin et al., 1985; Yau and Nakatani, 1985; McNaughton et al., 1986; Cervetto et al., 1989). When Ca* influx is blocked by closure of the channels in light, continued extrusion lowers the intracellular Ca” concentration with a time constant of 0.5 s. The concentration of Ca2+ thus provides a direct measure of the number of open channels during the recent past. Evidence that the drop in Ca2+ mediates the reduction of sensitivity in background light (adaptation) is provided by the finding that clamping the internal Ca2+ concentration blocks the gain reduction (Matthews et al., 1988; Nakatani and Yau, 1988a). The fall in Ca*+ concentration also speeds the recovery phase of the flash response, for recovery slows when Ca*+ buffers are put into a tranducing cell (Lamb et al.,
NWKMl 996
A
1 wj”
CSA
B
K
R
A C Ch E K PDE R Rh S T
Arrestin Cyclase Channel Exchange Rhodopsin kinase Phosphodiesterase Recoverin Rhodopsin S-modulin Transducin
photon
Hyperpolarization Figure
1. Molecular
Mechanism
of Visual
Transduction
(A) Schematic diagram of a retinal rod and the transducing molecules in the outer segment. The integral membrane protein rhodopsin lies embedded in the membranes of intracellular disks. Associated with the disk membranes are the peripheral membrane proteins transducin 0 and cGMP phosphodiesterase (PDE). In the surface membrane of the outer segment are the cCMP-activated cation channel and the Na+:Ca*+,K+exchanger. Soluble proteins arrestin, recoverin, s-modulin, and rhodopsin kinase are present in the cytosol. (B) Signal flow from pigment to cation channel. A photon isomerizes the chromophore of rhodopsin, which becomes enzymatically active (Rh’). Rh’ catalytically activates the GTP-binding protein transducin. T’ stimulates cCMP PDF, which hydrolyzes cGMP. The resulting fall in the level of cGMP closes the cation channel, interrupting the influxof Na+, Ca2’, and Mg2’. As a result, the cell membrane hyperpolarizes, causing a fall in the rate of release of transmitter from the synaptic terminal. Steps with appreciable amplificationtransducin activation, cGMP hydrolysis, and flux of cations through the open channel-are shown with bold arrows.
1986). The best-established biochemical correlate for these effects is the Ca*+ sensitivity of the rod’s guanylate cyclase, which synthesizes cGMP (Lolley and Racz, 1982; Koch and Stryer, 1988). Ca2+ effects on cyclase activity are mediated by the newly characterized protein recoverin (Lambrecht and Koch, 1991b; Dizhoor et al., 1991), which stimulates the cyclase at low Ca2+ levels, but fails to stimulate at the higher Ca*+ levels present in darkness. The scheme illustrated in Figure 1 is supported by extensive biochemical and physiological evidence, but it is incomplete. How is excitation shut off so that the dark state is restored? How is amplification in the enzymatic chain Rh*-T’-PDE” controlled? Where else might Ca2+ act? What loops and branches, other than those controlled by Ca2+, are hidden within the scheme? Rhodopsin
Shutoff
After a photon is absorbed, rhodopsin zymatically active state (metarhodopsin
enters the II, here
ende-
noted Rh’) within milliseconds. Since it is the trigger of the transduction cascade, Rh’ is the species whose activity is most highly amplified. If Rh’ were shut off in a single step, the active lifetime in different trials would be exponentially distributed, producing large variations in the size or duration of successive single photon responses. Contrary to this expectation, the rod’s response to a single photon is highly reproducible, as illustrated by the amplitude histogram in Figure 2B. The fit of the smooth curve to the experimental observations implies that the standard deviation of the quanta1 response amplitude was only about onefifth the mean. Although the mechanisms that shut rhodopsin off arequalitatively understood, it is not yet clear howthe reproducibility is achieved. We suggest below that feedback control of rhodopsin’s active lifetime may be involved. Whatever its molecular mechanism, reproducibility is a desirable property for a small signal that must be detected in noise. Rhodopsin’s enzymatic activity is terminated bytwo processes: ATP-dependent phosphorylation, which is catalyzed by rhodopsin kinase, and binding of the
Review: 997
Visual
Transduction
trials
I
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2. Kinetics
and Amplification
of Rod and Cone
1
2
PA
Responses
to Weak
Stimuli
(A) Dim flash responses of a rod and red-sensitive cone from the macaque retina, recorded by suction electrode. Change in outer segment membrane current, averaged from multiple trials, is plotted as a function of time. Flash timing is shown below. For the rod the flash caused an average of one photoisomerization per trial. At the peak response 3.3% of the channels in the outer segment closed. For the cone the flash caused an average of 35 photoisomerizations per trial, and one photoisomerization closed an estimated 0.36% of the channels. (6) Reproducibility of the rod response to a single photon. Amplitude histogram of a macaque rod’s responses to dim flashes calculated to give 0.46 photoisomerizations per trial on average. In individual trials there were failures (peak near 0 PA), single photon responses (peak near 0.8 PA), and some responses to two photons (peak near 1.6 PA). The theoretical curve was constructed assuming that the amplitude of the single photon response was Gaussian distributed with a mean of 0.81 pA and a standard deviation of 0.15 pA, that the probability of photon responses per trial was Poisson distributed with a mean of 0.46, and that the baseline noise in darkness was Gaussian with a standard deviation of 0.14 pA. The dark distribution, fitted by this baseline Gaussian, is shown in the inset. Modified from Baylor et al. (1984) and Schnapf et al. (1990).
protein arrestin to phosphorylated Rh’. Assays of light-triggered PDE activity show that the catalytic lifetime of Rh’ is greatly prolonged if phosphorylation is blocked by removing ATP (Liebman and Pugh, 1980) or rhodopsin kinase (Sitaramayya and Liebman, 1983), or by proteolytic removal of the phosphorylation sites at rhodopsin’s carboxyl terminus (Miller and Dratz, 1984). Physiological evidence for the quenching role of phosphorylation is provided by the observation that the flash response of an internally dialyzed outer segment fails to turn off if ATP is removed (Sather and Detwiler, 1987; Nakatani and Yau, 1988b; see Figure 3) or if rhodopsin kinase activity is blocked by the inhibitor sangivamycin (Palczewski et al., 1992). Phosphorylated Rh’ binds arrestin, a 48 kd protein that is the most abundant soluble protein in the cytosol. Arrestin blocks activation of transducin by competitively inhibiting its binding to phosphorylated Rh’ but it has a much lower affinity for unphosphorylated Rh’, or for phosphorylated rhodopsin whose chromophore is II-cis retinal (Kuhn et al., 1984). There are at least 7 phosphorylation sites on rhodopsin’s carboxyl terminus, but it is not clear whether fully phosphorylated rhodopsin still requires arrestin to complete its inactivation, or whether arrestin binds to partially
phosphorylated Rh’to block activation of transducin completely. Bennett and Sitaramayya (1988) reported that without arrestin binding, rhodopsin shutoff occurred slowly and required phosphorylation at 1216 sites, while in the presence of arrestin complete inactivation was achieved rapidly after the phosphorylation of only one site. The absence of multiple phosphorylations when arrestin was present suggests that binding of arrestin prevents further action of rhodopsin kinase. This would occur if arrestin, rhodopsin kinase, and transducin all recognized the same site on rhodopsin. In outer segments dialyzed with a patch pipette, phytic acid, an inhibitor of arrestin’s binding to Rh’, preferentially slowed the recovery of bright flash responses, in which complete phosphorylation of rhodopsin would be expected to require a long time (Figure 4; Palczewski et al., 1992). This result supports the notion that arrestin binds to partially phosphorylated Rh’ to shut it off completely. Although it is clear that phosphorylation lowers the enzymatic activity of Rh’, the relation between the number of phosphates added and the enzymatic activity of Rh’ is not known. Nathans (1987) and Liebman et al. (1987) suggested that progressive shutoff by multiple phosphorylations might extinguish rhodopsin’s
Neu”JIl 998
1 1 mMz;cplic -
0
2 a ET z 5 0
r
mhl-cyclic GMP t 0.5 mwGTPj A
1 muxyclic GMP + 0.5 mwGTP
Figure 3. Evidence That Generation of the Light Response Requires GTP and That Shutoff Requires ATP
+ 0.5 mwATP L--
Membrane current of an isolated rod outer segment was recorded with a suction electrode, and the end of the outer segment was knocked off with a glass probe to allow dialysis of the interior. The lower trace shows the timing of light flashes. Adding 1 mM cCMP opened the channels, allowing an inward current to flow. The first flash (-3.8) did not generate a response. When 0.5 mM GTP was added most of the lightsensitive channels closed because Rh’created by the first flash was still present. This response did not recover, and a second flash shut off more current. On addition of 0.5 mM ATP, the light-sensitive current recovered, and the responses to subsequent flashes were transient, as newly created Rh’ was shut off. The intensity of the last flash was raised, causing a larger responsethan the previous one. Reproduced in modified form from Nakatani and Yau (1988b) with the permission of the publisher.
