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Molecular mechanisms of vertebrate photoreceptor light adaptation
410
Molecular mechanisms of vertebrate photoreceptor EN Pugh Jr*, S Nikonovtand
TD LambS
An important recent advance in the understanding of vertebrate photoreceptor light adaptation has come from the discovery that as many as eight distinct molecular mechanisms may be involved, and the realization that one of the principal mechanisms is not dependent on calcium. Quantitative analysis of these mechanisms is providing new insights into the nature of rod photoreceptor light adaptation. Addresses
*tFM Kirby Center
for Molecular Ophthalmology, Department of Ophthalmology and Institute of Neurological Sciences, Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104-6069, USA *e-mail: [email protected] te-mail: [email protected] fDepartment of Physiology, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK; e-mail: TDLl @cam.ac.uk
Current Opinion
0 Elsevier
Science
in Neurobiology
Ltd ISSN
1999,
light adaptation
9:41 O-41 8
0959-4388
Abbreviations Arr arrestin lX$*+l, internal concentration of free calcium activated catalytic subunit of PDE activated moiety of G protein G GC guanylyl cyclase GCAP GC activating protein background intensity 43 half-activation concentration ‘612 PDE phosphodiesterase R* activated rhodopsin Ret recoverin RK rhodopsin kinase flash sensitivity SF dark-adapted SF sFD
Introduction Light adaptation refers to the ability of the visual system to adjust its performance to the ambient level of illumination. In a normal cycle of day and night, the illumination at the earth’s surface varies over 11 orders of magnitude [l], making light adaptation fundamentally important to the normal functioning of the vertebrate visual system. Much of the daily cycle of sensitivity adjustment in the vertebrate retina is managedby diurnal switching between rodand cone-dominated pathways. Individual cone photoreceptors have a prodigious capacity for light adaptation: their photocurrents never saturate in response to steady illumination [Z]. Thus, cones are the ultimate target for investigation of the full panoply of cellular mechanisms involved in vertebrate photoreceptor light adaptation. For the rod pathway, in contrast, the principal mechanisms of scotopic adaptation result from alterations in signal processing at postreceptoral stageswithin the retina [3], and the rods themselves adapt over only a modest
range ofsteady intensities before being driven into saturation. Nonetheless, because considerably more detail is currently known about the molecular processesin rods, we shall focus on these cells in this review. The mechanisms known or hypothesized to underlie light adaptation are shown in Figure 1, which depicts the rod phototransduction cascade. The contributions of several of these mechanisms to adaptation have recently been reviewed elsewhere [4-61, and the important role of calcium-independent processeshas been stressed [4,5].
Functional
characteristics
Desensitization
of light
adaptation
and acceleration
Two hallmarks of photoreceptor light adaptation are the reduction in sensitivity and the acceleration in response kinetics that occur in the presence of background illumination [7]. The dependence of these two parameters on background intensity, in salamander rods, is plotted in Figures 2b and Zc, respectively. The flash sensitivity (3,) is defined asthe peak amplitude of the dim-flash response divided by the flash intensity, and, according to convention, this parameter is plotted in normalized form as SF& D, where SFD is the dark-adapted sensitivity. Figure 2b shows that over roughly 2-3 decades of background intensity (I), flash sensitivity in salamander rods declines according to Weber’s Law - that is, roughly inversely with background intensity. Associated with this decline in sensitivity is an acceleration of the response kinetics, exemplified by the shortening of the time-topeak of the dim flash responseshown in Figure Zc. Range extension
by decline
in calcium
About ten years ago, a number of investigations established that when the internal calcium concentration ([Ca’+]i) is maintained near its resting (dark) level, rod and cone photoreceptors fail to show normal adaptational behavior and instead function over a very restricted range of intensities [8-131. Figure 2a plots the steadystate circulating current of salamander rods from several studies in which [Ca’+]i was free to change (open symbols) or was clamped (filled symbols). The results illustrate the severe range restriction imposed by blocking the normal decline in [Ca’+];, and, conversely, they show that the operating range is extended more than lOO-fold when [Ca’+]i is free to change. It is important to distinguish between the role of calcium in extending the photoreceptor’s operating range and any role it might have in modulating flash sensitivity. Figure 2a illustrates that in the absence of a change in [Ca”+]i, the rod would be driven into saturation by illumination of quite low intensity, and its sensitivity would thereby be drastically attenuated. The normal decline in [Ca2+li rescues the cell from this saturation, preventing its
Molecular
Figure
mechanisms
of vertebrate
photoreceptor
light
adaptation
Pugh, Nikonov
and Lamb
411
1
(a)
Cunent
Schematic of the rod phototransduction cascade. (a) The activation steps of the cascade. Following absorption of a photon (hv), the activated rhodopsin (R*) repeatedly contacts molecules of the heterotrimeric G protein (G), catalyzing the exchange of GDP for GTP, producing the active form G*cx (=Ga-GTP). Two G’cx subunits bind to the two inhibitory y subunits of the phosphodiesterase (E), thereby activating the corresponding a and 5 catalytic subunits, forming E*, which then catalyzes the hydrolysis of cGMP. The consequent reduction in the cytoplasmic concentration of cGMP leads to closure of the cyclic-nucleotide-gated channels and blockage of the inward flux of Na+ and Ca*+ (thereby reducing the circulating electrical current). The exchanger continues to pump Ca*+ out, so the cytoplasmic Ca*+ concentration declines, activating three ‘calcium feedback’ mechanisms, which are illustrated in this panel and in (b). Loss of Ca*+ from guanylyl cyclase activating proteins (GCAPs) allows them to bind to a cytoplasmic domain of the guanylyl cyclase (GC), increasing its activity; loss of Ca*+ from calmodulin (CM) causes it to dissociate from the channels, lowering the KIj2 of the channel for cGMP. The boxed symbols ‘a’ and ‘5’ are used throughout the text and in Table 1 to refer to the rate of synthesis of cGMP by GC and the rate constant of
sensitivity from being annihilated at low backgrounds, and thus extending its operating range. In other words, the decline in [Caz+]i does not actually cause the desensitization illustrated in Figure Zb,c - rather, it tends to do just the opposite.
Classification their effects
of molecular on operating
mechanisms by range and sensitivity
In keeping with the distinction made in the preceding paragraph, it is useful to classify the molecular mechanisms
Opinion
in Neurobiology
hydrolysis of cGMP by E’, respectively, and should not be confused with the protein subunit labels. (b) Inactivation of R*. At the dark concentration of [Ca”+]i (let3 side of diagram), most of the recoverin (Ret) is in the Ca*+-bound form at the membrane; Ret-2Ca forms a kinase (RK), blocking its activity. Thus, at , complex with rhodopsin resting Ca*+ levels, contact between R’ and uncomplexed RK occurs infrequently. When [Ca*+]i drops during the light response (arrows indicate progression of time), Ca *+ dissociates from Ret, which moves into solution. The concentration of free RK rapidly increases, increasing the frequency of interaction with R’, and leading to its rapid phosphorylation. Arrestin (Art) then binds, substantially quenching the R* activity. (c) Inactivation of G*a-E’. G’ is inactivated when the terminal phosphate of its bound GTP is hydrolyzed. Although the G protein has intrinsic GTPase activity, this capacity is only activated when the G* is bound to the y subunit of PDE (PDE-r) and when, in addition, the GTPase-activating protein or GAP factor (RGS9-G55) also binds. The resulting tetra-molecular complex, G*a-PDErRGS9-G55, rapidly hydrolyzes the GTP to GDP, returning the Ga subunit to its inactive form, so that the E* and G*a are simultaneously inactivated [59’,60].
underlying light adaptation according to their effects on the two fundamental characteristics illustrated in Figure 2: operating range and sensitivity. In the past, it has sometimes been assumed that a single class of mechanism and a single messenger (e.g. [CaZ+]i) might regulate all aspects of photoreceptor light adaptation; but, as we shall show, this is clearly not the case. In Table 1, we have gathered nine hypotheses about the molecular mechanisms underlying photoreceptor light
412
Sensory
Table
1
Mechanisms
systems
of light
adaptation
Mechanism/ hypothesis (as background increases)
in vertebrate
rod and cone
photoreceptors.
Protein mediating the effect
Predicted effect on operating range
Predicted effect on flash sensitivity
R*
Extends
Decreases
Yes
+, -I +
?
RK
Extends
Decreases
Yes
+, -, +
Recoverin
Increase in steady state cGMP hydrolysis rate constant (p)
PDE
Reduces
Decreases
No
-9 -10
Steady increase in cGMP synthesis rate (a)
GC
Extends
Increases
Yes
+, +, +
Transient increase in cGMP synthesis rate (AC&))
GC
No effect
Decreases
Yes
Decrease channels
in Kvs of for cGMP
cGMP-gated channel
Extends
Increases
cGMP-gated channel
Reduces
(fewer
Increase in Ca2+ buffering (B& of cytoplasm
Recoverin
Pigment
Cone photopigment
Decrease R* catalytic
in
Decrease R* lifetime
in (Q)
Response compression channels
Ca2+ mediated?
