Piecing together the timetable for visual transduction with transgenic animals

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Piecing together the timetable for visual transduction with transgenic animals Clint L Makino
y, Xiao-Hong Wen
z and Janis Lem§ Transgenic mice bearing null or functional mutations are being used to define the roles of specific elements in phototransduction and also to time the molecular interactions. Genetic manipulation of the collision frequency between rhodopsin and transducin molecules identified this parameter as rate-limiting for the photoresponse onset. Genetic interference with rhodopsin phosphorylation and arrestin binding, transducin shut-off and calcium feedback has revealed their respective roles in shaping the response waveform. The timetable for all of these molecular events determines the amplitude, kinetics and reproducibility of the photoresponse. Addresses Correspondence:
Department of Ophthalmology, Massachusetts Eye and Ear Infirmary and Harvard Medical School, 243 Charles Street, Boston, MA 02114, USA y e-mail: [email protected] z e-mail: [email protected] § Department of Ophthalmology, Program in Genetics and the Molecular Cardiology Research Institute, Tufts University School of Medicine and the New England Medical Center, 750 Washington Street, Boston, MA 02111, USA e-mail: [email protected]

Current Opinion in Neurobiology 2003, 13:404–412 This review comes from a themed issue on Sensory systems Edited by Clay Reid and King-Wai Yau 0959-4388/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S0959-4388(03)00091-6

Abbreviations A arrestin cGMP cyclic guanosine monophosphate GC guanylate cyclase GCAP guanylate cyclase activating protein Gb5 G protein b-subunit type 5 PPEF protein phosphatase with EF hands R rhodopsin R] photoactivated rhodopsin RGS9 regulator of G protein signaling type 9 T transducin

Introduction The task of encoding light into a neural signal by retinal photoreceptors may seem simple compared to the processing of visual information by downstream neurons. Nevertheless, the need to capture rapidly evolving visual scenes places stringent demands on phototransduction. The signal must be generated quickly and reproducibly. Current Opinion in Neurobiology 2003, 13:404–412

To maximize temporal resolution, the signal should be brief. But brevity comes at the expense of sensitivity because the size of the dim flash response depends upon how long the phototransduction process operates after photon absorption. Hence, rods generate relatively large, long-lived responses when photons are scarce, whereas cones give smaller, more brief responses when photons abound. What factors set the speed of phototransduction? What factors determine the duration of the response? How is reproducibility achieved? Here, by focusing on studies that use transgenic animals, we review the progress made towards answering these questions. Photon absorption by rhodopsin initiates a biochemical cascade within the rod outer segment that ultimately leads to the closure of ion channels. The underlying molecular events are summarized in Figure 1 [1–3]. Although phototransduction is similar in cones, the ensuing discussion refers to rods, except where specific reference is made to cones.

Timing the molecular events of phototransduction Photoactivated rhodopsin (R
) forms within a millisecond. That is fast relative to the time to the peak of the photoresponse, which occurs after a hundred milliseconds or more. Similarly, the cGMP-gated channel in the plasma membrane of the rod outer segment responds to changes in cyclic guanosine monophosphate concentration ([cGMP]i) on a millisecond time scale. Therefore, the rate-limiting step in the rising phase of the photoresponse must lie somewhere in-between. Candidate molecular events may be grouped into one of two broad categories: collisions or molecular processing. R
, transducin and phosphodiesterase diffuse randomly in or on the disk membrane. Physical contact is necessary for their interaction and some time passes before R
collides with each transducin and before transducin collides with phosphodiesterase. Additional time elapses during the rearrangements in molecular structure that constitute processing- nucleotide exchange on transducin and activation of phosphodiesterase by transducin. The slowest event determines the speed with which the photoresponse will appear. Calvert et al. [4] tested whether or not a molecular collision limited the rising phase by recording the electrical responses of single rods from heterozygous rhodopsin knockout (Rþ/) mice to brief flashes. Rþ/ rods inserted fewer rhodopsins into their outer segment disk membranes without changing the membrane densities of transducin or phosphodiesterase [4,5]. The accelerated rate of rise of the photon response www.current-opinion.com