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activity reproducibly. In the version of such a scheme that gives the best reproducibility, phosphorylations at n identical and independent sites each reduce the activity by the same amount. The expected ratio of the standard deviation to the mean active lifetime is then given by l/,/ii. To explain the constancy of the single photon effect shown in Figure 2B, this mechanism requires 25 phosphorylations, which is unrealistically large. If instead arrestin binds to singly phosphorylated rhodopsin to terminate its activity, as suggested by Bennett and Sitaramayya (1988), the active lifetime would vary much more widely, and the standard deviation would equal the mean. Reproducibility of the
single photon responsewould be improved if rhodopsin’s active lifetime were under feedback control. Complete recovery after a flash requires that the phosphate groups be removed from phosphorylated rhodopsin and that the II-cis retinal chromophore be replaced. The opsin phosphatase in the rod outer segment is of type 2A (Fowles et al., 1989). Phosphatase activity is blocked when arrestin is bound to phosphorylated Rh’, and arrestin does not dissociate until phosphorylated Rh’ has decayed to the inactive species phosphorylated metarhodopsin III, thus preventing reactivation (Palczewski et al., 1989). After removal of the phosphates, rhodopsin is regenerated
Figure 4. The Effect of Inhibiting Segment
of the Light
(A) Family of responses configuration. (6) Family of responses to phosphorylated Rh’. with the permission of
to flashes
the Action of increasing
of Arrestin intensity,
on the Recovery recorded
from
an isolated
Response outer
in Internally
segment
Dialyzed
by a patch
to the same flashes, recorded by a pipette containing 1 mM phytic acid, which inhibits Recovery of these responses was greatly prolonged. Reproduced in modified form from the publisher; copyright Cell Press.
pipette
Isolated
Rod Outer
in the whole-cell
the binding of arrestin Palczewski et al., (1992)
Review: 999
Visual
Transduction
by spontaneous combination of II-cis retinal with the protein opsin. Electrical recordings from rods support the idea that resumption of enzymatic activity following the initial burst is very improbable. The current noise in darkness is low, and little increase occurs after exposure to dim light (Baylor et al., 1980). “Trickleback” excitation becomes apparent only after exposure to bright lights (Lamb, 1980; Baylor et al., 1984) sufficient to reveal shutoff inefficiencies of 0.1% or less. Shutoff of the pigment’s enzyme activity in cones has not yet received much attention. The small size and short duration of their photon responses suggest that the cone pigment is shut off faster than rhodopsin. Similarly, cone transducin may also be shut off more rapidly than rod transducin. Shutoff
of Transducin
and
PDE
When Rh’ activates transducin, the exchange of GTP for GDP causes transducin to dissociate. The active T,GTP portion leaves the Tsu complex and stimulates PDE by binding to, and removing inhibition exerted by, they subunit. T, has intrinsic GTPase activity. Persistent doubts about whether CTP hydrolysis alone terminates the action of T’ have been countered by two kinds of recent evidence: When the hydrolysisresistant analog GTPyS is put into a transducing rod,
Figure vation
5. Microcalorimetric after a Flash
Measurement
of Transducin
Inacti-
The rate of heat release from a suspension of rod outer segments, which is proportional to the rate of CTP hydrolysis, is plotted as a function of time. Flashes were delivered at the times indicated bythearrows.TheGTPconcentrationwasZmM.ATPwasabsent, but Rh’ was inactivated by 50 mM NH20H. After a flash, the majority of the heat release was complete within 1 s. In traces a-c a second flash was delivered at increasing delay after the first, revealing that heat liberation from the second flash was reduced for about 10 s. This is attributed to the time required for tranducin to reassociate after the first flash. Reprinted in modified form from Vuongand Chabre (1990) Nature 346,71-74; Copyright 0 1990 Macmillan Magazines Limited.
the flash response fails to recover (Sather and Detwiler, 1987; Lamb and Matthews, 1988). In addition, eleganttime-resolved microcalorimetry byvuongand Chabre (1990, 1991) shows that, after a flash, GTP in the outer segment is hydrolyzed on a time scale shorter than that of the electrical response (see Figure 5). The rate of hydrolysis from the heat measurements is much faster than that from previous biochemical measurements of steady-state turnover because a step subsequent to GTP hydrolysis limits turnover rather than hydrolysis itself. A prime candidate for the ratelimiting step, which requires about 10 s, is reassociation of T, with TBr- The slowness of this reaction and the speed of Rh’ shutoff implies that T’ is allowed only a single round of PDE activation after a dim flash. Heat measurements reveal that after a flash, PDE’ falls with much the same time course as that of T (Vuong and Chabre, 1991). This indicates that hydrolysis of GTP on T’ is the rate-limiting step in PDE shutoff. Two mechanisms that may control the hydrolytic shutoff of T’-GTP have been proposed by Arshavsky et al. (1991), who measured light-stimulated GTPase activity by the release of labeled phosphate from GTP. At low concentrations of GTP, when all the hydrolysis should occur on T’ activated for the first time, two components of hydrolysis, with half-times of 1 and IO s, were resolved. The size of the fast component corresponded to the quantity of PDE in the outer segment, suggesting that the binding of T’-GTP to they subunit of PDE increases the rate of GTP hydrolysis IO-fold. This effect would increase the probability that GTP hydrolysis by T’occurs only after PDE is activated, thus helping to ensure that excitation from rhodopsin reaches PDE. A second mechanism of control was indicated by the finding that the fast GTPase activity, attributed to T,CTP-PDE,, was abolished by 5 PM cGMP, a concentration only slightly higher than that present in darkness. This result suggests that cGMP itself acts as a negative feedback control signal in the light response. Thus, as thecGMP level fell in the light, the GTPase activity would be disinhibited, reducing light-stimulated PDE activity and countering the fall in cGMP. Recently Arshavsky and Bownds (1992) have demonstrated that the isolated y subunit of PDE can accelerateGTP hydrolysis bytransducin and havesuggested that cGMP inhibits this effect by binding to noncatalytic sites on the a and B subunits of PDE. Another mechanism by which transducin’s activity may be controlled is covalent modification of its y subunit, which interacts with Rh’ to cause exchange of GTP for GDP on the a subunit of transducin. Farnesylation of they subunit causes a large increase in transducin’s activation by Rh’ (Fukada et al., 1990). Methylation, a potentially reversible modification, causesafurtherenhancementofthefarnesylatedsubunit’s activity (Ohguro et al., 1991; Perez-Sala et al., 1991). Enzymes that can methylate and demethylate the y subunit are present within the outer segment, and only the farnesylated subunit is able to be methylated (Perez-Sala et al., 1991).