Profile*
identity regulatory Ca2+-binding protein
of
Comment
References
Can be rejected; see text
KW71
Accelerates recovery
111 ,18,35, 40-42,43*,56*]
Principal Cap+independent factor
[14,34”1
GCAP1,2
Restores circulating current
[13-l
0, -1 +
GCAPl,2
Accelerates recovery
[34”1
Yes
+, +, +
Calmodulin
Restores circulating current
[26-31,32”1
Decreases
No
--
Saturation causes drastic decline in SF
L’,8,331
No effect
Increases
Yes
0, +, +
Extends
Decreases
No
+, -I 0
gain
-
-
1 ! 0
open)
bleaching
*The profile column calcium-dependence).
summarizes
the
characteristics
described
in the
three
preceding
Results decrease Ret-2Ca
-
columns
(i.e. operating
from in
Incapacitates rods, yet underlies much of cone light adaptation range,
6,23-251
sensitivity,
121
and
adaptation. The majority of these mechanisms have been considered previously and are reviewed in, for example, [4-61. For each potential mechanism, Table 1 lists three characteristics: first, the predicted effect on the cell’s operating range; second, the predicted effect on its flash sensitivity; and third, whether the mechanism is directly activated by the decline in [Ca’+]i. On the basis of these criteria, each mechanism can be assigned a three-component ‘profile’ (sixth column of Table 1). The hypotheses are arranged in sequence of the protein mediating the effect (second column of Table l), but in discussing them, we shall first consider those that underlie range extension, and then proceed to those governing sensitivity.
(Figure la). Many studies - including the seminal investigation of intact salamander rods by Hodgkin and Nunn [ 141 and the thorough investigation of truncated amphibian rods by Koutalos etal. [l&16] -have demonstrated that GC can be activated to at least 10 times its basal level by the normal decline in [Ca’+]i. The dark-adapted resting level of [Ca’+]i is 400-600 nM, but [Ca’+]i drops to about 30-50 nM when the cGMP-gated channels have been held closed for several seconds [17-211 under conditions that allow the Na+/CaZ+,K+ exchanger to operate normally [ZZ]. The decline in [Ca’+]i causes activation of GC, with a Z& near 100 nM and a Hill coefficient of -2, which is mediated by the calcium-binding proteins GCAPl and GCAPZ (for reviews, see [23X5]).
Principal
Calmodulin effect on cGMP-gated channels (Table 1, row 6) A second well understood mechanism of range extension is the calmodulin-dependent shift in the K1,2 of cyclicnucleotide-gated channels [‘Z--29]. In rod channels, this shift is effected by the binding of calmodulin to two binding sites on the amino- and carboxy-terminal cytoplasmic loops
mechanisms
of range
extension
Guanylyl cyclase (Table 1, row 4) The most thoroughly investigated molecular mechanism for range extension in phototransduction is the activation of particulate guanylyl cyclase (GC) by the decline in [Ca’+]i that accompanies the steady-state light response
Molecular
Figure
mechanisms
of vertebrate
photoreceptor
2
Circulating
(a)
Steady circulating current, sensitivity, and kinetics of salamander rods in the presence of background illumination. The smooth curves plotted through the data in parts (a,b) are empirical in character (i.e. they are not derived from theoretical analysis of the transduction cascade). (a) Normalized circulating current F, of salamander rods as a function of steady illumination, replotted from several investigations. The open symbols represent data obtained in Ringer’s solution; the filled triangles represent data obtained with [Ca*+]i clamped to the resting (dark) level. Open diamonds, figure 2 of 1141; filled down triangles and open down triangles, figure 2 of [8]; open up triangles, figure 9 of [16]; open circles and circles with embedded plus (S Nikonov, EN Pugh Jr, unpublished data). The smooth curve for the calcium-clamp data is F, = exp(-l/l,,), with Is = 7, while that for the Ringer’s data is F, = /n/(/n + I#), with Is = 425, n = 0.7. Note that (a) and (b) share a common abscissa (intensity axis), and that all intensities are expressed in photoisomerizations per second (ct, o-1). (b) Flash sensitivity, from two of the same studies. Symbols as in (a); in addition, the gray triangles were obtained with [Ca*+li clamped to the level reached during exposure to the steady background in Ringer’s, The curve drawn through the calcium-clamp data is the same as in (a); the curve through the Ringer’s data was calculated according to Weber’s law, SF/.&o = //(1+/c), with /c = 40; the ‘corrected’ curve was obtained by dividing the Weber-law curve by the curve for F, in panel (a). (c) Dimflash responses of a salamander rod obtained in the dark (top trace) or in the presence of a background of progressively higher intensity. Each trace was calculated by dividing the averaged raw response r(t) by the maximum response to obtain R(t) = r(t)/r,,,,; the normalized response R(t) was then scaled by dividing by the flash intensity (Q). These traces correspond to the circles with embedded plusses in (a) and (b). The smooth curve through the rising phase of the responses was computed with the pure activation model of reference [38], with amplification constant A = 0.06 s-2, and effective delay f,, = 55 ms.
5
increase
sensitivity
Both the steady-state activation of GC and the calmodulin-dependent decline in the Kl12 of the channels cause an increase in the sensitivity of the rod. Because both of these mechanisms contribute to range extension, their effect on sensitivity is paradoxical to the standard view
and
Lamb
413
current
0.5
Flash sensitivity
(b)
s -L SFD
10000 100 1000 I, steady illumination (ct, s.‘)
0
. . ,..., Q,
10
Normalized
(cl
2.0
t 1.0 * 0.0
dim-flash
responses
i--x
I
-\J--.n
4
0
I
1
1 2 Time from flash (s) Current
Opinion
J
3 in Neurobiology
of adaptation, which has led to the expectation that adaptational mechanisms that extend the operating range should necessarily decreasesensitivity. Although a rigorous proof that these two mechanisms increase flash sensitivity requires analysis beyond the scope of this review, the key idea is straightforward: by opening up more channels than would otherwise have been available, the rod’s circulating current is increased by both processes,so that a dim test flash (eliciting a fixed pulse E*(t) of activated phosphodiesterase catalytic subunits) generates a larger response; thus, although the fraction of channels closed is unchanged, the suppression of circulating current is larger.
Mechanisms actually
Nikonov
0.0
of the B subunit of the channel [30,31]. While the contribution of this mechanism to range extension is relatively small in rods [16,24], a related mechanism exists for cone channels that is much more powerful and that involves a calciumbinding protein distinct from calmodulin [32”]. These two mechanisms, cyclase activation and a shift in Kl12 of the channels, are thought to account for most (if not all) of the extension in the operating intensity range that takes place in rods when [Ca’+]i is free to drop. For primate rods, the range extension has been modeled in terms of GC activation alone [ 131. For amphibian rods, in which the range extension is greater, the behavior has been modeled with a combination of GC activation, a calmodulin-dependent shift in the K,lz of the channels, and a third mechanism identified as a reduction in the average number of phosphodiesterase (PDE) catalytic subunits activated (E*) per photoexcited rhodopsin (R*) at higher background intensities [16,24] (see Figure 1). Elucidation of the molecular basis for this last mechanism remains a focus of current research and will be discussed below.