The timing of phototransduction Makino, Wen and Lem 405

Figure 1

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The phototransduction cascade. (a) In darkness, a current circulates between the outer and the inner segments keeping the rod depolarized. Depolarization promotes the release of glutamate at the synapse. Within the outer segment, the visual pigment rhodopsin is packed in the membranes of hollow disks, which are stacked in register. Photon absorption by rhodopsin (white spot, right) attenuates the circulating current by causing ion channels in the plasma membrane to close. The rod hyperpolarizes and releases less glutamate. (b) Activation: photoexcited rhodopsin (R
) activates over a hundred transducin (T) molecules by catalyzing the exchange of a GTP for the GDP bound to T. Each activated T relieves a phosphodiesterase catalytic subunit (PDE) of an inhibition, allowing it to rapidly hydrolyze cGMP. Lowered [cGMP]i results in the closure of ion channels in the plasma membrane of the rod outer segment (ROS) curtailing the influx of Naþ and Ca2þ. (c) Shut-off and recovery: R
shuts off after phosphorylation by rhodopsin kinase (RK) and subsequent binding to arrestin (A). T ceases to interact with PDE after hydrolyzing its bound GTP. Regulator of G protein signaling type 9 (RGS9), which forms a complex with Gb5, facilitates the hydrolysis. Guanylate cyclase (GC) synthesizes cGMP enabling the channels to reopen. (d) Ca2þ mediated regulation: closure of cGMP-gated channels in response to light prevents the entry of Ca2þ. Continued extrusion by a Naþ/Kþ,Ca2þ exchanger (shown in blue) drives its concentration down to a low level. Low Ca2þ acts through recoverin (Rec) to promote shut-off of R
by RK, through guanylate cyclase activating proteins (GCAPs) to accelerate cGMP synthesis and through calmodulin (Cam) or a related protein to open the channel at a lower [cGMP]i.

was attributed to the increased lateral mobilities of R
and transducin (Figure 2). Accordingly, the collision rate between R
and transducin should vary with transducin concentration. Although heterozygous deletion of transducin (Tþ/) only produced a small change in its total expression in the retina, the large variance in Tþ/ rod sensitivity suggested that some rods lacked the full complement of transducin and their photon responses rose more gradually than normal [6]. Additional support for a proposed effect of transducin concentration on the photoresponse was provided by a recent study on light adapted rats [7]. Following exposure www.current-opinion.com

to bright light, most of the transducin in rat rods translocated from the outer segment to the inner segment where it was not accessible to R
molecules in the disk membrane [8–10]. The rate of rise of the flash response decreased in direct proportion to the fraction of transducin lost from the disks, just as predicted [7]. In addition, molecular collisions on the disk membrane limit the photoresponse recovery, as demonstrated by the fact that it too was accelerated in Rþ/ rods ([4,5,11]; Figure 2d). Consistent with this interpretation, expression of an extra gene(s) for rhodopsin (albeit a mutant form) on a wild type background slowed the photoresponse, Current Opinion in Neurobiology 2003, 13:404–412

406 Sensory systems

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The effects of molecular crowding on the disk membrane. (a) Rhodopsin molecules (red dots) occupy about one-fourth of the disk membrane surface of a normal rod (left). When one molecule is photoconverted to R
(green star), molecular crowding limits its mean free path of diffusion and obstructs it from finding a T (blue circles). In the Rþ/ rod (right), half of the rhodopsins are missing so R
and T enjoy greater mobility. The membrane surface sampled by the R
per unit time (area in yellow) enlarges so there are more collisions with T molecules. Collisions between T and PDE (black oval) are similarly affected. (b) If the collision between T and PDE were rate-limiting to the activation of phototransduction, then a higher collision rate would cause the photoresponse to appear sooner, because T activates PDE stoichiometrically (left). If the collision between R
and T were rate-limiting, a higher collision rate would cause the photoresponse to appear sooner and increase its rate of rise, because a single R
serially activates many T molecules (right). (c) The dim flash response of Rþ/ rods (red points, average  SEM) rises more steeply than that of wild type rods (blue points). The continuous line plots the wild type response multiplied by a factor of 1.7. Reproduced with permission from [4], copyright 2001 Macmillan Magazines Limited. (d) The dim flash response of Rþ/ rods (red trace) also recovered more rapidly than normal (blue trace). Reproduced with permission from [4], copyright 2001 Macmillan Magazines Limited.

