Visual Cascade. Introduction. Detection of a Single Photon by a Rod Photoreceptor. Secondary article. Mechanism. History.

Transcription

1 Wolfgang Baehr, University of Utah, Salt Lake City, Utah, USA Paul Liebman, University of Pennsylvania, Philadelphia, Pennsylvania, USA Vision begins in the outer segments of rod and cone photoreceptor cells when visual pigments absorb light photons and become activated. Some 10 4 to 10 5 cyclic nucleotide molecules are affected by activation of one rhodopsin molecule, so that signal generation requires enormous amplification through numerous molecules and mechanisms in the visual cascade. Introduction Vision begins in the outer segments of rod (dim light) and cone (bright light) photoreceptor cells of the retina where visual pigment molecules absorb photons and become activated. Individual activated pigment molecules carry the molecular activation signal to G proteins, which in turn activate the phosphodiesterase (PDE) enzyme to break down aqueous cyclic GMP (cgmp). This causes cgmp to be released from the plasma membrane ion channels where its binding sustains the inward-flowing cation current that keeps visual cells depolarized in darkness. The depolarization, as in other nerve cells, causes release of a neurotransmitter from the synaptic terminals of photoreceptors. The neurotransmitter signals other cells of the retina, a neural network that microprocesses visual images before sending the result to the brain via the optic nerve. Thus, light causes rod and cone synaptic transmission to be terminated by activating a chain of molecules that result in closure of the ion channels that conduct a darkness action current. Sight is surprisingly caused by termination of background activity by light! This article describes some details of how the rod and cone receptor molecules convey these functions, how the activations are each terminated after the light message is received, and how genetic failure in any one of the dozens of protein enzymes involved can cause defective vision. Detection of a Single Photon by a Rod Photoreceptor History Only a few years after the discovery (in the early 1900s) that light is not continuous but consists of a stream of electromagnetic particles (quanta or photons), calculation and experiment allowed biophysicists to demonstrate that human observers can detect single quanta within single molecules in single rod photoreceptor cells. Fifty years later, technology made it possible to record the tiny picoampere current change caused by absorption of a single photon in a single rod, confirming quantum sensitivity at first in toads and later in primates. Further demonstration of a match between the human behavioural dim light sensitivity spectrum and the test-tube absorption spectrum of rhodopsin made it clear that activation of single rhodopsin molecules causes rod vision. Communication theory, thermal noise theory, and consideration that some molecules must be affected by activation of one rhodopsin molecule made it clear that the initial photon-induced change in a single molecule must be amplified enormously to cause a reliable signal to be generated. This realization suggested a series of theories and experimental tests of how such a mechanism might work, with the eventual discovery of all the molecules and mechanisms comprising the visual cascade that are described in this article. Molecules absorb light through electromagnetic resonance of their chromophoric electrons with a specific frequency or wavelength of optical radiation passing within their absorption cross section. For rhodopsin, this absorption can only occur in the space of a few A 2 within the entire molecular physical cross section of 4000 A 2. Thus, light is absorbed with low probability by single molecules and this requires rods to contain 10 7 to 10 8 or more rhodopsin molecules to raise the cumulative probability of absorption to 50% or more. Once a photon is absorbed, its energy is converted to chemical change in rhodopsin (activation) with about 67% efficiency. The remaining 33% of photon absorptions cause heating of the environment and are thus lost to visual activation. Rods and cones. Introduction Secondary article Article Contents. Detection of a Single Photon by a Rod Photoreceptor. Activation of Rhodopsin by Light. Activation of the Visual G Protein, Transducin, by Rhodopsin. Activation of cgmp Phosphodiesterase by Transducin. The cgmp-gated Cation Channel and Rod Cell Hyperpolarization. Calcium and Recovery. Turning Off Rhodopsin by Rhodopsin Kinase and Arrestin. Turning Off Transducin by GTP Hydrolysis A corollary concern is to understand why rod vision functions over only the first 3 5 orders of magnitude of ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / 1