Pugh,
1.0
-
CC and calmodulin
light adaptation
Response
of desensitization
compression
(Table
1, row 7)
When the cGMP-activated current of the rod is reduced by steady illumination, the total output range of the cell -that is, the current available for modulation by an increment in illumination - is reduced. Compression of this kind haslong been understood to contribute to lossof
414
Sensory systems
flash sensitivity [7,33], but deserves clarification in terms of the phototransduction G-protein cascade. To illustrate the mechanism, suppose that output compression were the sole process causing the rod’s flash sensitivity to decreasewith increasing background intensity. In the context of the cascade schematic depicted in Figure 1, this would be equivalent to assumingthat E*(t), the transient number of additional phosphodiesterasecatalytic subunits activated by a given flash, is independent of background intensity [13]. With this assumption, analysis of the equations governing cGMP hydrolysis and synthesis [34”] showsthat a constant fraction of the open channels would be closed by a given flash, irrespective of background intensity. It is therefore possible to ‘correct’ responsesfor output compression simply by normalizing them with respect to the remaining circulating current, as was done originally by Torre et al. [35]. In terms of sensitivity, one may plot (SF/SFD)/F1, where FI is the circulating current remaining in the presence of a background (of intensity I) expressed as a fraction of the circulating current in darkness. In Figure Zb, rather than correcting the individual points, we have instead applied this correction to the curve, which therefore represents the underlying (or noncompressive) component of desensitization. Thus, the broken curve reveals the decreasein flash sensitivity that must be accounted for by molecular mechanisms within the transduction cascade. Intrinsic
‘gain’
of R* activity
(Table
1, row 1)
One hypothesis that has been proposed to account for decreased flash sensitivity in rods is that backgrounds (even dim ones) causea reduction in the ‘gain’ of the reactions coupling R* to activation of PDE [l&36,37], perhaps through the action of an asyet unidentified calcium-binding protein [36]. Two forms of this hypothesis are possible: either that the quantum efficiency for activation of R* by light is reduced, or that from the instant of its creation, an R* formed in the presence of a background is less effective in catalyzing the activation of the G protein to G* than one formed in dark-adapted conditions. We have found strong evidence against these hypotheses - and indeed against any hypothesis predicting a reduction in the ‘amplification constant’ (defined in [38]) - for background intensities that suppress up to 75% of the circulating current (S Nikonov, EN Pugh Jr, unpublished data). The evidence is straightforward; as illustrated in Figure Zc, the fractional suppressionof circulating current per unit flash intensity follows an invariant early trajectory, independent of background intensity. The invariance illustrated in Figure 2c contrasts with other recent reports [5,6,18,37]. However, similar invariance hasbeen reported in other studies [ 11,351,and very recently has been found in the responsesof human rods [39]. We suspect that the differences in interpretation between these different reports may have arisen because it had not previously been appreciated that acceleration of the inactivation reactions could diminish the responseat such early times, as discussedbelow.
Earlier
onset of recovery
In Figure Zc, the responsesof the rods ‘peel off’ from their common initial trajectory at progressively earlier times as the background is made more intense. This is consistent with the idea that the ‘lifetime’ of one or more of the cascade intermediates is shortened during exposure to steady light, a conclusion reached many years ago [7], before the G-protein cascade was known. In the context of the scheme of phototransduction shown in Figure 1, there are only three lifetimes that could be shortened: the effective lifetime of R*, the lifetime of the G*/E* complex, and the lifetime of free cGMP As there is no evidence for regulation of the G*/E* lifetime by calcium or by any other by-product of normal light exposure, this hypothesis will not be discussedfurther. However, there is a wealth of evidence for reduction of the other two lifetimes, as will be discussedin the next three sections. Calcium dependence of the lifetime disc-based intermediate
of a
Abundant physiological evidence supports the hypothesis that the lifetime and/or gain of one of the disc-associated intermediates in the rod transduction cascade is calcium-dependent, in a manner that would contribute to the decline in sensitivity in the presence of background illumination [11,18,35-37,40-42,43-l. A key piece of the evidence, obtained using a ‘step-flash’ paradigm, is illustrated in Figure 3. Normally, the time course of recovery from a bright flash is accelerated by prior light adaptation (Figure 3a); however, when [Ca2+li is clamped at the dark level for the duration of the light step, the recovery phase of the subsequent flash responsedoes not speed up (Figure 3b). Thus, the accelerated recovery in normal conditions is mediated by the decline in [Ca’+]i and not by molecular products of the light-driven reactions. The results of these step-flash experiments, in conjunction with the evidence cited above that the gain of the activation reactions is unchanged by light adaptation, show that the lifetime of a disc-associatedintermediate is shortenedby the lowered [Ca”];. Furthermore, becausethere is considerable evidence that the ‘dominant’ (i.e. rate-limiting) inactivation time constant of the disc-associatedreactionsis unaffected by light adaptation or by changesin [Ca2+li [34”,44,45], it follows that the mechanismunderlying the step-flasheffect is a reduction in a ‘nondominant’ lifetime. The likely mechanismis consideredin the following section. Reduction in R* lifetime (Table 1, row 2)
by steady-state
level of [Ca*+&
It has long been known that the catalytic activity of R* is reduced by phosphorylation by rhodopsin kinase (RK) and by subsequent arrestin (Arr) binding (Figure lb) [46]. These biochemical findings have received support from electrophysiological experiments on single rods [47] and have been confirmed by knocking out the genesencoding RK [48] and Arr [49] in mice. It has also been shown that
Molecular
mechanisms
of vertebrate
RK can exert an inhibitory effect on R* simply by binding to it, without phosphorylation [50]. In 1993, Kawamura [Sl] discovered that RK’s phosphorylation of R* in frog rods was calcium-dependent and that the effect was mediated by a calcium-binding protein that he named S-modulin, now known as recoverin (Ret) in mammalian rods. His observations have been confirmed and extended by several investigators [52,53,54’]. While the exact mechanism of recoverin’s action is not yet established, the in vitro evidence is consistent with the hypothesis that recoverin with two bound calcium ions (Ret-2Ca) binds to RK in a manner that prevents the latter from binding to and phosphorylating R* [S&53] (Figure 1b). Although the action of Ret and its calcium dependence in sihc remain matters of debate [SS’], it has been hypothesized that regulation of RK by Ret might be the mechanism mediating acceleration of the recovery of responsesto saturating flashesdelivered in adapted conditions [40-42]. The simplest molecular mechanismwould be that Ret changes the effective or mean lifetime (zn) of R* by controlling the amount of RK available to bind to and/or phosphorylate R*; this hypothesis can be formulated as TR0~l/mfree, where RKfre, is the concentration of RK that is not complexed with Ret-ZCa. From a straightforward model of the binding reactions involving calcium, Ret and RK, we predict a three- to fourfold increase in IX,,, (Figure 4c), and a comparable decline in 2~ (Figure 4b), when a salamander rod light adapts. This hypothesis is able to account for much of the desensitization observed in the step-flash paradigm (Figure 4a,b). Further support for the hypothesis comes from the finding that rod responsesin Ret knockout mice fail to show any acceleration of recovery in the step-flash paradigm[43’]. Thus, in rodslacking Ret, the recovery from a saturating flash appearsequivalent to that observed when [Ca*+]i is prevented from changing (i.e. asin Figure 3b). Dynamic
decrease
in R* lifetime
(Table
1, row 2)
The decline in [Caz+]i elicited by a test flash may trigger a dynamic reduction in R* lifetime. For moderate to bright flashes,this will be seenasa reduction in the mean R* lifetime (TR) measured in a traditional adaptation experiment or in a step-flash paradigm [4OA2], but at the single-photon level, the distribution of R* lifetimes may be shifted to shorter values [56’]. This dynamic feedback effect is predicted to be largest under dark-adapted conditions because the fraction of RK with Ret bound is then largest, so that the initial lifetime of R* is greatest. The dynamic effect on R* lifetime should decrease in the presence of higherintensity backgrounds becausethe RK is then mostly in its free state (compare Figure 2a with Figure 4~). Decreased cGMP lifetime caused PDE activity (Table 1, row 3)
by increased
photoreceptor
light
adaptation
Pugh, Nikonov
and Lamb
415
Figure 3
(a)
Light
-40 0
5 Time
10
(s)
Current
15 Opinion
in Neurobiology
Step-flash paradigm. Responses to steady illumination of different intensity are followed by a bright flash of fixed intensity, either (a) in Ringer’s solution or (b) in calcium-clamping solution, presented from -5 to 7.5 s. Backgrounds delivered 0, 66, 320 and 1400 photoisomerizations per second; bright flashes delivered 11000 photoisomerizations. Under control conditions (in Ringer’s), the flash responses exhibit accelerated recovery upon exposure to backgrounds; when [Ca*+li is clamped to the dark level, exposure to the same backgrounds causes no acceleration. Note that the rod was jumped back into Ringer’s solution immediately after delivery of the bright flash in the calcium-clamp condition. Reproduced with permission from figure 8 of Ill].
increases(Figure la), increasingthe steady rate constant p of cGMP hydrolysis above its value in darkness (Pdark).As shown by Nikonov et al. [34”], the reciprocal of p acts as a time constant in the rate equation governing the transient decrement in cGMP elicited by a dim flash, so that when PDE is activated, the recovery is acceleratedand the peak of the flash response is reduced. The measurements of Hodgkin and Nunn [14], and of many subsequentinvestigators, show that in a salamanderrod, p increaseslO-ZO-fold between the dark-adapted state and backgroundsthat produce 3000-5000 photoisomerizationsper second. It follows that the increase in p contributes materially both to the speededrecovery and to the desensitizationof the dim-flash responseshownin Figure Zc. Indeed, our analysisshowsthat the increasein p - a calcium-independent phenomenonis the most significant factor in the noncompressivedecrease in flash sensitivity. A quantitative description of this effect is not trivial, in part becausethe calcium-dependent feedback relation involving GC producesan intrinsically second-order character to the response;the interested reader is referred to equation (20) in [34”].