Neuron 1000
:;a2+ /
Open channels 4
Exchange
* Outside
Figure6 Flow Transduction
of
Ca2+ Signals
in Visual
Ca2+ enters the outer segment through open channels and is extruded by the Nat: Ca2+,K+ exchanger. Ca2+ inhibits cyclase activity by binding to the protein recoverin (R) and blocking its stimulation of cyclase. Ca2+also stimulatestheactivityof PDE, bya mechanism that remains to be determined. Possibilities are prolonging the lifetime of PDE’ by inhibiting reassociation of the y subunit thatturns it off, inhibiting hydrolysis of CTP on T’, and inhibiting shutoff of Rh’. GTP
Cyclase-R
*
cG;vlP
PDE*
)5’-GMP
0 T
Sites
of Action
of Ca2+
It is clear that the light-induced fail in intracellular Ca2+ accelerates recovery of the light response and is a key signal for light adaptation, but the sites at which Ca2+ acts are only partly understood (see Figure 6). The effect on the synthetic enzyme guanylate cyclase is the best documented. Cyclase activity increases over 5-fold when Ca2+ is lowered from 300 nM, the dark level, to about 10 nM, the level reached in bright light. This effect is highly cooperative, with a Hill coefficient of 3 (Koch and Stryer, 1988; Dizhoor et al., 1991). The effect of Caz+ is mediated by recoverin, a soluble 23 kd Ca*+-binding protein that binds to the cyclase and stimulates it when Ca*+ is low (Lambrecht and Koch, 1991b; Dizhoor et al., 1991; Ray et al., 1992). Phosphorylation is reported to increase recoverin’s activity 2-fold when Ca2+ is reduced to levels below 100 nM (Lambrecht and Koch, 1991a). Thiswould reinforcethe stimulation of cyclase activity. Recoverin shows sequence homologies with visinin, a cone Ca2+-binding protein of unknown function. New evidence reveals that Ca*+ prolongs PDE’s activation by light. Kawamura and Murakami (1991) internally perfused truncated rod outer segments with solutions that supported a light response but minimized the effect of cyclase activity. The response to a flash became briefer when internal Ca2+ was lowered from 1 PM to 30 nM. Biochemical assays of light-triggered PDE activity confirmed that the Ca*+effect was exerted on PDE rather than the cyclase. The Ca*+ sensitivity appears to be mediated by a soluble 26 kd protein, dubbed S-modulin, which in the presence of Ca2+ binds to the disk membranes to prolong PDE’s activation by a flash. These findings leave open the question of where in the chain Rh*-T*-PDE’ Ca2+ acts (see Figure 6). In principle, light-induced PDE activity might be modified by changing the reaction rate or active lifetime of the species PDE’, T’, or Rh’. The fact that the
early rising phase of the flash response is invariant during adaptation (Baylor and liodgkin, 1974; Lamb, 1984) points to an effect on lifetime rather than reaction rate. At PDE’, Ca:+ might SLOW the reassociation of the inhibitory y subunit that terminates activity. At T’, Ca2+ might inhibit CTPase activity, while at Rh’ it might inhibit shutoff. A Ca2+ effect on shutoff of Rh* seems particularly interesting, since it could make the activity of Rh” reproducible. Shutoff of an individual Rh’, which might otherwise fluctuate widely, would occur at a time dictated by a stereotyped fall in Ca2+ concentration. Control of the Rh’ lifetime could be achieved if Ca*+ inhibited the phosphorylation of Rh’ or the binding of arrestin to phosphorylated Rh”. Huppertz et al. (1990) report that the Caz+ binding capacity of protein extracts from rods is halved when they are depleted of arrestin, and they conclude that arrestin binds Ca*+ in a first-order manner with a Kd of 2 bM. Their quantities are in rough agreement with measurementsof Ca*+ buffering in single rods by Lagnado et al. (1992), who found a buffer with a, capacity of about 200 f.rM and a Kd of 1 PM. In contrast, Palczewski and Hargrave (199l)found no evidence that Ca*+ binds to purified arrestin. The sites of action of Ca2+ remain an important area for further work. In addition, it will be important to determine the relative contributions of the different Ca*+ effects. How would adaptation be affected if the action of recoverin, or S-modulin, were selectively blocked? Channel
Control
Closure of the cationic channels during the response to light is adequately explained by removal of the cGMP that holds them open in darkness, and it is not necessary to suppose that any other messenger directs channel closure on a short time scale. Thus, when the hydrolysis-resistant cGMP analog 8-BrcGMP was put into a transducing rod, the cationic channels were locked in the open state and the light
Review: 1001
Visual
Transduction
response was severely attenuated as expected from very limited cGMP hydrolysis (Zimmerman et al., 1985). This result does not, however, rule out regulation of the channel’s responsiveness to cCMP on a longer time scale. Indeed, the need for regulation of the channel is suggested by the fact that even in darkness only about I%-2% of the channels are open (reviewed in Yau and Baylor, 1989). The large number of channels available to be activated put a rod in danger of a catastrophic influx of cations from a modest rise in cGMP concentration. Alterations in the channel’s cCMP affinity would be a powerful potential site for regulation, because channel activation appears to require the binding of three or more ligand molecules. At the low cGMP concentrations present in darkness, a 2-fold change in channel affinity would produce an 8-fold change in activation. Hints of such a control is provided by the wide variations in the cGMP sensitivity of excised patches from one rod to the next and from one laboratory to another. Gordon and Zimmerman (1990, ARVO, abstract) suggest that such changes in affinity may depend on an ATP-dependent phosphorylation. Conclusion Three major accomplishments have marked recent research on signal flow in visual transduction. First, cGMP has been identified as the diffusible messenger that controls the transduction channels and brings information to them from the disks. Second, the enzymatic mechanisms that amplify signals issuing from single rhodopsin molecules have been elucidated. Third, Ca*+ has been identified as a key messenger for light adaptation and response shutoff. Not so well understood is how the explosive amplification of the enzyme cascade is controlled. This control adjusts the gain in changing background light levels, produces a reproducible single photon effect, and gives the different performance characteristics of rods and cones. Such a control might also conserve metabolic energy in rods during daylight, when their response is saturated and GTP would otherwise be wastefully consumed at high rate. This regulation might operate on a much slower time scale than the light response. Immediate problems in the control of transduction concern how rhodopsin and transducin are shut off and how Ca2+ acts to stimulate lighttriggered PDE activity. It seems safe to guess that new loops and branches will be discovered within this intricate mechanism and that many of the outer segment proteins in search of a function will turn out to be controllers of the cascade. We may also anticipate the characterization of new messengers that measure the state of the system on time scales longer than those of cGMP and Ca*+. One candidate substance may be Mgti, which enters the outer segment in darkness and should fall with a time constant of tens of minutes in the light. Finally, it will be interesting to see whether similar mechanisms of gain control have
been adopted strategies for
in other cells signal amplification.
that
use
homologous
Acknowledgments This work was supported by grants EY01543 and EY05750 from the National Eye Institute, byafellowship from the Human Frontiers in Science Program, and by research awards from the Retina Research Foundation and Alcon Research Institute. We thank Professor Lubert Stryer and Drs. Steven Devries and Clint Makino for comments on the manuscript. References Arshavsky, V. Y., and Bownds, M. D. (1992). Deactivation toreceptor C protein is regulated by its target enzyme cCMP. Nature, in press.
of phoand by
Arshavsky, V. Y., Gray-Keller, M. P., and Bownds, M. D. (1991). ~GMPsuppressesGTPaseactivityofaportionoftransducinequimolar to phosphodiesterase in frog rod outer segments. Lightinduced cCMP decreases as a putative feedback mechanism of the photoresponse. J. Biol. Chem. 266, 18530-18537. Baylor, D. A., and Hodgkin, A. L. (1974). Changes and sensitivity in turtle photoreceptors. J. Physiol.