2 light intensity above single-photon sensitivity. Behavioural evidence indicates that cone vision covers most of the remaining 5 7 orders of magnitude of visual sensation (pupillary constriction attenuates little more than one log unit of this range). Are cones unable to respond to single photons? The answer is that rods and cones each respond to single photons but that cones respond with about 100- fold lower cellular amplification and with briefer persistence to photon absorption. Further amplification differences occur in the smooth switchover from use of rod to cone cell types and vision that is coupled, at appropriate light intensity, to corollary switches within retinal neuron microprocessing circuits that trade off sensitivity for spatial resolution in the visual image before it is sent on to the brain. Though differing in morphology, cones are built molecularly much like rods, with nearly identical numbers of pigment and intermediary signal transduction molecules. Activation of Rhodopsin by Light The rod photoreceptor molecules In the 1870s rhodopsin was extracted from the retina using bile salt, a predecessor of modern detergents (Ku hne, 1878). It is an integral membrane protein, permanently embedded among the lipids of the rod disc membranes. It is a member of the very large family of seven transmembrane helix receptors, so called because seven a-helical coils of peptide structure thread their way back and forth across the lipid membrane. These include receptors for most hormones, neurotransmitters and odorants. All such receptors function by activating G proteins and are therefore called G protein-coupled receptors (GPCR). Rhodopsin is a rose-coloured molecule (it absorbs green light and transmits red and violet). Its colour results from the protonated Schiff base linkage of 11-cis-retinal, the aldehyde of vitamin A, to the colourless apoprotein opsin (Wald, 1968). Although retinal itself absorbs only near ultraviolet light (and vitamin A (retinol) only far UV), the spontaneous addition of a proton to the carbon nitrogen double bond linking the two molecules, together with critical placements of intramolecular negative charges, results in the large red shift in the absorption spectrum characteristic of intact rhodopsin. Cone pigments differ from those of rods only in their protein primary sequence, which includes determinants for different absorption colours, faster photobleaching, and possibly weaker or briefer interaction with their G proteins that would decrease signal amplification. Formation of R* Absorbed photons whose energy is not lost to heat cause stereoisomeric conversion of inactive 11-cis retinal to the active all-trans configuration within about 35 femtoseconds. Retinal is converted from a steric shape that fits its surroundings perfectly in the inactive rhodopsin to a misfit shape, requiring at first small local changes in packing and charge distribution, and then more global and distant changes in internal configuration of amino acid side-chains and tertiary structure until a final equilibrium is reached. These changes are identified by a series of precisely measurable changes in colour of the molecule (by visible light spectroscopy) that evolve in time from picoseconds to milliseconds. Ultimately, light-activated rhodopsin becomes colourless, so that the whole process of colour loss is called bleaching. The last pair of spectral and protein conformational states that appear on the time scale of vision are called metarhodopsin I (MI) and metarhodopsin II (MII). Conversion of MI to MII has long been known to involve a large increase in thermodynamic entropy which is ascribed to its substantial change in molecular structure compared to all previous bleaching steps. This structure change has now been given more detailed shape through a variety of physical measurements. MI to MII conversion is the critical step that allows rhodopsin s conversion from a posture of protected inactivity to that of a catalyst that produces the first stage of amplification