steady
In the presenceof steady background illumination, the mean number of active phosphodiesterase catalytic subunits
Transient
increase
in GC activity (Table
1, row 5)
During any flash response,[Caz+]i will undergo a transient decrease, because channels close while the Na+/Caz+,K+
416
Sensory
systems
Figure 4 (a) Reduction
in recovery
r
time
2.0 0
z s s
0 1 .o
0.0 LL
(b) Predicted
(c)
Free [Ca*+]i,
1.0 g * s ‘g 2 u
decline
0.6
-
0.6
-
0.4
-
0.2
-
o.oL.7
.-I-
recoverin,
rhodopsin
kinase
-..
a ’
0
in zn
’
’
10
100
I, steady
illumination
’
’
’
1000
AA
30
Theoretical account of the step-flash acceleration effect shown in Figure 3. The account is based on the hypotheses that the effective lifetime of R* in any state of adaptation is inversely proportional to the amount of free rhodopsin kinase (in = 1 IRK&, and that the dominant (rate-limiting) inactivation time constant zo of the disc-associated reactions is the lifetime of the G*-E* complex (Figure 1 c); ‘ho is estimated as the increase in the time to 50% recovery (ATSoN) per e-fold change in the intensity of saturating flashes [34**,40,44,45]. (a) Reduction in ATsmr from the step-flash paradigm. Filled symbols are taken from Figure 3 (see also [l 1 I); open symbols are from the same cell whose data are shown in Figure 2c. The traces replot the theoretical prediction derived below in (b,c) using AT,,&,= In(RK free,dark/Rl(free), where the dominant time constant is zo = 1 .g s and 1.5 s for the two cells. Diffences in 7o between cells [34**,40,45] will result in different magnitudes in AT,,, even if the underlying dependence of RK,,, on steady illumination is the same. (b) Comparison of experimental results and prediction, in normalized form. The symbols plot the calculated reduction of the effective lifetime of R*, computed as q$zR,dark = exp(-ATSo&,). The smooth curve was computed with the model described in (c), assuming ‘in = l/R/& Given that RK& is predicted to increase about threefold over the rod’s operating range, in accordingly decreases threefold. (c) Concentrations of free calcium ([Ca*+]i), free recoverin (Reef,,) and free rhodopsin kinase (RK&, as a function of steady background intensity, predicted by a model of the reactions involving recoverin. The [Ca*+]i curve was calculated with equations (11) and (12) and the parameters of Table 2 in reference [34”], and the assumption that the steady-state circulating current follows the curve illustrated in Figure 2a. The concentrations of free Ret and RK were computed with an equilibrium binding model of the reaction scheme for ReclRK interaction provided in 1531, using the binding constants given in references [53,6 1 I.
10000
(@ s-‘)
Increased Current
Opinion
in Neurobiology
exchanger continues to pump calcium out. As a consequence, the activity of GC will transiently rise, causing the cGMP level to recover more rapidly than it would have otherwise. The electrical response will therefore also recover more rapidly, and its peak will be truncated, causing a decreasein flash sensitivity (which is measuredat the peak). Comparison of responsesobtained in control conditions and in calcium-clamped (or calcium-buffered) conditions indicates that this feedback mechanismappears to reduce the peak amplitude of the dim flash responseby threefold or more in salamander rods [8,11,34”]. We say ‘appears’ because the calcium feedback mechanism involving recoverin may also make a dynamic contribution (see previous section). Our analysis indicates that the transient calcium-mediated increasein GC activity should have little effect on the shape of the sensitivity versus background relation, and electrophysiological experiments have indeed already shown this [8]. Thus, when [Ca’+]i is clamped to the level set by each background in Ringer’s solution then, although the absolute sensitivity is higher, the normalized sensitivity S,/S,D has approximately the samedependence on background intensity (gray symbols in Figure 2b) asobtained when [Ca2+li is free to change.
calcium
buffering
(Table
1, row 8)
The intrinsic calcium buffer in the photoreceptor [.57]serves both to retard and reduce the transient decreasein [Ca’+]i that occurs during the responseto a flash, and therefore leadsto an increasein flash sensitivity, in the sameway that calcium-clamping or calcium-buffering does. Of the known calcium-binding proteins in the rod (e.g. recoverin, calmodulin, GCAPs), by far the most abundant is recoverin, which may be present in concentrations exceeding 30 pM [51,53]. Our calculations(S Nikonov, EN Pugh Jr, unpublished data) indicate that the cytoplasmic calcium-buffering power contributed by recoverin is dependent on background intensity through the decline of [Ca’+]i, increasingby a factor of about two over the intensity operating range of the salamander rod. The elevated calcium-buffering power will slow the transient decline in [Ca’+]i during the flash response,and will thereby serve to sensitize the responsein the presence of modestto strongbackgrounds.This effect will need to be taken into consideration in any comprehensive account of transduction and adaptation. Cones: adaptation
and pigment
bleaching
(Table 1, row 9)
Light adaptation in cone photoreceptors appearsto use the same molecular mechanismsas in rods, with at least one important addition - in cones,adaptation at higher intensities is mediated by pigment bleaching [Z]. Imagine a steady background intensity that bleaches 90% of the cone pigment, leaving 10%functional. A lo-fold increasein intensity will reduce the available pigment by a factor of 10 (to l%),
Molecular
mechanisms
of vertebrate
so that a given test flash will generate just a tenth of the previous number of R*s. Thus, in this high-intensity regime, cones will exhibit Weber-law adaptation purely as a result of pigment depletion. Perhaps one of the main differences between the two classes of photoreceptor is that cones can function perfectly well with massive fractions of their total pigment complement in the bleached state, whereas the responses of rods are eliminated by bleaches of as little as 1% (i.e. when 99% of the pigment remains available) [58].
10.
photoreceptor
Nakatani
K, Yau K-W:
sensitivity 11.
Fain GL,
Lamb
as the messenger
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and recommended
Papers of particular have been highlighted
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Tamura
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We thank Vadim Arshavsky and Roth Anne Eatock for helpful comments on the manuscript. Our work is supported by the National Institutes of Health (EY-02660) and The Wellcome Trust (034792).
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14.
To understand the molecular mechanisms underlying photoreceptor light adaptation, a fundamental distinction needs to be made between the effect of these mechanisms on the cell’s operating range and on its sensitivity. In the past, it has often been assumed or expected that all molecular mechanisms of light adaptation would extend the operating range, decrease the sensitivity, and be mediated by the decline in [Ca’+]i - such mechanisms would be assigned a (+, -, +) profile in Table 1. Clearly, the situation is far more complex, because, of the eight viable mechanisms, only one has this profile, and at least six distinct profiles are seen. Furthermore, the dominant mechanism of flash desensitization - the increased hydrolytic activity of PDE caused by the background - is independent of calcium concentration. While additional mechanisms of adaptation may yet be discovered (particularly in cones), a key goal of future research must be to obtain a fully quantitative understanding of the distinctive contributions of the known mechanisms.
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Molecular mechanisms of vertebrate photoreceptor EN Pugh Jr*, S Nikonovtand
TD LambS
An important recent advance in the understanding of vertebrate photoreceptor light adaptation has come from the discovery that as many as eight distinct molecular mechanisms may be involved, and the realization that one of the principal mechanisms is not dependent on calcium. Quantitative analysis of these mechanisms is providing new insights into the nature of rod photoreceptor light adaptation. Addresses
*tFM Kirby Center
for Molecular Ophthalmology, Department of Ophthalmology and Institute of Neurological Sciences, Stellar-Chance Laboratories, University of Pennsylvania, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104-6069, USA *e-mail: [email protected] te-mail: [email protected] fDepartment of Physiology, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK; e-mail: TDLl @cam.ac.uk
Current Opinion
0 Elsevier
Science
in Neurobiology
Ltd ISSN
1999,
light adaptation
9:41 O-41 8
0959-4388
Abbreviations Arr arrestin lX$*+l, internal concentration of free calcium activated catalytic subunit of PDE activated moiety of G protein G GC guanylyl cyclase GCAP GC activating protein background intensity 43 half-activation concentration ‘612 PDE phosphodiesterase R* activated rhodopsin Ret recoverin RK rhodopsin kinase flash sensitivity SF dark-adapted SF sFD
Introduction Light adaptation refers to the ability of the visual system to adjust its performance to the ambient level of illumination. In a normal cycle of day and night, the illumination at the earth’s surface varies over 11 orders of magnitude [l], making light adaptation fundamentally important to the normal functioning of the vertebrate visual system. Much of the daily cycle of sensitivity adjustment in the vertebrate retina is managedby diurnal switching between rodand cone-dominated pathways. Individual cone photoreceptors have a prodigious capacity for light adaptation: their photocurrents never saturate in response to steady illumination [Z]. Thus, cones are the ultimate target for investigation of the full panoply of cellular mechanisms involved in vertebrate photoreceptor light adaptation. For the rod pathway, in contrast, the principal mechanisms of scotopic adaptation result from alterations in signal processing at postreceptoral stageswithin the retina [3], and the rods themselves adapt over only a modest
range ofsteady intensities before being driven into saturation. Nonetheless, because considerably more detail is currently known about the molecular processesin rods, we shall focus on these cells in this review. The mechanisms known or hypothesized to underlie light adaptation are shown in Figure 1, which depicts the rod phototransduction cascade. The contributions of several of these mechanisms to adaptation have recently been reviewed elsewhere [4-61, and the important role of calcium-independent processeshas been stressed [4,5].