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Baylor, D. A., Matthews, C., and Yau, K.-W. (1980). Two components of electrical dark noise in toad retinal rod outer segments. J. Physiol. 309, 591-621. Baylor, D. A., Nunn, B. J., and Schnapf, J. (1984). The photocurrent, noise and spectral sensitivity of rods of the monkey Macaca fascicularis. J. Physiol. 357, 575-607. Bennett, N., and Sitaramayya, A. (1988). Inactivation of photoexcited rhodopsin in retinal rods: the rolesof rhodopsin kinase and 48-kDa protein (arrestin). Biochemistry 27, 1710-1715. Cervetto, L., Lagnado, L., Perry, R. J., Robinson, D. W., and McNaughton, P. A. (1989). Extrusion of calcium from rod outer segments isdriven by both sodium and potassium gradients. Nature 337, 740-743. Dizhoor, A. M., Ray, S., Kumar, ley, D., Walsh, K. A., Philipov, L. (1991). Recoverin: a calcium guanylate cyclase. Science 257,
S., Niemi, G., Spencer, M., BrolP. P., Hurley, J. B., and Stryer, sensitive activator of retinal rod 915-918.
Fesenko, E. E., Kolesnikov, S. S., and Lyubarsky, A. L. (1985). Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature 373, 310-313. Forti, S., Menini, A., Rispoli, C., and Torre, V. (1989). Kinetics of phototransduction in retinal rods of the newt Tritorus cristatus J. Physiol. 479, 265-295. Fowles, C., Akhtar, M., and Cohen, phorylation and dephosphorylation tases of bovine rod outer segments.
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Fukada, Y., Takao, T., Ohguro, H., Yoshizawa, T., Akino, T., and Shimonishi, Y. (1990). Farnesylated y-subunit of photoreceptor C protein indispensable for CTP binding. Nature 346, 658-660. Hecht, S., Shlaer, S., and Pirenne, M. H. (1942). Energy, and vision. J. Gen. Physiol. 25, 819-840.
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Hodgkin, A. L., McNaughton, P. A., and Nunn, B. 1. (1985). The ionic selectivity and calcium dependence of the light-sensitive pathway in toad rods. J. Physiol. 358, 447-468. Huppertz, B., Weyand, capacity of cytoplasmic mainly due to arrestin.
I., and Bauer, P. J. (1990). Ca2+ binding proteins from rod photoreceptors is J. Biol. Chem. 265, 9470-9475.
Hurley, J. B. (1987). Molecular of vertebrate photoreceptors.
properties of the cGMP cascade Annu. Rev. Physiol. 49, 793-812.
Kawamura, S.,and Murakami,M. (1991).Calcium-dependent ulation of cyclic CMP phosphodiesterase by a protein retinal rods. Nature 349, 420-423.
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Koch, K. W., and Stryer, L. (1988). Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions. Nature 334, 64-66.
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Kuhn, H., Hall, S. W., and Wilden, U. (1984). Light-induced binding of 48-kDa protein to photoreceptor membranes is highly enhanced by phosphorylation of rhodopsin. FEBS Lett. 776,473478. Lagnado, L., Cervetto, L., and McNaughton, P. A. (1992). Calcium homeostasis in the outer segments of retinal rods from the tiger salamander. J. Physiol., in press. Lamb, T. D. (1980). rods by bleaching.
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Lamb, T. D. (1984). Effects photocurrents. J. Physiol.
events
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induced
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Lamb, T. D., and Matthews, H. R. (1988). Incorporation of analogues of CTP and GDP into rod photoreceptors isolated from the tiger salamander. J. Physiol. 407, 463-487. Lamb, T. D., and Pugh, E. N. (1990). Physiology and adaptation in rod and cone photoreceptors. rosci. 2, 3-13.
of transduction Seminars Neu-
Lamb, T. D., and Pugh, E. N. (1992). A quantitative theactivation steps involved in phototransduction photoreceptors. J. Physiol. 449, 719-757.
account of in amphibian
Lamb, T. D., Matthews, H. R., and Torre, V. (1986). Incorporation of calcium buffers into salamander retinal rods: a rejection of the calcium hypothesis of phototransduction. J. Physiol. 372, 315-349. Lambrecht, H.-C., and Koch, K.-W. (1991a). Phosphorylation recoverin, the calcium-sensitive activator of photoreceptor nylyl cyclase. FEBS Lett. 294, 207-209. Lambrecht, ing protein photoreceptor Liebman, reversal receptor
of gua-
H.-G., and Koch, K.-W. (1991b). A 26 kd calcium bindfrom bovine rod outer segments as modulator of guanylate cyclase. EMBO J. 10, 793-798.
P. A., and Pugh, E. N. J. (1980). of cyclic CMP phosphodiesterase membranes. Nature 287, 734-736.
ATP mediates rapid activation in visual
Liebman, P. A., Parker, K. R., and Dratz, E. A. (198;3.The molecular mechanism of visual excitation and its relation to the structure and composition of the rod outer segment. Annu. Rev. Physiol. 49, 765-791. Lolley, R. N., and Racz, E. (1982). Calcium modulation of cyclic GMP synthesis in rat visual cells. Vision Res. 22, 148-1486. Matthews, H. R., Murphy, R. L., Fain, G. L., and Lamb,T. D. (1988). Photoreceptor light adaptation is mediated by cytoplasmic calcium concentration. Nature 334, 67-69. McNaughton, P. A. (1991). Light response ceptors. Physiol. Rev. 70, 847883.
of vertebrate
photore-
McNaughton, P. A., Cervetto, L., and Nunn, B. J. (1986). Measurement of the intracellular free calcium concentration in salamander rods. Nature 322, 261-263. Miller, J. L., and Dratz, E. A. (1984). Phosphorylation at sites near rhodopsin’s carboxyl terminus regulates light initiated cGMP hydrolysis. Vision Res. 24, 1509-1521. Nakatani, K., and Yau, K.-W. (1988a). Calcium and light tion in retinal rods and cones. Nature 334, 69-71.
adapta-
Nakatani, K., and Yau, K.-W. (198813). Guanosine 3’-S-cyclic monophosphate-activated conductance studied in a truncated rod outer segment of the toad. J. Physiol. 395, 731-753. Nathans, 1. (1987). Molecular Rev. Neurosci. 70, 163-194.
biology
of visual
pigments.