of the visual signal on rod membranes. The function of MII (or R*) catalysis is to cause a fold acceleration of the spontaneous rate of loss of GDP from inactive G protein and its rapid replacement by GTP (see below) until MII is inactivated by phosphorylation and arrestin binding. The rhodopsin gene was cloned in the 1980s (Nathans and Hogness, 1983), and the first mutation linked to disease was identified in 1990 (Dryja et al., 1990). To date, over 100 mutations in the human gene have been associated with recessive and dominant retinitis pigmentosa, as well as congenital stationary night blindness (CSNB)(see also Table 1). Transgenic mice mimicking human retinitis pigmentosa have been generated. Mutant mice in whom the rhodopsin gene has been knocked out by gene replacement techniques are unable to form disc membranes, indicating a structural role for rhodopsin in addition to that as photoreceptor. Activation of the Visual G Protein, Transducin, by Rhodopsin The G protein, transducin Transducin, first identified in 1979 (Godchaux and Zimmerman, 1979), is the visual member of another ubiquitous family of proteins, the G proteins, and therefore termed G t. Like other G proteins, G t consists of three subunits, G ta,g tb and G tg, and is attached to the disc 2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group /

3 Table 1 The components of the visual cascade in rods and cones Component Subunits Calculated molecular weight (approximate) Amount/ Rho Activity/function Disease Locus Rhodopsin Rho Light reception Catalyst of G tα (GTP)/ Mg 2+ formation RP CSNB 3q21-24 Blue pigment (cone) Light reception Tritanopia 7q Green pigment (cone) Light reception Colour Xq28 blindness Red pigment (cone) Light reception Colour blindness Xq28 Transducin Transducin (cone) Phosphodiesterase (PDE) Phosphodiesterase (PDE c ) (cone) Cation channel Cation channel (cone) G tα G t G tγ G c tα G c t3 G c tγ PDE α PDE PDE γ PDE δ Activator of rod PDE CSNB 3p21 1pter-p Activator rod of cone PDE PDE α' PDE γ ' CN61 CN cgmp hydrolysis RP RP cgmp hydrolysis cgmp-gated cation channel CN cgmp-gated cation channel Exchanger NCKX Exchanges 4 Na + in vs 1Ca 2+ /1K + out; light-insensitive Guanylate cyclase 1 Guanylate cyclase 2 (rod and cone?) Guanylate cyclaseactivating protein 1 Guanylate cyclase activating protein 2 Guanylate cyclaseactivating protein 3 Recoverin (S-modulin) Arrestin RP Rod monochromacy 1p13 12p1 17q21 5q31.2-q34 4p q21.1 2q35-q36 17q21 12p13 4p14-4p12-c 16q13 2q q22 GC Produces cgmp LCA-1 17p13.1 GC Produces cgmp Xq22 GCAP Mediates Ca 2+ sensitivity of GC1 GCAP Mediates Ca 2+ sensitivity of GC1, GC2 GCAP Mediates Ca 2+ sensitivity of GC1, GC2 Cone dystrophy 6p21.1 6p21.1 3q13.1 Rec Ca 2+ sensor 17p13.1 Arr Binds to phosphorylated Rho CSNB 2q24-q37 continued ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / 3

4 Table 1 continued Calculated Component Subunits molecular weight (approximate) Amount/ Rho Activity/function Disease Locus X-arrestin X-arr xq (cone) Phosducin Pdc Binds to G t,α 1q25-q32 R-Kinase RGS9 RK (GRK1) Phosphorylates Rho CSNB 13q34 RGS Accelerates GTP hydrolysis of G t,α -GTP Abbreviations: RP, retinitis pigmentosa; LCA, Leber congenital amaurosis; CSNB, congenital stationary night blindness; p, short arm of human chromosomes; q, long arm. For details and references to mutations in genes of the visual cascade leading to disease of the retina, we refer the interested reader to RetNet/ 17q24 membrane surface through the lipid chains that form part of the a- and g-subunits. Rods and cones have distinct G t subunits. The G tb subunit appears to be always tightly associated with the G tg subunit. The first crystal structure of G ta appeared in 1993 (Noel et al., 1993). There are now some eight different G protein structures in various combinations with nucleotides, with G bg subunits and with specific RGS (regulators of G protein signalling) proteins. of activation Both rhodopsin and G t are in constant Brownian motion, diffusing randomly and colliding endlessly with each other in two-dimensional space along the rod disc membrane surface. Only upon MII formation do they interact specifically and for long enough to cause release of GDP from G t. This allows tighter binding of nucleotide-free G t to MII. The MIIG t complex then awaits the arrival of GTP and Mg 2+ from the adjacent cellular cytoplasm. GTP with Mg 2+ binds to