presumably because of overcrowding of rhodopsin molecules in the disk membranes [12]. Collisions between rhodopsin and rhodopsin kinase or between transducin and regulator of G protein signaling type 9 (RGS9) could limit response recovery, as all four proteins are disk membrane associated. Some evidence suggests that the collision between rhodopsin and rhodopsin kinase is ratelimiting. Heterozygous rhodopsin kinase knockout rods contained half the normal concentration of rhodopsin kinase and their flash responses took longer to recover [13]. Although mice overexpressing rhodopsin kinase have not yet been reported, the condition was achieved functionally by knockout of recoverin, which is thought to bind a fraction of the rhodopsin kinase in darkness and lower its effective concentration. Rods from recoverin knockout mice had a faster flash response recovery than rods from wild type mice [14,15]. Current Opinion in Neurobiology 2003, 13:404–412

The binding of arrestin to multiply phosphorylated R
fully quenches R
activity. Collisions between phosphorylated R
and arrestin are thought to occur rapidly, because a 70% decrease in arrestin in heterozygous arrestin knockout (Aþ/) rods had no effect on photoresponse recovery [16]. Binding occurred near the peak of the photon response. As the outer segment content of arrestin is less than that of rhodopsin, exposure to very bright light could cause nearly all of the arrestins in the outer segment to bind R
s. Subsequently, the collision frequency between R
and free arrestins would decline substantially. To prevent arrestin depletion from prolonging the photoresponse, arrestin mobilizes from the inner to the outer segment [17]. Confirmation of these ideas about arrestin awaits the determination of the outer segment concentrations of free arrestin in Aþ/ and wild type animals. Interestingly, the intracellular www.current-opinion.com

The timing of phototransduction Makino, Wen and Lem 407

movement of arrestin is not dependent upon R
phosphorylation [18]. Other evidence argues that the time scale for transducin shut-off may be comparable to that for R
shut-off. When transducin shut-off was slowed tenfold by deletion of RGS9 (RGS9/), the photon response diverged from its normal course near the response peak, suggesting that in wild type rods transducins had begun to shut off at approximately the same time as R
. Furthermore, if transducin were to shut off much faster than R
, then slowing transducin shut-off during the rising phase of the photoresponse would increase the rate of rise and the amplitude of the quantal response [19]. No such changes were observed in RGS9/ rods, consistent with a slow shut-off of transducin [20]. Decreasing RGS9 concentration should then prolong the photoresponse. RGS9þ/ mice were not helpful in this regard because their RGS9 levels and flash response kinetics were normal [20]. Another approach may be to alter the membrane concentration of RGS9 by targeting R9AP, the membrane anchor for RGS9 [21]. The low affinity of transducin for RGS9 in the absence of phosphodiesterase ensures that transducin will shut down only after having carried out its role in phototransduction. The need for phosphodiesterase was demonstrated by the slow photoresponse recovery in phosphodiesterase g-subunit knockout rods expressing a transgene for a mutant g-subunit that failed to promote RGS9 binding to transducin [22]. Ultimately, the photoresponse returns to baseline upon recovery of [cGMP]i. The concentration of cGMP reflects