Ohguro, H., Fukada, Y.,Takao,T., Shimonishi,Y.,Yoshizawa,T., and Akino, T. (1991). Carboxyl methylation and farnesylation transducin y-subunit synergistically enhance its coupling metarhodopsin II. EMBO J. 70, 3669-3674. Palczewski, K., and Hargrave, P. A. (1991). Studies ing to arrestin. J. Biol. Chem. 266, 4201-4206.
of ligand
Annu.
of with bind-
Palcrewski, K., McDowell, J. H., Jakes, S., Ingebritsen, T. S., and Hargrave, P. A. (1989). Regulation of rhodopsin dephosphorylation by arrestin. J. Biol. Chem. 264, 15770-15773. Palczewski,
K., Rispoli,
C., and
Detwiler,
P. B. (1992).
The influ-
ence of arrestin (48K protein) and transduction. Neuron 8, 117-126.
rhodopsin
kinase
on visual
Perez-Sala, D., Tan, E. W., Canada, F. J., and Rando, R. R. (1991). Methylation and demethylation reactions of guanine nucleotide-binding proteins of retinal rod outer segments. Proc. Natl. Acad. Sci. USA 88, 3043-3046. Ray, S., Zozulya, S., Niemi, C. A., Flaherty, K. M., Brolley, D., Dizhoor, A. M., McKay, D. B., Hurley, J., and Stayer, L. (1992). Cloning, expression, and crystallization of recoverin, a calcium sensor in vision. Proc. Natl. Acad. Sci. USA, in press. Sather, W.A., manipulation ments. Proc.
and Detwiler, P. B. (1987). Intracellular of phototransduction in detached Natl. Acad. Sci. USA 84, 9290-9294.
biochemical rod outer seg-
Schnapf, J. L., Nunn, B. J., Meister, M., and Baylor, Visual transduction in cones of the monkey Macaca J. Physiol. 427, 681-713. Sitaramayya, A., and Liebman, rhodopsin and quenchingofcyclic vation by ATP at weak bleaches.
P. A. (1983). Phosphorylation GMP phosphodiesteraseactiJ. Biol. Chem. 258,12106-12109.
Sneyd, J., and Tranchina, D. (1989). Phototransduction an inverse problem in enzyme kinetics. Buil. Math. 784. Stryer, L. (1986). Cyclic rosci. 9, 87-119. Stryer, L. (1991). 266, 10711-10714.
GMP
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Vuong, T. M., and Chabre, transducin by endogenous
cascade
excitation
D. A. (1990). iascicularis.
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in cones: Biol. 57,749-
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J. Biol. Chem.
M. (1990). Subsecond deactivation of GTP hydrolysis. Nature 346, 71-74.
Vuong, T. M., and Chabre, M. (1991). Deactivation kinetics of the transduction cascade of vision. Proc. Nail. Acad. Sci. USA 88, 9813-9817. Yau, K.-W., ductanceof 289-327.
and Baylor, D. A. (1989). Cyclic GMP-activated retinal photoreceptor cells. Annu. Rev. Neurosci.
Yau, K.-W., and Nakatani, cytoplasmic free calcium 313, 579-582.
con72,
K. (1985). Light-induced reduction of in retinal rod outer segment. Nature
Zimmerman, A. L., Yamanaka, G., Eckstein, F., Baylor, D.A., and Stryer, L. (1985). Interaction of hydrolysis-resistant analogs of cyclic CMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments. Proc. Natl. Acad. Sci. USA 82, 8813-8817.
Vol. 8, 995-1002,
June, 1992, Copyright
0 1992 by Cell Press
Signal Flow in Visual Transduction
Leon Lagnado and Denis Baylor The Department of Neurobiology Sherman Fairchild Science Center Stanford University School of Medicine Stanford, California 94305
Over the past decade our understanding of visual transduction has advanced at a rapid pace, revealing molecular strategies for intracellular signaling that are exploited in a wide range of other cells, including olfactory receptor neurons, pinealocytes, retinal bipolar cells, and hepatocytes. Retinal photoreceptors have proved especially valuable in attacking signal transduction mechanisms because of their experimental advantages. The outer segment, the specialized organelle in which phototransduction occurs, can readily be obtained in quantity. The natural stimulus, light, is easily controlled. In photoreceptors, cGMP has been identified as the ligand controlling the ion channels that generate the light-evoked membrane hyperpolarization (Fesenko et al., 1985). Ca*+, once a candidate ligand for the channels, is now known to be important in regulating the sensitivity of the photoreceptor. The mechanism of transduction appears similar in the rods, which mediate vision at low light levels, and in the cones, which operate at higher light levels. Key questions now concern the molecular mechanics of individual components of the transduction apparatus and the way in which thecomponents function in concert to produce the light response. Our review focuses on the latter aspect and attempts to present recent advances as well as current problems. Visual transduction is the subject of a number of recent reviews (Liebman et al., 1987; Stryer, 1986,199l; Lamb and Pugh, 1990; Hurley, 1987; Signal
McNaughton, Flow
from
1991). Visual
Pigment
to Ion
Channels
Figure 1 diagrams the pathway bywhich signals originating in the visual pigment modulate the membrane channels that generate the rod’s electrical response. Absorption of light isomerizes rhodopsin’s retinal chromophore from the II-cis to the all-trans configuration, causing the protein to become enzymatically active. Active rhodopsin (Rh*) catalyzes the replacement of GDP by CTP on the G protein transducin (T). T-GTP activates cCMP phosphodiesterase (PDE), which hydrolyzes cGMP. In darkness cGMP binds to the ion channels and holds them open. Light-triggered hydrolysis of cGMP allows the channels to close, interrupting an inward current of Na+, CaZ+, and Mg 2+. The resulting membrane hyperpolarization lowers the rate of transmitter release from the synaptic terminal, triggering responses in second-order cells.