the exact site from which GDP has departed, resulting in internal conformational changes in G t that cause separation of the G ta /GTP from the G tbg subunit and from MII. MII, still active, is then freed, to serially activate other copies of G protein upon diffusional collision. This repetitive process continues at a protein collisional frequency set by diffusion on a membrane surface (for rods about 5000 collisions/second per MII at the prevalent G t concentration of 5000 G t per mm 2 of membrane surface). Thus, protein interdiffusion on rod disc membranes occurs at the rate of one square micron per second and this rate controls the overall speed of the first stage of visual amplification. Subsequent signal transmission and amplification steps occur at a faster rate and are thus not rate limiting. Mutations in the G ta subunit gene have been associated with congenital stationary night blindness of the Nougaret type. Activation of cgmp Phosphodiesterase by Transducin cgmp phosphodiesterase (PDE) degrades the internal messenger of phototransduction, cgmp. This step constitutes the second catalytic interface of the visual cascade in which single photon absorptions are again amplified (gain 2 in Figure 1). PDE enzymatic activity in rods was identified in the early 1970s, and isolated first from frog (1975) and then from bovine tissue (1979). (For references see Liebman et al., 1987). Like G t, PDE is a peripheral rod disc membrane heterotrimeric complex. Components Photoreceptor PDE belongs to the PDE6 subtype of the large and heterogeneous PDE superfamily that is found only in rods and cones as two distinct but closely related versions. Each type of photoreceptor PDE (see Table 1) consists of two large catalytic subunits (PDE a and PDE b in rods, two identical PDE a in cones), and two different cellspecific inhibitory subunits (PDE g and PDE g ). A fourth putative subunit, PDE d, identical for rods and cones, has been identified, but its precise role in regulation of PDE remains to be determined. The large subunits PDE a and PDE b are responsible for the rod membrane association of the PDE holoenzyme through their C-terminal lipid 4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group /

5 Ca GCAP 2+ GC Ca + Ca Ca 2+ K +,Ca 2+ Photon R Ca 4Na + NCKX Arr P P ase 11-cisretinal RK Rec R* T GDP γ α Gain 1 T γ T + α GAP rgs9 γ GC* PDE α γ GCAP GTP Na + cgmp Ca 2+ CaM CaM α CNG open Cations Pdc GTP PDE* γ T α α γ Gain 2 CNG closed GTP GMP Disc membrane Cytoplasm IPM Figure 1 Model of rod phototransduction. Rhodopsin serially activates many copies of the G protein transducin (G t ) (gain 1), and the a subunit of G t,in turn, activates a cgmp-specific phosphodiesterase (PDE), which degrades cytoplasmic cgmp with rapid turnover (gain 2). The drop in [cgmp] causes cgmp-gated cation (CGC) channels in the plasma membrane to close, which produces hyperpolarization of the outer segment plasma membrane. Closure of cation channels prevents entry of cations, and cytoplasmic [Ca 2 1 ] drops owing to its continued extrusion by the light-independent exchanger, also located in the plasma membrane. In a negative feedback loop, the drop in [Ca 2 1 ] causes stimulation of a guanylate cyclase (GC) by one or several specific Ca 2 1 -binding proteins, termed guanylate cyclase-activating proteins (GCAPs). Sites where regulatory proteins (phosducin, Pdc; arrestin, Arr; recoverin, Rec) interact with components of the cascade are indicated by arrows. A simplified movie version of the Visual Cascade can be seen at insight.med.utah.edu (click first on webvision, then click on photoreceptors/transduction). A computer-generated simulation of the lateral diffusional activation of components of the visual cascade can be seen at modifications. Rod PDE a and PDE b are catalytically inactive when individually expressed. In the dark, the activity of PDE is minimized by its (inhibitory) PDE g subunit. This keeps cgmp at a low level but sufficiently high to sustain a small dark current through its binding to a small fraction of the cation channels. During phototransduction, PDE is activated through dislocation of the PDE g subunit by its activator G ta - (GTP) to form PDE*. PDE* is a complex of G ta (GTP) with PDE ab and PDE g. PDE* destroys cgmp as quickly as it can diffuse (maximal rate, several thousand cgmp hydrolysed per second per PDE* at high cgmp levels, several hundred