Functional
characteristics
Desensitization
of light
adaptation
and acceleration
Two hallmarks of photoreceptor light adaptation are the reduction in sensitivity and the acceleration in response kinetics that occur in the presence of background illumination [7]. The dependence of these two parameters on background intensity, in salamander rods, is plotted in Figures 2b and Zc, respectively. The flash sensitivity (3,) is defined asthe peak amplitude of the dim-flash response divided by the flash intensity, and, according to convention, this parameter is plotted in normalized form as SF& D, where SFD is the dark-adapted sensitivity. Figure 2b shows that over roughly 2-3 decades of background intensity (I), flash sensitivity in salamander rods declines according to Weber’s Law - that is, roughly inversely with background intensity. Associated with this decline in sensitivity is an acceleration of the response kinetics, exemplified by the shortening of the time-topeak of the dim flash responseshown in Figure Zc. Range extension
by decline
in calcium
About ten years ago, a number of investigations established that when the internal calcium concentration ([Ca’+]i) is maintained near its resting (dark) level, rod and cone photoreceptors fail to show normal adaptational behavior and instead function over a very restricted range of intensities [8-131. Figure 2a plots the steadystate circulating current of salamander rods from several studies in which [Ca’+]i was free to change (open symbols) or was clamped (filled symbols). The results illustrate the severe range restriction imposed by blocking the normal decline in [Ca’+];, and, conversely, they show that the operating range is extended more than lOO-fold when [Ca’+]i is free to change. It is important to distinguish between the role of calcium in extending the photoreceptor’s operating range and any role it might have in modulating flash sensitivity. Figure 2a illustrates that in the absence of a change in [Ca”+]i, the rod would be driven into saturation by illumination of quite low intensity, and its sensitivity would thereby be drastically attenuated. The normal decline in [Ca2+li rescues the cell from this saturation, preventing its
Molecular
Figure
mechanisms
of vertebrate
photoreceptor
light
adaptation
Pugh, Nikonov
and Lamb
411
1
(a)
Cunent
Schematic of the rod phototransduction cascade. (a) The activation steps of the cascade. Following absorption of a photon (hv), the activated rhodopsin (R*) repeatedly contacts molecules of the heterotrimeric G protein (G), catalyzing the exchange of GDP for GTP, producing the active form G*cx (=Ga-GTP). Two G’cx subunits bind to the two inhibitory y subunits of the phosphodiesterase (E), thereby activating the corresponding a and 5 catalytic subunits, forming E*, which then catalyzes the hydrolysis of cGMP. The consequent reduction in the cytoplasmic concentration of cGMP leads to closure of the cyclic-nucleotide-gated channels and blockage of the inward flux of Na+ and Ca*+ (thereby reducing the circulating electrical current). The exchanger continues to pump Ca*+ out, so the cytoplasmic Ca*+ concentration declines, activating three ‘calcium feedback’ mechanisms, which are illustrated in this panel and in (b). Loss of Ca*+ from guanylyl cyclase activating proteins (GCAPs) allows them to bind to a cytoplasmic domain of the guanylyl cyclase (GC), increasing its activity; loss of Ca*+ from calmodulin (CM) causes it to dissociate from the channels, lowering the KIj2 of the channel for cGMP. The boxed symbols ‘a’ and ‘5’ are used throughout the text and in Table 1 to refer to the rate of synthesis of cGMP by GC and the rate constant of
sensitivity from being annihilated at low backgrounds, and thus extending its operating range. In other words, the decline in [Caz+]i does not actually cause the desensitization illustrated in Figure Zb,c - rather, it tends to do just the opposite.
Classification their effects
of molecular on operating
mechanisms by range and sensitivity
In keeping with the distinction made in the preceding paragraph, it is useful to classify the molecular mechanisms
Opinion
in Neurobiology
hydrolysis of cGMP by E’, respectively, and should not be confused with the protein subunit labels. (b) Inactivation of R*. At the dark concentration of [Ca”+]i (let3 side of diagram), most of the recoverin (Ret) is in the Ca*+-bound form at the membrane; Ret-2Ca forms a kinase (RK), blocking its activity. Thus, at , complex with rhodopsin resting Ca*+ levels, contact between R’ and uncomplexed RK occurs infrequently. When [Ca*+]i drops during the light response (arrows indicate progression of time), Ca *+ dissociates from Ret, which moves into solution. The concentration of free RK rapidly increases, increasing the frequency of interaction with R’, and leading to its rapid phosphorylation. Arrestin (Art) then binds, substantially quenching the R* activity. (c) Inactivation of G*a-E’. G’ is inactivated when the terminal phosphate of its bound GTP is hydrolyzed. Although the G protein has intrinsic GTPase activity, this capacity is only activated when the G* is bound to the y subunit of PDE (PDE-r) and when, in addition, the GTPase-activating protein or GAP factor (RGS9-G55) also binds. The resulting tetra-molecular complex, G*a-PDErRGS9-G55, rapidly hydrolyzes the GTP to GDP, returning the Ga subunit to its inactive form, so that the E* and G*a are simultaneously inactivated [59’,60].
underlying light adaptation according to their effects on the two fundamental characteristics illustrated in Figure 2: operating range and sensitivity. In the past, it has sometimes been assumed that a single class of mechanism and a single messenger (e.g. [CaZ+]i) might regulate all aspects of photoreceptor light adaptation; but, as we shall show, this is clearly not the case. In Table 1, we have gathered nine hypotheses about the molecular mechanisms underlying photoreceptor light
412
Sensory
Table
1
Mechanisms
systems
of light
adaptation
Mechanism/ hypothesis (as background increases)
in vertebrate
rod and cone
photoreceptors.
Protein mediating the effect
Predicted effect on operating range
Predicted effect on flash sensitivity
R*
Extends
Decreases
Yes
+, -I +
?
RK
Extends
Decreases
Yes
+, -, +
Recoverin
Increase in steady state cGMP hydrolysis rate constant (p)
PDE
Reduces
Decreases
No
-9 -10
Steady increase in cGMP synthesis rate (a)
GC
Extends
Increases
Yes
+, +, +
Transient increase in cGMP synthesis rate (AC&))
GC
No effect
Decreases
Yes
Decrease channels
in Kvs of for cGMP
cGMP-gated channel
Extends
Increases
cGMP-gated channel
Reduces
(fewer
Increase in Ca2+ buffering (B& of cytoplasm
Recoverin
Pigment
Cone photopigment
Decrease R* catalytic
in
Decrease R* lifetime
in (Q)
Response compression channels
Ca2+ mediated?
Profile*
identity regulatory Ca2+-binding protein
of
Comment
References
Can be rejected; see text
KW71
Accelerates recovery
111 ,18,35, 40-42,43*,56*]
Principal Cap+independent factor
[14,34”1
GCAP1,2
Restores circulating current
[13-l
0, -1 +
GCAPl,2
Accelerates recovery
[34”1
Yes
+, +, +
Calmodulin
Restores circulating current
[26-31,32”1
Decreases
No
--
Saturation causes drastic decline in SF
L’,8,331
No effect
Increases
Yes
0, +, +
Extends
Decreases
No
+, -I 0
gain
-
-
1 ! 0
open)
bleaching
*The profile column calcium-dependence).
summarizes
the
characteristics
described
in the
three
preceding
Results decrease Ret-2Ca
-
columns
(i.e. operating
from in
Incapacitates rods, yet underlies much of cone light adaptation range,
6,23-251
sensitivity,
121
and
adaptation. The majority of these mechanisms have been considered previously and are reviewed in, for example, [4-61. For each potential mechanism, Table 1 lists three characteristics: first, the predicted effect on the cell’s operating range; second, the predicted effect on its flash sensitivity; and third, whether the mechanism is directly activated by the decline in [Ca’+]i. On the basis of these criteria, each mechanism can be assigned a three-component ‘profile’ (sixth column of Table 1). The hypotheses are arranged in sequence of the protein mediating the effect (second column of Table l), but in discussing them, we shall first consider those that underlie range extension, and then proceed to those governing sensitivity.