Review
Psychophysical experiments by Hecht et al. (1942) showed that human rods register single photon absorptions, which change only 1 of the 108rhodopsin molecules in the cell, and that a coincidence of single quanta1 absorptions in 5 rods reaches consciousness. The electrical response to absorption of a single photon is highly amplified (see upper trace in Figure 2A). At the peak of the response, 500 channels close; this is 3% of the number open in darkness. The response blocks the entry of several hundred thousand cations. The steps shown in Figure 1 account for this amplification. One photoisomerized rhodopsin activates hundreds of copies of transducin within a fraction of a second. Additional gain results from the powerful hydrolytic activity of PDE and the sizeable ion fluxes through individual open channels. A quantitative treatment, based on the known properties of the individual steps shown in Figure 1, accounts for the rising phase of the flash response over awide range of intensity (Lamb and Pugh, 1992), and models of the entire response have also been advanced (Forti et al., 1989; Sneyd and Tranchina, 1989). The single photon response of cones is too small to measure directly, but the dim flash response (lower trace in Figure 2A) is a scaled up replica of the single photon effect. This response is briefer than the rod’s and is diphasic; the oscillation is characteristic of a system with a negative feedback that operates after a delay. If the the large gain of the tranduction mechanism were constant, a steady background light of moderate intensity would close all the channels in the outer segment, preventing further changes in light intensity from being encoded. Instead, again control automatically reduces the sensitivity of transduction in background light so that some channels remain open and available for modulation by changes in light intensity. The intracellular Ca2+ concentration tracks the number of open channels and controls the gain of transduction. The Ca2+ level in the cell is determined by the balance of Ca2+ entry through the channels in the surface membrane and extrusion through the Na+: Ca2+,K+ exchanger (Hodgkin et al., 1985; Yau and Nakatani, 1985; McNaughton et al., 1986; Cervetto et al., 1989). When Ca* influx is blocked by closure of the channels in light, continued extrusion lowers the intracellular Ca” concentration with a time constant of 0.5 s. The concentration of Ca2+ thus provides a direct measure of the number of open channels during the recent past. Evidence that the drop in Ca2+ mediates the reduction of sensitivity in background light (adaptation) is provided by the finding that clamping the internal Ca2+ concentration blocks the gain reduction (Matthews et al., 1988; Nakatani and Yau, 1988a). The fall in Ca*+ concentration also speeds the recovery phase of the flash response, for recovery slows when Ca*+ buffers are put into a tranducing cell (Lamb et al.,
NWKMl 996
A
1 wj”
CSA
B
K
R
A C Ch E K PDE R Rh S T
Arrestin Cyclase Channel Exchange Rhodopsin kinase Phosphodiesterase Recoverin Rhodopsin S-modulin Transducin
photon
Hyperpolarization Figure
1. Molecular
Mechanism
of Visual
Transduction
(A) Schematic diagram of a retinal rod and the transducing molecules in the outer segment. The integral membrane protein rhodopsin lies embedded in the membranes of intracellular disks. Associated with the disk membranes are the peripheral membrane proteins transducin 0 and cGMP phosphodiesterase (PDE). In the surface membrane of the outer segment are the cCMP-activated cation channel and the Na+:Ca*+,K+exchanger. Soluble proteins arrestin, recoverin, s-modulin, and rhodopsin kinase are present in the cytosol. (B) Signal flow from pigment to cation channel. A photon isomerizes the chromophore of rhodopsin, which becomes enzymatically active (Rh’). Rh’ catalytically activates the GTP-binding protein transducin. T’ stimulates cCMP PDF, which hydrolyzes cGMP. The resulting fall in the level of cGMP closes the cation channel, interrupting the influxof Na+, Ca2’, and Mg2’. As a result, the cell membrane hyperpolarizes, causing a fall in the rate of release of transmitter from the synaptic terminal. Steps with appreciable amplificationtransducin activation, cGMP hydrolysis, and flux of cations through the open channel-are shown with bold arrows.
1986). The best-established biochemical correlate for these effects is the Ca*+ sensitivity of the rod’s guanylate cyclase, which synthesizes cGMP (Lolley and Racz, 1982; Koch and Stryer, 1988). Ca2+ effects on cyclase activity are mediated by the newly characterized protein recoverin (Lambrecht and Koch, 1991b; Dizhoor et al., 1991), which stimulates the cyclase at low Ca2+ levels, but fails to stimulate at the higher Ca*+ levels present in darkness. The scheme illustrated in Figure 1 is supported by extensive biochemical and physiological evidence, but it is incomplete. How is excitation shut off so that the dark state is restored? How is amplification in the enzymatic chain Rh*-T’-PDE” controlled? Where else might Ca2+ act? What loops and branches, other than those controlled by Ca2+, are hidden within the scheme? Rhodopsin
Shutoff
After a photon is absorbed, rhodopsin zymatically active state (metarhodopsin
enters the II, here
ende-
noted Rh’) within milliseconds. Since it is the trigger of the transduction cascade, Rh’ is the species whose activity is most highly amplified. If Rh’ were shut off in a single step, the active lifetime in different trials would be exponentially distributed, producing large variations in the size or duration of successive single photon responses. Contrary to this expectation, the rod’s response to a single photon is highly reproducible, as illustrated by the amplitude histogram in Figure 2B. The fit of the smooth curve to the experimental observations implies that the standard deviation of the quanta1 response amplitude was only about onefifth the mean. Although the mechanisms that shut rhodopsin off arequalitatively understood, it is not yet clear howthe reproducibility is achieved. We suggest below that feedback control of rhodopsin’s active lifetime may be involved. Whatever its molecular mechanism, reproducibility is a desirable property for a small signal that must be detected in noise. Rhodopsin’s enzymatic activity is terminated bytwo processes: ATP-dependent phosphorylation, which is catalyzed by rhodopsin kinase, and binding of the
Review: 997
Visual
Transduction
trials
I
I
I
0
0.5
1.0
-1
0
seconds Figure
2. Kinetics
and Amplification
of Rod and Cone
1
2
PA
Responses
to Weak
Stimuli
(A) Dim flash responses of a rod and red-sensitive cone from the macaque retina, recorded by suction electrode. Change in outer segment membrane current, averaged from multiple trials, is plotted as a function of time. Flash timing is shown below. For the rod the flash caused an average of one photoisomerization per trial. At the peak response 3.3% of the channels in the outer segment closed. For the cone the flash caused an average of 35 photoisomerizations per trial, and one photoisomerization closed an estimated 0.36% of the channels. (6) Reproducibility of the rod response to a single photon. Amplitude histogram of a macaque rod’s responses to dim flashes calculated to give 0.46 photoisomerizations per trial on average. In individual trials there were failures (peak near 0 PA), single photon responses (peak near 0.8 PA), and some responses to two photons (peak near 1.6 PA). The theoretical curve was constructed assuming that the amplitude of the single photon response was Gaussian distributed with a mean of 0.81 pA and a standard deviation of 0.15 pA, that the probability of photon responses per trial was Poisson distributed with a mean of 0.46, and that the baseline noise in darkness was Gaussian with a standard deviation of 0.14 pA. The dark distribution, fitted by this baseline Gaussian, is shown in the inset. Modified from Baylor et al. (1984) and Schnapf et al. (1990).
protein arrestin to phosphorylated Rh’. Assays of light-triggered PDE activity show that the catalytic lifetime of Rh’ is greatly prolonged if phosphorylation is blocked by removing ATP (Liebman and Pugh, 1980) or rhodopsin kinase (Sitaramayya and Liebman, 1983), or by proteolytic removal of the phosphorylation sites at rhodopsin’s carboxyl terminus (Miller and Dratz, 1984). Physiological evidence for the quenching role of phosphorylation is provided by the observation that the flash response of an internally dialyzed outer segment fails to turn off if ATP is removed (Sather and Detwiler, 1987; Nakatani and Yau, 1988b; see Figure 3) or if rhodopsin kinase activity is blocked by the inhibitor sangivamycin (Palczewski et al., 1992). Phosphorylated Rh’ binds arrestin, a 48 kd protein that is the most abundant soluble protein in the cytosol. Arrestin blocks activation of transducin by competitively inhibiting its binding to phosphorylated Rh’ but it has a much lower affinity for unphosphorylated Rh’, or for phosphorylated rhodopsin whose chromophore is II-cis retinal (Kuhn et al., 1984). There are at least 7 phosphorylation sites on rhodopsin’s carboxyl terminus, but it is not clear whether fully phosphorylated rhodopsin still requires arrestin to complete its inactivation, or whether arrestin binds to partially
phosphorylated Rh’to block activation of transducin completely. Bennett and Sitaramayya (1988) reported that without arrestin binding, rhodopsin shutoff occurred slowly and required phosphorylation at 1216 sites, while in the presence of arrestin complete inactivation was achieved rapidly after the phosphorylation of only one site. The absence of multiple phosphorylations when arrestin was present suggests that binding of arrestin prevents further action of rhodopsin kinase. This would occur if arrestin, rhodopsin kinase, and transducin all recognized the same site on rhodopsin. In outer segments dialyzed with a patch pipette, phytic acid, an inhibitor of arrestin’s binding to Rh’, preferentially slowed the recovery of bright flash responses, in which complete phosphorylation of rhodopsin would be expected to require a long time (Figure 4; Palczewski et al., 1992). This result supports the notion that arrestin binds to partially phosphorylated Rh’ to shut it off completely. Although it is clear that phosphorylation lowers the enzymatic activity of Rh’, the relation between the number of phosphates added and the enzymatic activity of Rh’ is not known. Nathans (1987) and Liebman et al. (1987) suggested that progressive shutoff by multiple phosphorylations might extinguish rhodopsin’s
Neu”JIl 998
1 1 mMz;cplic -
0
2 a ET z 5 0
r
mhl-cyclic GMP t 0.5 mwGTPj A
1 muxyclic GMP + 0.5 mwGTP
Figure 3. Evidence That Generation of the Light Response Requires GTP and That Shutoff Requires ATP
+ 0.5 mwATP L--
Membrane current of an isolated rod outer segment was recorded with a suction electrode, and the end of the outer segment was knocked off with a glass probe to allow dialysis of the interior. The lower trace shows the timing of light flashes. Adding 1 mM cCMP opened the channels, allowing an inward current to flow. The first flash (-3.8) did not generate a response. When 0.5 mM GTP was added most of the lightsensitive channels closed because Rh’created by the first flash was still present. This response did not recover, and a second flash shut off more current. On addition of 0.5 mM ATP, the light-sensitive current recovered, and the responses to subsequent flashes were transient, as newly created Rh’ was shut off. The intensity of the last flash was raised, causing a larger responsethan the previous one. Reproduced in modified form from Nakatani and Yau (1988b) with the permission of the publisher.