cgmp per second at lower physiological cgmp levels). This powerful (enzymatic) amplification step leads to rapid depletion of cytoplasmic cgmp, dissociation of cgmp from the cation channel, and closure of these cgmp-gated cation channels located in the plasma membrane with hyperpolarization of the rod and termination of synaptic transmission. Defects in genes encoding PDEa and PDEb are associated with recessive RP in humans. Several naturally occurring animal models in which the PDEb subunit is nonfunctional are known (for example, rd mouse, rcd1 Irish setter). Surprisingly, mutant mice in which the gene encoding the inhibitory PDEg subunit is knocked out, display a phenotype resembling that of the rd mouse (inactive ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / 5

6 PDE). This suggests that the presence of PDE g during retinal development is essential for the formation of an active PDE. The cgmp-gated Cation Channel and Rod Cell Hyperpolarization Cyclic nucleotide-gated (CNG) channels and hyperpolarization play important roles in vision, olfaction and other sensory transduction systems. The gating mechanism in rod plasma membranes was identified in 1985 (Fesenko et al., 1985), and the channel subunits were cloned in (for review see Kaupp, 1995). Components Molecular cloning showed that CNG channels are structurally related to voltage-gated ion channels. The rod cgmp-gated channel is composed of two homologous subunits, CNG1 (a subunit) and CNG4 (b subunit), and both are thought to form a heterotetramer. Each subunit contains six putative membrane-spanning segments, a cgmp-binding domain, a voltage sensor-like motif and a pore region. Each subunit can form cgmp-gated channels when expressed individually. CNG4 contains a calmodulin-binding site and an extended N-terminal region that is identical in sequence to a glutamic acid-rich protein (GARP). GARP was independently identified earlier and only later recognized to be part of CNG4. The cgmp-gated cation channel of the rod photoreceptor is the final target of the visual cascade. The allosteric binding of 3 4 cgmp molecules required for full opening of each four-subunit channel allows removal of a single cgmp to act as a sudden off switch, enhancing the sensitivity of detection when levels of cgmp drop by a small amount. Hundreds of channels then act in an ensemble to statistically smooth and faithfully follow the kinetics of the visual cascade response of the rod to a single photon. The affinity of cgmp for the channel may also be modulated by Ca 2 1 /calmodulin and other factors. The corresponding cone channel subunits are encoded by distinct genes and are also modulated by Ca 2 1. The biophysical effect of closure of the channel is to hyperpolarize the photoreceptor cell. Mutations in the rod CNG1 gene have been associated with retinitis pigmentosa. Mutations in the cone CNG3 gene lead to rod monochromacy (total colour blindness). The CNG3 gene has been knocked out in mouse leading to a cone-specific degeneration. Calcium and Recovery Levels of Ca 2 1 ions are key to regulation of phototransduction and recovery of the dark state. In addition to modulation of the CNG channel, there are two more Ca 2 1 -sensitive interfaces in the cascade: (1) Ca 2 1 binding to the protein recoverin downregulates R* phosphorylation by rhodopsin kinase; (2) guanylate cyclase is stimulated in the absence of Ca 2 1 by photoreceptorspecific Ca 2 1 -binding proteins (for a detailed review see Polans et al., 1996). Components The major players involved in restoration of cgmp levels are guanylate cyclase (GC), the enzyme that produces cgmp; the Ca 2 1 -inhibited GC-activating proteins (GCAPs); and the Na 1 /Ca 2 1 /K 1 exchanger (NCKX). Two retina-specific, membrane-associated GCs (GC1 and GC2) were identified and cloned in the 1990s. To date, three GCAPs (GCAP1, GCAP2, GCAP3) have been identified and sequenced (see Table 1). GC1 and GCAP1 are present in rod and cone OS, while GCAP2 is mostly found in rods, cone inner segments, and several cell types of the inner retina. GCAP3 is retina-specific, but its precise subcellular location is yet unknown. The light-insensitive NCKX is a single-subunit membrane protein complex found only in photoreceptors. A significant fraction of the rod NCKX copurifies with the CGC channel, suggesting that the channel may interact with the exchanger under certain conditions (for a detailed review of components see Palczewski et al., 2000). In dark-adapted rods, [Ca 2 1 ] is high ( 500 nmol L 2 1 ), [cgmp] is high (1 10 mmol L 2 1 ), both GC and PDE activities are low, and a fraction of the cation channels are open (Figure 1). Following phototransduction and cgmp hydrolysis, cation channels in the plasma membrane close, influx of cations, including Ca 2 1, is reduced, and [Ca 2 1 ] drops owing to continuous extrusion by the lightinsensitive exchanger (NCKX). The Ca 2 1 -free forms of GCAP(s) are able to stimulate GC, and [cgmp] rises again if (1) MII is quenched and (2) PDE is re-inhibited. As a result, the cation channels re-open, Ca 2+ levels are replenished, and finally GC is deactivated. The mechanism of GCAP/GC interaction is not well understood. It is thought that GCAPs are globular proteins, that form a compact (inactive) structure in the Ca 2 1 -charged form and a much less compact structure (active form) when in 6 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group /

7 the Ca 2 1 -free form. The interaction with GCs occurs on the intracellular kinase-like domain. The binding sites for GCAPs are at least partially overlapping. Mutations in the GC1 gene lead to severe degeneration of rod and cone photoreceptors in human and animal models (Leber congenital amaurosis type I, cone/rod dystrophy, rd chicken). A missense mutation in the GCAP1 gene has recently been associated with a dominant cone dystrophy (for details see Table 1). Turning Off Rhodopsin by Rhodopsin Kinase and Arrestin Components Rhodopsin kinase (RK) and arrestin (S-antigen, 48K protein) (Arr) were identified in the 1970s and early 1980s. RK contains a lipid modification at its C-terminus that mediates membrane attachment. Phosphorylation of R* by RK is thought to be inhibited by the Ca 2+ -bound form of recoverin (Rec), a protein discovered in This prolongs the action of dim light, resulting in prolonged amplification in this environment. Rods and cones bear the same RK and Rec but have different Arr genes (for review see Palczewski and Saari, 1997). History The discovery of an amplified visual cascade immediately brought into focus a new problem. MII (or R*) remains active for more than a minute after a pulse of light both in the test tube and in the eye before its spontaneous decay to form inactive opsin and all-trans retinal. Yet the rod can recover from one stimulus in time to be reactivated by a new stimulus within a fraction of a second. In biochemical terms, G t and PDE activations are known to continue to accumulate as long as rhodopsin remains active. This prolongs the effect of even weak light so that it appears like a longer-lasting or saturating stimulus that would confuse any newly introduced stimulus while it lasts. The missing link, an R* quenching mechanism that prevents continuation of MII activity, was proposed in 1979, completely proved during , and found to consist of phosphorylation of MII by RK and ATP. Although RK had already been discovered to phosphorylate bleached rhodopsin, the function of this phosphorylation remained unknown for about a decade until its rediscovery with the mechanism of rod phototransduction (a detailed account is given in Liebman and Pugh, 1982). Phosphorylation of receptors by specific receptor kinases (GRKs) is now known to regulate many different seven transmembrane helix receptors. It is thought to be a general mechanism for turning off and/or reducing activity levels of signal transduction activated by hormones, pheromones and neurotransmitters throughout the body and brain. s Detailed work showed that rhodopsin is very rapidly and multiply phosphorylated at several serine and threonine residues in its C-terminus. Both phosphorylation and quenching of transduction activity were shown to require rhodopsin kinase. Proteolytic removal of rhodopsin s phosphorylation sites prevents quenching of transduction by ATP. Arrestin may bind phosphorylated rhodopsin to complete the quenching action of ATP. It probably sterically blocks the access of G protein to the activating surface of MII, thus arresting further R* activity. Other unbleached rhodopsin molecules are responsible for absorbing subsequent photons while arrestin-blocked molecules slowly make their way back into the visual cycle after MII decay, dephosphorylation and regeneration by a new molecule of 11-cis-retinal. Mutations in