the balance between synthesis and hydrolysis. Two membrane-bound cyclases, guanylate cyclase-E (GC-E) and -F (GC-F), are responsible for cGMP synthesis. Experiments aimed at defining the functions of each cyclase have only just begun. Elimination of GC-E in mice delayed the time to peak of the dim flash response, but quickened the overall recovery and even caused an undershoot, where for a short time, there were more cGMP-gated channels open than before the flash [23]. Both cyclases are subject to Ca2þ feedback regulation through guanylate cyclase activating proteins (GCAPs) (Figure 1d). Knockout of GCAPs 1 and 2 (GCAPs/) revealed that the Ca2þ sensitivity and rapidity of feedback onto cGMP synthesis enabled it to limit the size and duration of the single photon response (Figure 3a; [24,25]). Importantly, the feedback also reduced the continuous noise by dampening the effects of thermal phosphodiesterase activations. Addition of a Ca2þ buffer to GCAP/ rods had little effect on the flash response, indicating that the Ca2þ feedback onto rhodopsin kinase and the cGMP-gated channel operate mainly during adaptation to steady light. Restoring GCAP1 but not GCAP2 to GCAP/ rods conferred normal recovery kinetics (Figure 3b; [24,26]). Although GCAP2 alone did provide Ca2þ feedback, it appeared to operate rather slowly so its function remains unclear. The acceleration in flash response recovery in steady light arises predominantly from the increase in steady-state cGMP hydrolysis [27]. Mutations in opsin, such as G90D, which increase basal phosphodiesterase activity also speed up the flash response recovery [11,28]. As the basal rate of cGMP hydrolysis in darkness originates from

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Ca2þ-dependent feedback onto cGMP synthesis. Traces show averaged flash responses: wild type (bold traces), GCAPs/ (thin continuous traces) and expression of a transgene for GCAP1 on a GCAPs/ background (G1T4þ, GCAPs/, dashed trace), to a flash given at time ¼ 0 s. (a) In the absence of GCAPs, the single photon response climbed for a longer period. The peak height grew fourfold and the integration time increased 2.7-fold. Reproduced from [25]. (b) Responses to different flash strengths were scaled to the same height to compare dim flash response kinetics. Kinetics returned to normal upon expression of a transgene for GCAP1 in GCAPs/ rods. Transgene expression was probably variable because in other rods (not shown), restoration of response kinetics was incomplete. Courtesy of F Rieke and P Detwiler, University of Washington. www.current-opinion.com

Current Opinion in Neurobiology 2003, 13:404–412

408 Sensory systems

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The timing of shut-off and recovery events for the single photon response. (a) The calculated time course for the guanylate cyclase activity relative to that at rest (broken line) lagged the rising phase of the photoresponse (continuous line) and exhibited a more complex recovery. Reproduced from [25]. (b) The approximate onset of each shut-off and recovery event is marked with an arrow.

spontaneous activations of phosphodiesterase [29], it will vary with phosphodiesterase concentration. We predict that responses of rods that express lower levels of phosphodiesterase might recover slowly. By comparing the photoresponses of normal rods to those of rods deficient in R
phosphorylation [13,30,31], rods lacking arrestin [16] and rods lacking GCAPs [24,25], it becomes possible to construct a timeline for the events that control photoresponse recovery (Figure 4). Ca2þ feedback onto guanylate cyclase kicks in early after the flash, initially increasing the rate of cGMP synthesis. About 60 ms later, guanylate cyclase activity begins to decline. It falls to a sub-basal rate and then slowly returns to the basal rate. Phosphorylation of R
commences approximately 100 ms after the flash. Additional phosphates are added over the next 30-50 ms whereupon arrestin binds to the phosphorylated R
. RGS9-mediated transducin shut-off occurs at about the same time as arrestin binding.

Reproducibility of the photon response A stochastic, one-step shut-off of R
would give rise to highly variable quantal responses and preclude the rod from counting single photons accurately. So to achieve a reproducible response, rods use a multi-step shut-off, although the requisite number of steps is controversial [32–34]. Recordings of rods expressing mutant, phosphorylation deficient rhodopsins on a R/ background demonstrated that three phosphorylation sites were required for reproducible termination of the flash response. Current Opinion in Neurobiology 2003, 13:404–412

More than three sites conferred little improvement, though (Figure 5; [31]). Three was an unexpectedly low number and implicated the involvement of feedback. Reproducibility was maintained after removal of Ca2þ feedback onto cGMP synthesis [34]. One hypothesis could be that the light-induced fall in Ca2þ gradually inhibits an initially rapid dephosphorylation of R
before arrestin binding. An exclusive role for the Ca2þ-dependent phosphatase, protein phosphatase with EF hands (PPEF-2) seems unlikely as the knockout failed to change the ratio of the ensemble variance to the mean of the dim flash response [35]. Rods also express PPEF-1, however, which could compensate for the loss of PPEF-2. Mice lacking both phosphatases have been produced, but the reproducibility of their responses has not yet been ascertained.