-
-40
-80
:
-120
I L
I
I I
0
!
I I
I
100
1
200 Time
I
300
fs)
activity reproducibly. In the version of such a scheme that gives the best reproducibility, phosphorylations at n identical and independent sites each reduce the activity by the same amount. The expected ratio of the standard deviation to the mean active lifetime is then given by l/,/ii. To explain the constancy of the single photon effect shown in Figure 2B, this mechanism requires 25 phosphorylations, which is unrealistically large. If instead arrestin binds to singly phosphorylated rhodopsin to terminate its activity, as suggested by Bennett and Sitaramayya (1988), the active lifetime would vary much more widely, and the standard deviation would equal the mean. Reproducibility of the
single photon responsewould be improved if rhodopsin’s active lifetime were under feedback control. Complete recovery after a flash requires that the phosphate groups be removed from phosphorylated rhodopsin and that the II-cis retinal chromophore be replaced. The opsin phosphatase in the rod outer segment is of type 2A (Fowles et al., 1989). Phosphatase activity is blocked when arrestin is bound to phosphorylated Rh’, and arrestin does not dissociate until phosphorylated Rh’ has decayed to the inactive species phosphorylated metarhodopsin III, thus preventing reactivation (Palczewski et al., 1989). After removal of the phosphates, rhodopsin is regenerated
Figure 4. The Effect of Inhibiting Segment
of the Light
(A) Family of responses configuration. (6) Family of responses to phosphorylated Rh’. with the permission of
to flashes
the Action of increasing
of Arrestin intensity,
on the Recovery recorded
from
an isolated
Response outer
in Internally
segment
Dialyzed
by a patch
to the same flashes, recorded by a pipette containing 1 mM phytic acid, which inhibits Recovery of these responses was greatly prolonged. Reproduced in modified form from the publisher; copyright Cell Press.
pipette
Isolated
Rod Outer
in the whole-cell
the binding of arrestin Palczewski et al., (1992)
Review: 999
Visual
Transduction
by spontaneous combination of II-cis retinal with the protein opsin. Electrical recordings from rods support the idea that resumption of enzymatic activity following the initial burst is very improbable. The current noise in darkness is low, and little increase occurs after exposure to dim light (Baylor et al., 1980). “Trickleback” excitation becomes apparent only after exposure to bright lights (Lamb, 1980; Baylor et al., 1984) sufficient to reveal shutoff inefficiencies of 0.1% or less. Shutoff of the pigment’s enzyme activity in cones has not yet received much attention. The small size and short duration of their photon responses suggest that the cone pigment is shut off faster than rhodopsin. Similarly, cone transducin may also be shut off more rapidly than rod transducin. Shutoff
of Transducin
and
PDE
When Rh’ activates transducin, the exchange of GTP for GDP causes transducin to dissociate. The active T,GTP portion leaves the Tsu complex and stimulates PDE by binding to, and removing inhibition exerted by, they subunit. T, has intrinsic GTPase activity. Persistent doubts about whether CTP hydrolysis alone terminates the action of T’ have been countered by two kinds of recent evidence: When the hydrolysisresistant analog GTPyS is put into a transducing rod,
Figure vation
5. Microcalorimetric after a Flash
Measurement
of Transducin
Inacti-
The rate of heat release from a suspension of rod outer segments, which is proportional to the rate of CTP hydrolysis, is plotted as a function of time. Flashes were delivered at the times indicated bythearrows.TheGTPconcentrationwasZmM.ATPwasabsent, but Rh’ was inactivated by 50 mM NH20H. After a flash, the majority of the heat release was complete within 1 s. In traces a-c a second flash was delivered at increasing delay after the first, revealing that heat liberation from the second flash was reduced for about 10 s. This is attributed to the time required for tranducin to reassociate after the first flash. Reprinted in modified form from Vuongand Chabre (1990) Nature 346,71-74; Copyright 0 1990 Macmillan Magazines Limited.
the flash response fails to recover (Sather and Detwiler, 1987; Lamb and Matthews, 1988). In addition, eleganttime-resolved microcalorimetry byvuongand Chabre (1990, 1991) shows that, after a flash, GTP in the outer segment is hydrolyzed on a time scale shorter than that of the electrical response (see Figure 5). The rate of hydrolysis from the heat measurements is much faster than that from previous biochemical measurements of steady-state turnover because a step subsequent to GTP hydrolysis limits turnover rather than hydrolysis itself. A prime candidate for the ratelimiting step, which requires about 10 s, is reassociation of T, with TBr- The slowness of this reaction and the speed of Rh’ shutoff implies that T’ is allowed only a single round of PDE activation after a dim flash. Heat measurements reveal that after a flash, PDE’ falls with much the same time course as that of T (Vuong and Chabre, 1991). This indicates that hydrolysis of GTP on T’ is the rate-limiting step in PDE shutoff. Two mechanisms that may control the hydrolytic shutoff of T’-GTP have been proposed by Arshavsky et al. (1991), who measured light-stimulated GTPase activity by the release of labeled phosphate from GTP. At low concentrations of GTP, when all the hydrolysis should occur on T’ activated for the first time, two components of hydrolysis, with half-times of 1 and IO s, were resolved. The size of the fast component corresponded to the quantity of PDE in the outer segment, suggesting that the binding of T’-GTP to they subunit of PDE increases the rate of GTP hydrolysis IO-fold. This effect would increase the probability that GTP hydrolysis by T’occurs only after PDE is activated, thus helping to ensure that excitation from rhodopsin reaches PDE. A second mechanism of control was indicated by the finding that the fast GTPase activity, attributed to T,CTP-PDE,, was abolished by 5 PM cGMP, a concentration only slightly higher than that present in darkness. This result suggests that cGMP itself acts as a negative feedback control signal in the light response. Thus, as thecGMP level fell in the light, the GTPase activity would be disinhibited, reducing light-stimulated PDE activity and countering the fall in cGMP. Recently Arshavsky and Bownds (1992) have demonstrated that the isolated y subunit of PDE can accelerateGTP hydrolysis bytransducin and havesuggested that cGMP inhibits this effect by binding to noncatalytic sites on the a and B subunits of PDE. Another mechanism by which transducin’s activity may be controlled is covalent modification of its y subunit, which interacts with Rh’ to cause exchange of GTP for GDP on the a subunit of transducin. Farnesylation of they subunit causes a large increase in transducin’s activation by Rh’ (Fukada et al., 1990). Methylation, a potentially reversible modification, causesafurtherenhancementofthefarnesylatedsubunit’s activity (Ohguro et al., 1991; Perez-Sala et al., 1991). Enzymes that can methylate and demethylate the y subunit are present within the outer segment, and only the farnesylated subunit is able to be methylated (Perez-Sala et al., 1991).