(Figure la). Many studies - including the seminal investigation of intact salamander rods by Hodgkin and Nunn [ 141 and the thorough investigation of truncated amphibian rods by Koutalos etal. [l&16] -have demonstrated that GC can be activated to at least 10 times its basal level by the normal decline in [Ca’+]i. The dark-adapted resting level of [Ca’+]i is 400-600 nM, but [Ca’+]i drops to about 30-50 nM when the cGMP-gated channels have been held closed for several seconds [17-211 under conditions that allow the Na+/CaZ+,K+ exchanger to operate normally [ZZ]. The decline in [Ca’+]i causes activation of GC, with a Z& near 100 nM and a Hill coefficient of -2, which is mediated by the calcium-binding proteins GCAPl and GCAPZ (for reviews, see [23X5]).
Principal
Calmodulin effect on cGMP-gated channels (Table 1, row 6) A second well understood mechanism of range extension is the calmodulin-dependent shift in the K1,2 of cyclicnucleotide-gated channels [‘Z--29]. In rod channels, this shift is effected by the binding of calmodulin to two binding sites on the amino- and carboxy-terminal cytoplasmic loops
mechanisms
of range
extension
Guanylyl cyclase (Table 1, row 4) The most thoroughly investigated molecular mechanism for range extension in phototransduction is the activation of particulate guanylyl cyclase (GC) by the decline in [Ca’+]i that accompanies the steady-state light response
Molecular
Figure
mechanisms
of vertebrate
photoreceptor
2
Circulating
(a)
Steady circulating current, sensitivity, and kinetics of salamander rods in the presence of background illumination. The smooth curves plotted through the data in parts (a,b) are empirical in character (i.e. they are not derived from theoretical analysis of the transduction cascade). (a) Normalized circulating current F, of salamander rods as a function of steady illumination, replotted from several investigations. The open symbols represent data obtained in Ringer’s solution; the filled triangles represent data obtained with [Ca*+]i clamped to the resting (dark) level. Open diamonds, figure 2 of 1141; filled down triangles and open down triangles, figure 2 of [8]; open up triangles, figure 9 of [16]; open circles and circles with embedded plus (S Nikonov, EN Pugh Jr, unpublished data). The smooth curve for the calcium-clamp data is F, = exp(-l/l,,), with Is = 7, while that for the Ringer’s data is F, = /n/(/n + I#), with Is = 425, n = 0.7. Note that (a) and (b) share a common abscissa (intensity axis), and that all intensities are expressed in photoisomerizations per second (ct, o-1). (b) Flash sensitivity, from two of the same studies. Symbols as in (a); in addition, the gray triangles were obtained with [Ca*+li clamped to the level reached during exposure to the steady background in Ringer’s, The curve drawn through the calcium-clamp data is the same as in (a); the curve through the Ringer’s data was calculated according to Weber’s law, SF/.&o = //(1+/c), with /c = 40; the ‘corrected’ curve was obtained by dividing the Weber-law curve by the curve for F, in panel (a). (c) Dimflash responses of a salamander rod obtained in the dark (top trace) or in the presence of a background of progressively higher intensity. Each trace was calculated by dividing the averaged raw response r(t) by the maximum response to obtain R(t) = r(t)/r,,,,; the normalized response R(t) was then scaled by dividing by the flash intensity (Q). These traces correspond to the circles with embedded plusses in (a) and (b). The smooth curve through the rising phase of the responses was computed with the pure activation model of reference [38], with amplification constant A = 0.06 s-2, and effective delay f,, = 55 ms.
5
increase
sensitivity
Both the steady-state activation of GC and the calmodulin-dependent decline in the Kl12 of the channels cause an increase in the sensitivity of the rod. Because both of these mechanisms contribute to range extension, their effect on sensitivity is paradoxical to the standard view
and
Lamb
413
current
0.5
Flash sensitivity
(b)
s -L SFD
10000 100 1000 I, steady illumination (ct, s.‘)
0
. . ,..., Q,
10
Normalized
(cl
2.0
t 1.0 * 0.0
dim-flash
responses
i--x
I
-\J--.n
4
0
I
1
1 2 Time from flash (s) Current
Opinion
J
3 in Neurobiology
of adaptation, which has led to the expectation that adaptational mechanisms that extend the operating range should necessarily decreasesensitivity. Although a rigorous proof that these two mechanisms increase flash sensitivity requires analysis beyond the scope of this review, the key idea is straightforward: by opening up more channels than would otherwise have been available, the rod’s circulating current is increased by both processes,so that a dim test flash (eliciting a fixed pulse E*(t) of activated phosphodiesterase catalytic subunits) generates a larger response; thus, although the fraction of channels closed is unchanged, the suppression of circulating current is larger.
Mechanisms actually
Nikonov
0.0
of the B subunit of the channel [30,31]. While the contribution of this mechanism to range extension is relatively small in rods [16,24], a related mechanism exists for cone channels that is much more powerful and that involves a calciumbinding protein distinct from calmodulin [32”]. These two mechanisms, cyclase activation and a shift in Kl12 of the channels, are thought to account for most (if not all) of the extension in the operating intensity range that takes place in rods when [Ca’+]i is free to drop. For primate rods, the range extension has been modeled in terms of GC activation alone [ 131. For amphibian rods, in which the range extension is greater, the behavior has been modeled with a combination of GC activation, a calmodulin-dependent shift in the K,lz of the channels, and a third mechanism identified as a reduction in the average number of phosphodiesterase (PDE) catalytic subunits activated (E*) per photoexcited rhodopsin (R*) at higher background intensities [16,24] (see Figure 1). Elucidation of the molecular basis for this last mechanism remains a focus of current research and will be discussed below.
Pugh,
1.0
-
CC and calmodulin
light adaptation
Response
of desensitization
compression
(Table
1, row 7)
When the cGMP-activated current of the rod is reduced by steady illumination, the total output range of the cell -that is, the current available for modulation by an increment in illumination - is reduced. Compression of this kind haslong been understood to contribute to lossof
414
Sensory systems
flash sensitivity [7,33], but deserves clarification in terms of the phototransduction G-protein cascade. To illustrate the mechanism, suppose that output compression were the sole process causing the rod’s flash sensitivity to decreasewith increasing background intensity. In the context of the cascade schematic depicted in Figure 1, this would be equivalent to assumingthat E*(t), the transient number of additional phosphodiesterasecatalytic subunits activated by a given flash, is independent of background intensity [13]. With this assumption, analysis of the equations governing cGMP hydrolysis and synthesis [34”] showsthat a constant fraction of the open channels would be closed by a given flash, irrespective of background intensity. It is therefore possible to ‘correct’ responsesfor output compression simply by normalizing them with respect to the remaining circulating current, as was done originally by Torre et al. [35]. In terms of sensitivity, one may plot (SF/SFD)/F1, where FI is the circulating current remaining in the presence of a background (of intensity I) expressed as a fraction of the circulating current in darkness. In Figure Zb, rather than correcting the individual points, we have instead applied this correction to the curve, which therefore represents the underlying (or noncompressive) component of desensitization. Thus, the broken curve reveals the decreasein flash sensitivity that must be accounted for by molecular mechanisms within the transduction cascade. Intrinsic
‘gain’
of R* activity
(Table
1, row 1)
One hypothesis that has been proposed to account for decreased flash sensitivity in rods is that backgrounds (even dim ones) causea reduction in the ‘gain’ of the reactions coupling R* to activation of PDE [l&36,37], perhaps through the action of an asyet unidentified calcium-binding protein [36]. Two forms of this hypothesis are possible: either that the quantum efficiency for activation of R* by light is reduced, or that from the instant of its creation, an R* formed in the presence of a background is less effective in catalyzing the activation of the G protein to G* than one formed in dark-adapted conditions. We have found strong evidence against these hypotheses - and indeed against any hypothesis predicting a reduction in the ‘amplification constant’ (defined in [38]) - for background intensities that suppress up to 75% of the circulating current (S Nikonov, EN Pugh Jr, unpublished data). The evidence is straightforward; as illustrated in Figure Zc, the fractional suppressionof circulating current per unit flash intensity follows an invariant early trajectory, independent of background intensity. The invariance illustrated in Figure 2c contrasts with other recent reports [5,6,18,37]. However, similar invariance hasbeen reported in other studies [ 11,351,and very recently has been found in the responsesof human rods [39]. We suspect that the differences in interpretation between these different reports may have arisen because it had not previously been appreciated that acceleration of the inactivation reactions could diminish the responseat such early times, as discussedbelow.