Source of biological variation Differences in the expression levels of key proteins would produce biological variation in the sensitivity and kinetics of the photoresponse. Remarkably, the variation observed for responses across rods from the same retina and across individuals of a species is quite low. Times to peak and integration times of the dim flash response typically show standard errors of less than 10%, for example in monkey [36], rabbit, cow and cat [37] and mouse (by many investigators). Put another way, rods are extraordinarily competent at expressing the proper levels of proteins. Microspectrophotometric measurements of specific absorbance attest to the uniform expression of rhodopsin in individual rods of nearly all vertebrates [38,39]. www.current-opinion.com

The timing of phototransduction Makino, Wen and Lem 409 Figure 5

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Proper shut-off of rhodopsin requires multiple phosphorylations. (a) Putative phosphorylation sites in mouse rod opsin are marked with red circles along with their position numbers in the sequence. Phosphorylation was verified by mass spectrometry at all positions except 336 and 340 [61–63]. Adapted from [61], copyright 2002 American Society for Biochemistry and Molecular Biology. (b) Averaged single photon responses of rods containing rhodopsins with fewer than three carboxy terminal phosphorylation sites recovered slowly. In individual trials, response lifetimes were exponentially distributed with a mean duration of several seconds. In the completely substituted mutant (CSM), all serines and threonines highlighted in (a) were substituted with alanines. S338/CSM and S334/S338/CSM had the same substitutions except at position 338 and at positions 334 and 338, respectively. The wild type response is shown with the bold trace. Reproduced with permission from [31]. (c) Averaged single photon responses of rods with more than three phosphorylation sites on rhodopsin recovered somewhat more slowly than normal, but response reproducibility in individual trials was preserved. The recovery rate seemed to depend more on the number of phosphorylation sites rather than their positions. The reduced rate of rise seen in S334/S338/CSM and in the serine triple mutant (STM) may reflect rhodopsin hyperexpression or compensatory changes in the phototransduction machinery. Labels designate serine to alanine mutations. In STM, substitutions were made at positions 334, 338, 343. The wild type response is shown in bold. Reproduced with permission from [31]. www.current-opinion.com

Current Opinion in Neurobiology 2003, 13:404–412

410 Sensory systems

Gene expression How do rods coordinate their gene expression? Changes in the patterns of expression in transgenic animals reveal some clues. Disruption of one rhodopsin allele halved the protein content, but expression increased with the addition of transgenes for opsin [11,40]. We propose that upregulation of rhodopsin is not possible in Rþ/ rods because the gene is already translated and transcribed at the cell’s maximal rate. Perhaps this is to be expected, given the rod’s prodigious production of ten million copies per day. In contrast, there was compensatory upregulation in Tþ/ [6] and RGS9þ/ [20] mice. Arrestin fell 70% in Aþ/ mice [16], perhaps because the rate of production could not keep up with the rate of degradation. The levels or activities of some proteins are interdependent. Proper folding and/or protection from degradation of RGS9/Gb5 (G protein b subunit type 5) and phosphodiesterase depend upon co-expression of all subunits [20,41,42]. That interdependence is not universal, however. Increases or decreases in transducin a-subunit expression had no effect on the levels of the transducin b-subunit [6,43]. A fall in one protein can sometimes raise another; for example, phosducin levels are higher after heterozygous deletion of either rhodopsin [5] or the transducin a-subunit [6]. Unraveling the regulation of protein expression stands as a challenge for future investigations. Methods for screening RNA for a large number of retinal genes [44,45] should prove invaluable to the discovery of other relationships; however, RNA and protein levels do not necessarily correspond, for example, Gb5 in the RGS9þ/ mouse [20]. It is important to note that such variables complicate the interpretation of a transgenic phenotype and underscore the importance of checking protein levels carefully.