Neuron 1000
:;a2+ /
Open channels 4
Exchange
* Outside
Figure6 Flow Transduction
of
Ca2+ Signals
in Visual
Ca2+ enters the outer segment through open channels and is extruded by the Nat: Ca2+,K+ exchanger. Ca2+ inhibits cyclase activity by binding to the protein recoverin (R) and blocking its stimulation of cyclase. Ca2+also stimulatestheactivityof PDE, bya mechanism that remains to be determined. Possibilities are prolonging the lifetime of PDE’ by inhibiting reassociation of the y subunit thatturns it off, inhibiting hydrolysis of CTP on T’, and inhibiting shutoff of Rh’. GTP
Cyclase-R
*
cG;vlP
PDE*
)5’-GMP
0 T
Sites
of Action
of Ca2+
It is clear that the light-induced fail in intracellular Ca2+ accelerates recovery of the light response and is a key signal for light adaptation, but the sites at which Ca2+ acts are only partly understood (see Figure 6). The effect on the synthetic enzyme guanylate cyclase is the best documented. Cyclase activity increases over 5-fold when Ca2+ is lowered from 300 nM, the dark level, to about 10 nM, the level reached in bright light. This effect is highly cooperative, with a Hill coefficient of 3 (Koch and Stryer, 1988; Dizhoor et al., 1991). The effect of Caz+ is mediated by recoverin, a soluble 23 kd Ca*+-binding protein that binds to the cyclase and stimulates it when Ca*+ is low (Lambrecht and Koch, 1991b; Dizhoor et al., 1991; Ray et al., 1992). Phosphorylation is reported to increase recoverin’s activity 2-fold when Ca2+ is reduced to levels below 100 nM (Lambrecht and Koch, 1991a). Thiswould reinforcethe stimulation of cyclase activity. Recoverin shows sequence homologies with visinin, a cone Ca2+-binding protein of unknown function. New evidence reveals that Ca*+ prolongs PDE’s activation by light. Kawamura and Murakami (1991) internally perfused truncated rod outer segments with solutions that supported a light response but minimized the effect of cyclase activity. The response to a flash became briefer when internal Ca2+ was lowered from 1 PM to 30 nM. Biochemical assays of light-triggered PDE activity confirmed that the Ca*+effect was exerted on PDE rather than the cyclase. The Ca*+ sensitivity appears to be mediated by a soluble 26 kd protein, dubbed S-modulin, which in the presence of Ca2+ binds to the disk membranes to prolong PDE’s activation by a flash. These findings leave open the question of where in the chain Rh*-T*-PDE’ Ca2+ acts (see Figure 6). In principle, light-induced PDE activity might be modified by changing the reaction rate or active lifetime of the species PDE’, T’, or Rh’. The fact that the
early rising phase of the flash response is invariant during adaptation (Baylor and liodgkin, 1974; Lamb, 1984) points to an effect on lifetime rather than reaction rate. At PDE’, Ca:+ might SLOW the reassociation of the inhibitory y subunit that terminates activity. At T’, Ca2+ might inhibit CTPase activity, while at Rh’ it might inhibit shutoff. A Ca2+ effect on shutoff of Rh* seems particularly interesting, since it could make the activity of Rh” reproducible. Shutoff of an individual Rh’, which might otherwise fluctuate widely, would occur at a time dictated by a stereotyped fall in Ca2+ concentration. Control of the Rh’ lifetime could be achieved if Ca*+ inhibited the phosphorylation of Rh’ or the binding of arrestin to phosphorylated Rh”. Huppertz et al. (1990) report that the Caz+ binding capacity of protein extracts from rods is halved when they are depleted of arrestin, and they conclude that arrestin binds Ca*+ in a first-order manner with a Kd of 2 bM. Their quantities are in rough agreement with measurementsof Ca*+ buffering in single rods by Lagnado et al. (1992), who found a buffer with a, capacity of about 200 f.rM and a Kd of 1 PM. In contrast, Palczewski and Hargrave (199l)found no evidence that Ca*+ binds to purified arrestin. The sites of action of Ca2+ remain an important area for further work. In addition, it will be important to determine the relative contributions of the different Ca*+ effects. How would adaptation be affected if the action of recoverin, or S-modulin, were selectively blocked? Channel
Control
Closure of the cationic channels during the response to light is adequately explained by removal of the cGMP that holds them open in darkness, and it is not necessary to suppose that any other messenger directs channel closure on a short time scale. Thus, when the hydrolysis-resistant cGMP analog 8-BrcGMP was put into a transducing rod, the cationic channels were locked in the open state and the light
Review: 1001
Visual
Transduction
response was severely attenuated as expected from very limited cGMP hydrolysis (Zimmerman et al., 1985). This result does not, however, rule out regulation of the channel’s responsiveness to cCMP on a longer time scale. Indeed, the need for regulation of the channel is suggested by the fact that even in darkness only about I%-2% of the channels are open (reviewed in Yau and Baylor, 1989). The large number of channels available to be activated put a rod in danger of a catastrophic influx of cations from a modest rise in cGMP concentration. Alterations in the channel’s cCMP affinity would be a powerful potential site for regulation, because channel activation appears to require the binding of three or more ligand molecules. At the low cGMP concentrations present in darkness, a 2-fold change in channel affinity would produce an 8-fold change in activation. Hints of such a control is provided by the wide variations in the cGMP sensitivity of excised patches from one rod to the next and from one laboratory to another. Gordon and Zimmerman (1990, ARVO, abstract) suggest that such changes in affinity may depend on an ATP-dependent phosphorylation. Conclusion Three major accomplishments have marked recent research on signal flow in visual transduction. First, cGMP has been identified as the diffusible messenger that controls the transduction channels and brings information to them from the disks. Second, the enzymatic mechanisms that amplify signals issuing from single rhodopsin molecules have been elucidated. Third, Ca*+ has been identified as a key messenger for light adaptation and response shutoff. Not so well understood is how the explosive amplification of the enzyme cascade is controlled. This control adjusts the gain in changing background light levels, produces a reproducible single photon effect, and gives the different performance characteristics of rods and cones. Such a control might also conserve metabolic energy in rods during daylight, when their response is saturated and GTP would otherwise be wastefully consumed at high rate. This regulation might operate on a much slower time scale than the light response. Immediate problems in the control of transduction concern how rhodopsin and transducin are shut off and how Ca2+ acts to stimulate lighttriggered PDE activity. It seems safe to guess that new loops and branches will be discovered within this intricate mechanism and that many of the outer segment proteins in search of a function will turn out to be controllers of the cascade. We may also anticipate the characterization of new messengers that measure the state of the system on time scales longer than those of cGMP and Ca*+. One candidate substance may be Mgti, which enters the outer segment in darkness and should fall with a time constant of tens of minutes in the light. Finally, it will be interesting to see whether similar mechanisms of gain control have
been adopted strategies for
in other cells signal amplification.
that
use
homologous
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