the RK and Arr genes have been associated with Ogouchi disease (recessive CSNB) characterized by delayed photoreceptor recovery. Mice in which the Arr and RK genes have been knocked out simulate the human disease. Turning Off Transducin by GTP Hydrolysis It was noted in the 1980s that hydrolysis of GTP bound to G ta was slow in vitro but dependent on membrane concentration, indicating the presence at high membrane concentrations of a factor that accelerates GTPase activity. This factor, termed GTPase-activating protein (GAP), was finally identified and cloned in the 1990s (He et al., 1998). It was found to be identical with RGS9, a member of the large family of regulators of G protein signalling (RGS). A splice variant of RGS9 (RGS9-1) was found exclusively in the retina while another variant (RGS9-2) and other RGS proteins are present mostly in neurons of the brain. RGS9 is tightly membrane associated but does not contain conventional membrane-binding domains like lipid attachment. The recombinant protein accelerates GTPase activity of transducin nearly 50-fold. An essential cofactor for RGS9 GAP activity is the inhibitory subunit of PDE, PDEg, a feature distinguishing RGS9 from other RGS ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / 7

8 proteins and other factors. RGS9 is present in rods and cones but appears to be present at much higher concentrations in cones, possibly contributing to the much faster response time seen in cones. RGS9 was recently found to be tightly associated with the long splice variant of a G protein b-subunit, Gb5. References Dryja TP, McGee TL, Reichel E et al. (1990) A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 343: Fesenko EE, Kolesnikov SS and Lyubarski AL (1985) Induction by cgmp of cationic conductance in plasma membrane of retinal rod outer segment. Nature 313: Godchaux W III and Zimmerman WF (1979) Membrane-dependent guanine nucleotide binding and GTPase activities of soluble protein from bovine rod cell outer segments. Journal of Biological Chemistry 254: He W, Cowan CW and Wensel TG (1998) RGS9, a GTPase accelerator for phototransduction. Neuron 20: Kaupp UB (1995) Family of cyclic nucleotide gated ion channels. Current Opinion in Neurobiology 5: Ku hne W (1878) U ber den Sehpurpur. In: Untersuchungen aus dem Physiologischen Institut der Universita t Heidelberg, pp Liebman PA and Pugh EN Jr (1982) Gain, speed and sensitivity of GTP binding vs PDE activation in visual excitation. Vision Research 22: Liebman PA, Parker KR and DratzEA (1987) The molecular mechanism of visual excitation and its relation to the structure and composition of the rod outer segment. Annual Review of Physiology 49: Nathans J and Hogness DS (1983) Isolation, sequence analysis, and intron exon arrangement of the gene encoding bovine rhodopsin. Cell 34: Noel JP, Hamm HE and Sigler PB (1993) The 2.2 A crystal structure of transducin-a complexed with GTPgS. Nature 366: Palczewski K and Saari JC (1997) Activation and inactivation steps in the visual transduction pathway. Current Opinion in Neurobiology 7: Palczewski K, Polans AS, Baehr W and Ames JB (2000) Calcium binding proteins in the retina: Structure, function, and the etiology of human disease. BioEssays (in press). Polans A, Baehr W and Palczewski K (1996) Turned on by Ca 2+! The physiology and pathology of Ca 2+ -binding proteins in the retina. Trends in Neuroscience 19: Wald G (1968) The molecular basis of visual excitation. Nature 219: Further Reading Fain GL, Matthews HR and Cornwall MC (1996) Dark adaptation in vertebrate photoreceptors. Trends in Neuroscience 19: Hurley JB (1994) Termination of photoreceptor responses. Current Opinion in Neurobiology 4: Koutalos Y and Yau KW (1996) Regulation of sensitivity in vertebrate rod photoreceptors by calcium. Trends in Neuroscience 19: Liebman PA, Parker KR and DratzEA (1987) The molecular mechanism of visual excitation and its relation to the structure and composition of the rod outer segment. Annual Review of Physiology 49: Palczewski K, Polans AS, Baehr W and Ames JB (2000) Calcium binding proteins in the retina: Structure, function, and the etiology of human disease. BioEssays (in press). Polans A, Baehr W and Palczewski K (1996) Turned on by Ca 2 1! The physiology and pathology of Ca 2 1 -binding proteins in the retina. Trends in Neuroscience 19: ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group /