Earlier
onset of recovery
In Figure Zc, the responsesof the rods ‘peel off’ from their common initial trajectory at progressively earlier times as the background is made more intense. This is consistent with the idea that the ‘lifetime’ of one or more of the cascade intermediates is shortened during exposure to steady light, a conclusion reached many years ago [7], before the G-protein cascade was known. In the context of the scheme of phototransduction shown in Figure 1, there are only three lifetimes that could be shortened: the effective lifetime of R*, the lifetime of the G*/E* complex, and the lifetime of free cGMP As there is no evidence for regulation of the G*/E* lifetime by calcium or by any other by-product of normal light exposure, this hypothesis will not be discussedfurther. However, there is a wealth of evidence for reduction of the other two lifetimes, as will be discussedin the next three sections. Calcium dependence of the lifetime disc-based intermediate
of a
Abundant physiological evidence supports the hypothesis that the lifetime and/or gain of one of the disc-associated intermediates in the rod transduction cascade is calcium-dependent, in a manner that would contribute to the decline in sensitivity in the presence of background illumination [11,18,35-37,40-42,43-l. A key piece of the evidence, obtained using a ‘step-flash’ paradigm, is illustrated in Figure 3. Normally, the time course of recovery from a bright flash is accelerated by prior light adaptation (Figure 3a); however, when [Ca2+li is clamped at the dark level for the duration of the light step, the recovery phase of the subsequent flash responsedoes not speed up (Figure 3b). Thus, the accelerated recovery in normal conditions is mediated by the decline in [Ca’+]i and not by molecular products of the light-driven reactions. The results of these step-flash experiments, in conjunction with the evidence cited above that the gain of the activation reactions is unchanged by light adaptation, show that the lifetime of a disc-associatedintermediate is shortenedby the lowered [Ca”];. Furthermore, becausethere is considerable evidence that the ‘dominant’ (i.e. rate-limiting) inactivation time constant of the disc-associatedreactionsis unaffected by light adaptation or by changesin [Ca2+li [34”,44,45], it follows that the mechanismunderlying the step-flasheffect is a reduction in a ‘nondominant’ lifetime. The likely mechanismis consideredin the following section. Reduction in R* lifetime (Table 1, row 2)
by steady-state
level of [Ca*+&
It has long been known that the catalytic activity of R* is reduced by phosphorylation by rhodopsin kinase (RK) and by subsequent arrestin (Arr) binding (Figure lb) [46]. These biochemical findings have received support from electrophysiological experiments on single rods [47] and have been confirmed by knocking out the genesencoding RK [48] and Arr [49] in mice. It has also been shown that
Molecular
mechanisms
of vertebrate
RK can exert an inhibitory effect on R* simply by binding to it, without phosphorylation [50]. In 1993, Kawamura [Sl] discovered that RK’s phosphorylation of R* in frog rods was calcium-dependent and that the effect was mediated by a calcium-binding protein that he named S-modulin, now known as recoverin (Ret) in mammalian rods. His observations have been confirmed and extended by several investigators [52,53,54’]. While the exact mechanism of recoverin’s action is not yet established, the in vitro evidence is consistent with the hypothesis that recoverin with two bound calcium ions (Ret-2Ca) binds to RK in a manner that prevents the latter from binding to and phosphorylating R* [S&53] (Figure 1b). Although the action of Ret and its calcium dependence in sihc remain matters of debate [SS’], it has been hypothesized that regulation of RK by Ret might be the mechanism mediating acceleration of the recovery of responsesto saturating flashesdelivered in adapted conditions [40-42]. The simplest molecular mechanismwould be that Ret changes the effective or mean lifetime (zn) of R* by controlling the amount of RK available to bind to and/or phosphorylate R*; this hypothesis can be formulated as TR0~l/mfree, where RKfre, is the concentration of RK that is not complexed with Ret-ZCa. From a straightforward model of the binding reactions involving calcium, Ret and RK, we predict a three- to fourfold increase in IX,,, (Figure 4c), and a comparable decline in 2~ (Figure 4b), when a salamander rod light adapts. This hypothesis is able to account for much of the desensitization observed in the step-flash paradigm (Figure 4a,b). Further support for the hypothesis comes from the finding that rod responsesin Ret knockout mice fail to show any acceleration of recovery in the step-flash paradigm[43’]. Thus, in rodslacking Ret, the recovery from a saturating flash appearsequivalent to that observed when [Ca*+]i is prevented from changing (i.e. asin Figure 3b). Dynamic
decrease
in R* lifetime
(Table
1, row 2)
The decline in [Caz+]i elicited by a test flash may trigger a dynamic reduction in R* lifetime. For moderate to bright flashes,this will be seenasa reduction in the mean R* lifetime (TR) measured in a traditional adaptation experiment or in a step-flash paradigm [4OA2], but at the single-photon level, the distribution of R* lifetimes may be shifted to shorter values [56’]. This dynamic feedback effect is predicted to be largest under dark-adapted conditions because the fraction of RK with Ret bound is then largest, so that the initial lifetime of R* is greatest. The dynamic effect on R* lifetime should decrease in the presence of higherintensity backgrounds becausethe RK is then mostly in its free state (compare Figure 2a with Figure 4~). Decreased cGMP lifetime caused PDE activity (Table 1, row 3)
by increased
photoreceptor
light
adaptation
Pugh, Nikonov
and Lamb
415
Figure 3
(a)
Light
-40 0
5 Time
10
(s)
Current
15 Opinion
in Neurobiology
Step-flash paradigm. Responses to steady illumination of different intensity are followed by a bright flash of fixed intensity, either (a) in Ringer’s solution or (b) in calcium-clamping solution, presented from -5 to 7.5 s. Backgrounds delivered 0, 66, 320 and 1400 photoisomerizations per second; bright flashes delivered 11000 photoisomerizations. Under control conditions (in Ringer’s), the flash responses exhibit accelerated recovery upon exposure to backgrounds; when [Ca*+li is clamped to the dark level, exposure to the same backgrounds causes no acceleration. Note that the rod was jumped back into Ringer’s solution immediately after delivery of the bright flash in the calcium-clamp condition. Reproduced with permission from figure 8 of Ill].
increases(Figure la), increasingthe steady rate constant p of cGMP hydrolysis above its value in darkness (Pdark).As shown by Nikonov et al. [34”], the reciprocal of p acts as a time constant in the rate equation governing the transient decrement in cGMP elicited by a dim flash, so that when PDE is activated, the recovery is acceleratedand the peak of the flash response is reduced. The measurements of Hodgkin and Nunn [14], and of many subsequentinvestigators, show that in a salamanderrod, p increaseslO-ZO-fold between the dark-adapted state and backgroundsthat produce 3000-5000 photoisomerizationsper second. It follows that the increase in p contributes materially both to the speededrecovery and to the desensitizationof the dim-flash responseshownin Figure Zc. Indeed, our analysisshowsthat the increasein p - a calcium-independent phenomenonis the most significant factor in the noncompressivedecrease in flash sensitivity. A quantitative description of this effect is not trivial, in part becausethe calcium-dependent feedback relation involving GC producesan intrinsically second-order character to the response;the interested reader is referred to equation (20) in [34”].
steady
In the presenceof steady background illumination, the mean number of active phosphodiesterase catalytic subunits
Transient
increase
in GC activity (Table
1, row 5)
During any flash response,[Caz+]i will undergo a transient decrease, because channels close while the Na+/Caz+,K+
416
Sensory
systems
Figure 4 (a) Reduction
in recovery
r
time
2.0 0
z s s
0 1 .o
0.0 LL
(b) Predicted
(c)
Free [Ca*+]i,
1.0 g * s ‘g 2 u
decline
0.6
-
0.6
-
0.4
-
0.2
-
o.oL.7
.-I-
recoverin,
rhodopsin
kinase
-..
a ’
0
in zn
’
’
10
100
I, steady
illumination
’
’
’
1000
AA
30
Theoretical account of the step-flash acceleration effect shown in Figure 3. The account is based on the hypotheses that the effective lifetime of R* in any state of adaptation is inversely proportional to the amount of free rhodopsin kinase (in = 1 IRK&, and that the dominant (rate-limiting) inactivation time constant zo of the disc-associated reactions is the lifetime of the G*-E* complex (Figure 1 c); ‘ho is estimated as the increase in the time to 50% recovery (ATSoN) per e-fold change in the intensity of saturating flashes [34**,40,44,45]. (a) Reduction in ATsmr from the step-flash paradigm. Filled symbols are taken from Figure 3 (see also [l 1 I); open symbols are from the same cell whose data are shown in Figure 2c. The traces replot the theoretical prediction derived below in (b,c) using AT,,&,= In(RK free,dark/Rl(free), where the dominant time constant is zo = 1 .g s and 1.5 s for the two cells. Diffences in 7o between cells [34**,40,45] will result in different magnitudes in AT,,, even if the underlying dependence of RK,,, on steady illumination is the same. (b) Comparison of experimental results and prediction, in normalized form. The symbols plot the calculated reduction of the effective lifetime of R*, computed as q$zR,dark = exp(-ATSo&,). The smooth curve was computed with the model described in (c), assuming ‘in = l/R/& Given that RK& is predicted to increase about threefold over the rod’s operating range, in accordingly decreases threefold. (c) Concentrations of free calcium ([Ca*+]i), free recoverin (Reef,,) and free rhodopsin kinase (RK&, as a function of steady background intensity, predicted by a model of the reactions involving recoverin. The [Ca*+]i curve was calculated with equations (11) and (12) and the parameters of Table 2 in reference [34”], and the assumption that the steady-state circulating current follows the curve illustrated in Figure 2a. The concentrations of free Ret and RK were computed with an equilibrium binding model of the reaction scheme for ReclRK interaction provided in 1531, using the binding constants given in references [53,6 1 I.