Cones One advantage of a concentration dependent system is the relative ease with which it can be modified on an evolutionary as well as a physiological time scale [46]. Certain fish living at great oceanic depths pack more rhodopsin into their rod outer segments to improve photon capture [47,48]. Although protein crowding would be expected to produce slow photoresponses, such a trade-off may be acceptable in this ecological niche. Do cones lower their pigment density to reduce flash sensitivity and speed up their photoresponses? Apparently not; the pigment concentration in cones and rods is similar [39]. Lowering pigment density may not be a viable option in cones because it seems to compromise outer segment structure [5,11,49,50]. Instead, differences in the concentrations of other proteins may be involved. RGS9 deletion slowed the cone flash response recovery [51] and RGS9 is expressed at higher levels in cones than in rods [52]. It will be very interesting to find out whether or not the RGS9 knockout has a greater impact on cone than on rod photoresponses. Rhodopsin Current Opinion in Neurobiology 2003, 13:404–412

kinase was found to be important for the timely recovery of mouse cone responses [53]. This came as a surprise, because in other species cones express a distinct kinase [54,55]. Levels of rhodopsin kinase and recoverin in cones, as well as the importance of kinase type, have yet to be determined.

Conclusions In the future, the application of emerging genetic technologies to photoreceptors including inducible [56] and suppressible promoters [57], antisense knockdown [58], and ribozymes [59,60] will be useful in evaluating other factors that affect the photoresponse kinetics of rods and cones, and in exploring changes that occur during light adaptation.

Acknowledgements We thank V Govardovskii and T Isayama for helpful comments on an earlier version of the manuscript. The authors are supported by grants from the National Eye Institute.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Fain GL, Matthews HR, Cornwall MC, Koutalos Y: Adaptation in vertebrate photoreceptors. Physiol Rev 2001, 81:117-151.

2.

Pugh EN Jr, Lamb TD: Phototransduction in vertebrate rods and cones: molecular mechanisms of amplification, recovery and light adaptation. In Handbook of Biological Physics, vol 3. Edited by Stavenga DG, DeGrip WJ, Pugh EN Jr. Amsterdam: Elsevier Science; 2000:183-255.

3.

Roof D, Makino CL: The structure and function of retinal photoreceptors. In Principles and Practice of Ophthalmology, vol 3, edn 2. Edited by Jakobiec FA, Albert DM. Philadelphia: Saunders; 2000:1624-1673.

4. 

Calvert PD, Govardovskii VI, Krasnoperova N, Anderson RE, Lem J, Makino CL: Membrane protein diffusion sets the speed of rod phototransduction. Nature 2001, 411:90-94. The authors found that heterozygous rhodopsin knockout mice expressed 50% less rhodopsin in their disk membranes. The accompanying relief of protein crowding on the membrane accelerated the single photon response kinetics, demonstrating that phototransduction speed is limited by the rates at which membrane proteins encounter one another. 5.

Lem J, Krasnoperova NV, Calvert PD, Kosaras B, Cameron DA, Nicolo´ M, Makino CL, Sidman RL: Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc Natl Acad Sci USA 1999, 96:736-741.

6.

Calvert PD, Krasnoperova NV, Lyubarsky AL, Isayama T, Nicolo´ M, Kosaras B, Wong G, Gannon KS, Margolskee RF, Sidman RL et al.: Phototransduction in transgenic mice after targeted deletion of the rod transducin a-subunit. Proc Natl Acad Sci USA 2000, 97:13913-13918.

7.

Sokolov M, Lyubarsky AL, Strissel KJ, Savchenko AB, Govardovski VI, Pugh EN Jr, Arshavsky VY: Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation. Neuron 2002, 34:95-106.

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Brann MR, Cohen LV: Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina. Science 1987, 235:585-587.

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