10000
(@ s-‘)
Increased Current
Opinion
in Neurobiology
exchanger continues to pump calcium out. As a consequence, the activity of GC will transiently rise, causing the cGMP level to recover more rapidly than it would have otherwise. The electrical response will therefore also recover more rapidly, and its peak will be truncated, causing a decreasein flash sensitivity (which is measuredat the peak). Comparison of responsesobtained in control conditions and in calcium-clamped (or calcium-buffered) conditions indicates that this feedback mechanismappears to reduce the peak amplitude of the dim flash responseby threefold or more in salamander rods [8,11,34”]. We say ‘appears’ because the calcium feedback mechanism involving recoverin may also make a dynamic contribution (see previous section). Our analysis indicates that the transient calcium-mediated increasein GC activity should have little effect on the shape of the sensitivity versus background relation, and electrophysiological experiments have indeed already shown this [8]. Thus, when [Ca’+]i is clamped to the level set by each background in Ringer’s solution then, although the absolute sensitivity is higher, the normalized sensitivity S,/S,D has approximately the samedependence on background intensity (gray symbols in Figure 2b) asobtained when [Ca2+li is free to change.
calcium
buffering
(Table
1, row 8)
The intrinsic calcium buffer in the photoreceptor [.57]serves both to retard and reduce the transient decreasein [Ca’+]i that occurs during the responseto a flash, and therefore leadsto an increasein flash sensitivity, in the sameway that calcium-clamping or calcium-buffering does. Of the known calcium-binding proteins in the rod (e.g. recoverin, calmodulin, GCAPs), by far the most abundant is recoverin, which may be present in concentrations exceeding 30 pM [51,53]. Our calculations(S Nikonov, EN Pugh Jr, unpublished data) indicate that the cytoplasmic calcium-buffering power contributed by recoverin is dependent on background intensity through the decline of [Ca’+]i, increasingby a factor of about two over the intensity operating range of the salamander rod. The elevated calcium-buffering power will slow the transient decline in [Ca’+]i during the flash response,and will thereby serve to sensitize the responsein the presence of modestto strongbackgrounds.This effect will need to be taken into consideration in any comprehensive account of transduction and adaptation. Cones: adaptation
and pigment
bleaching
(Table 1, row 9)
Light adaptation in cone photoreceptors appearsto use the same molecular mechanismsas in rods, with at least one important addition - in cones,adaptation at higher intensities is mediated by pigment bleaching [Z]. Imagine a steady background intensity that bleaches 90% of the cone pigment, leaving 10%functional. A lo-fold increasein intensity will reduce the available pigment by a factor of 10 (to l%),
Molecular
mechanisms
of vertebrate
so that a given test flash will generate just a tenth of the previous number of R*s. Thus, in this high-intensity regime, cones will exhibit Weber-law adaptation purely as a result of pigment depletion. Perhaps one of the main differences between the two classes of photoreceptor is that cones can function perfectly well with massive fractions of their total pigment complement in the bleached state, whereas the responses of rods are eliminated by bleaches of as little as 1% (i.e. when 99% of the pigment remains available) [58].
10.
photoreceptor
Nakatani
K, Yau K-W:
sensitivity 11.
Fain GL,
Lamb
as the messenger
rods. 12.
and recommended
Papers of particular have been highlighted
interest, as:
published
within
reading the annual
13.
15
Matthews
Tamura
17.
18.
Hodgkin
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Burkhardt
Koutalos
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R, Enroth-Cugell controls. Prog Retinal MD,
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Characterization of retinal rod outer segments.
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ST, Younger
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JP, Owen
WG:
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ST, Owen
WG:
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Sampath AP, Matthews HR, Cornwall MC, Fain GL: Bleached pigment produces a maintained decrease in outer segment in salamander rode. J Gen Physiol1998, 111:53-64. L, McNaughton PA: Electrogenic J Membr 6io/1990,113:177-191.
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W, Palczewski K: Turned on by Ca*+: the and pathology of (%*+-binding proteins in the retina. Neurosci 1996, 19:547-554.
Polans
A, Baehr
Y, Yau K-W: Regulation of sensitivity photoreceptors by calcium. Trends Neurosci
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EN Jr, Duda
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A, Sharma RK: Photoreceptor Biosci Reports 1997,17:429-473.
RS: Modulation of the cGMP-gated channel cells by calmodulin. Nature 1993, 361:76-79.
of md
Hsu YT, Molday RS: Interaction of calmodulin with the cyclic GMPgated channel of md photoreceptor cells. Modulation of activity, affinity purification, and localization. J Biol Chem 1994.
RL, Fain GL, Lamb
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and sensitivity
TY, llling
1994,91 Bauer
30.
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TD: Photoreceptor light calcium concentration.
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K, Yau K-W: Calcium and light adaptation Nature 1988,334:69-71.
and cones.
in
M, Molday
LL, Hsu
YT, Yau K-W,
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RS:
Subunit 2 (or beta) of retinal rod cGMP-gated cation channel is a component of the 240-kDa channel-associated pmtein and mediates Ca*+-calmodulin modulation. Proc Nat/ Acad Sci USA
of vertebrate light activation. Curr Opin
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AL: Changes in time scale J Physioll974,242:729-750.
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PB, Gray-Keller
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VA: What are the mechanisms of Behav Brain Sci 1995,18:415-424.
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TD: Light adaptation a role for cytoplasmic
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BJ: Control
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adaptation and photopigment bleaching in in situ in the retina of the turtle. I Neurosci
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We thank Vadim Arshavsky and Roth Anne Eatock for helpful comments on the manuscript. Our work is supported by the National Institutes of Health (EY-02660) and The Wellcome Trust (034792).
in retinal
calcium
salamander
Acknowledgements
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19%9,409:525-548.
14.
To understand the molecular mechanisms underlying photoreceptor light adaptation, a fundamental distinction needs to be made between the effect of these mechanisms on the cell’s operating range and on its sensitivity. In the past, it has often been assumed or expected that all molecular mechanisms of light adaptation would extend the operating range, decrease the sensitivity, and be mediated by the decline in [Ca’+]i - such mechanisms would be assigned a (+, -, +) profile in Table 1. Clearly, the situation is far more complex, because, of the eight viable mechanisms, only one has this profile, and at least six distinct profiles are seen. Furthermore, the dominant mechanism of flash desensitization - the increased hydrolytic activity of PDE caused by the background - is independent of calcium concentration. While additional mechanisms of adaptation may yet be discovered (particularly in cones), a key goal of future research must be to obtain a fully quantitative understanding of the distinctive contributions of the known mechanisms.
Pugh,
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TI, Korenbrot JI: In intact cone photoreceptors, a Gas+dependent, diffusible factor modulates the cGMP-gated ion channels differently than in rods. J Gen fhysioll998, Rebrik
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112:537-548. Cone photoreceptor currents were recorded with a suction pipette after electropermeabilization of the currents inner segment membrane. The authors report that cGMP activation of channels was modulated fivefold by a novel calcium-dependent diffusible factor. This modulation is substantially larger than that effected by calmodulin in rods. 33.
Dowling JE, Ripps H: Visual J Gen Physiol19?0,56:491-520.
34.
Nikonov
**
adapted
adaptation
111:7-37. This paper provides a rigorous investigation of the inactivation steps in vertebrate phototransduction through a combination of suction pipette experiments on isolated rods and mathematical analysis. The authors develop predictions for the electrical response kinetics of rod photoreceptors when calcium is either free to change or is clamped at its resting level. 35.
Torre V, Matthews
HR,
Acad 36.
SC; USA
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