Vertebrate Rod Photoreceptors Research Articles

Dynamic Behavior of Rod Photoreceptor Disks

Eukaryotic cells use membrane organelles, like the endoplasmic reticulum or the Golgi, to carry out different functions. Vertebrate rod photoreceptors use hundreds of membrane sacs (the disks) for the detection of light. We have used fluorescent tracers and single cell imaging to study the properties of rod photoreceptor disks. Labeling of intact rod photoreceptors with membrane markers and polar tracers revealed communication between intradiskal and extracellular space. Internalized tracers moved along the length of the rod outer segment, indicating communication between the disks as well. This communication involved the exchange of both membrane and aqueous phase and had a time constant in the order of minutes. The communication pathway uses ∼2% of the available membrane disk area and does not allow the passage of molecules larger than 10 kDa. It was possible to load the intradiskal space with fluorescent Ca 2+ and pH dyes, which reported an intradiskal Ca 2+ concentration in the order of 1 μM and an acidic pH 6.5, both of them significantly different than intracellular and extracellular Ca 2+ concentrations and pH. The results suggest that the rod photoreceptor disks are not discrete, passive sacs but rather comprise an active cellular organelle. The communication between disks may be important for membrane remodeling as well as for providing access to the intradiskal space of the whole outer segment.

(1)H and (13)C MAS NMR evidence for pronounced ligand-protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin.

Rhodopsin is a member of the superfamily of G-protein-coupled receptors. This seven alpha-helix transmembrane protein is the visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In the active binding site of this protein the ligand or chromophore, 11-cis-retinal, is covalently bound via a protonated Schiff base to lysine residue 296. Here we present the complete (1)H and (13)C assignments of the 11-cis-retinylidene chromophore in its ligand-binding site determined with ultra high field magic angle spinning NMR. Native bovine opsin was regenerated with 99% enriched uniformly (13)C-labeled 11-cis-retinal. From the labeled pigment, (13)C carbon chemical shifts could be obtained by using two-dimensional radio frequency-driven dipolar recoupling in a solid-state magic angle spinning homonuclear correlation experiment. The (1)H chemical shifts were assigned by two-dimensional heteronuclear ((1)H-(13)C) dipolar correlation spectroscopy with phase-modulated Lee-Goldburg homonuclear (1)H decoupling applied during the t(1) period. The data indicate nonbonding interactions between the protons of the methyl groups of the retinylidene ionone ring and the protein. These nonbonding interactions are attributed to nearby aromatic acid residues Phe-208, Phe-212, and Trp-265 that are in close contact with, respectively, H-16/H-17 and H-18. Furthermore, binding of the chromophore involves a chiral selection of the ring conformation, resulting in equatorial and axial positions for CH(3)-16 and CH(3)-17.

Calcium Diffusion Coefficient in Rod Photoreceptor Outer Segments

Calcium (Ca 2+) modulates several of the enzymatic pathways that mediate phototransduction in the outer segments of vertebrate rod photoreceptors. Ca 2+ enters the rod outer segment through cationic channels kept open by cyclic GMP (cGMP) and is pumped out by a Na +/Ca 2+,K + exchanger. Light initiates a biochemical cascade, which leads to closure of the cGMP-gated channels, and a concomitant decline in the concentration of Ca 2+. This decline mediates the recovery from stimulation by light and underlies the adaptation of the cell to background light. The speed with which the decline in the Ca 2+ concentration propagates through the rod outer segment depends on the Ca 2+ diffusion coefficient. We have used the fluorescent Ca 2+ indicator fluo-3 and confocal microscopy to measure the profile of the Ca 2+ concentration after stimulation of the rod photoreceptor by light. From these measurements, we have obtained a value of 15 ± 1 μm 2s −1 for the radial Ca 2+ diffusion coefficient. This value is consistent with the effect of a low-affinity, immobile buffer reported to be present in the rod outer segment (L. Lagnado, L. Cervetto, and P. A. McNaughton, 1992, J. Physiol. 455:111–142) and with a buffering capacity of ∼20 for rods in darkness (S. Nikonov, N. Engheta, and E. N. Pugh, Jr., 1998, J. Gen. Physiol. 111:7–37). This value suggests that diffusion provides a significant delay for the radial propagation of the decline in the concentration of Ca 2+. Also, because of baffling by the disks, the longitudinal Ca 2+ diffusion coefficient will be in the order of 2 μm 2s −1, which is much smaller than the longitudinal cGMP diffusion coefficient (30–60 μm 2s −1; Y. Koutalos, K. Nakatani, and K.-W. Yau, 1995, Biophys. J. 68:373–382). Therefore, the longitudinal decline of Ca 2+ lags behind the longitudinal spread of excitation by cGMP.

Two temporal phases of light adaptation in retinal rods.

Vertebrate rod photoreceptors adjust their sensitivity as they adapt during exposure to steady light. Light adaptation prevents the rod from saturating and significantly extends its dynamic range. We examined the time course of the onset of light adaptation in bullfrog rods and compared it with the projected onset of feedback reactions thought to underlie light adaptation on the molecular level. We found that adaptation developed in two distinct temporal phases: (1) a fast phase that operated within seconds after the onset of illumination, which is consistent with most previous reports of a 1-2-s time constant for the onset of adaptation; and (2) a slow phase that engaged over tens of seconds of continuous illumination. The fast phase desensitized the rods as much as 80-fold, and was observed at every light intensity tested. The slow phase was observed only at light intensities that suppressed more than half of the dark current. It provided an additional sensitivity loss of up to 40-fold before the rod saturated. Thus, rods achieved a total degree of adaptation of approximately 3,000-fold. Although the fast adaptation is likely to originate from the well characterized Ca(2+)-dependent feedback mechanisms regulating the activities of several phototransduction cascade components, the molecular mechanism underlying slow adaptation is unclear. We tested the hypothesis that the slow adaptation phase is mediated by cGMP dissociation from noncatalytic binding sites on the cGMP phosphodiesterase, which has been shown to reduce the lifetime of activated phosphodiesterase in vitro. Although cGMP dissociated from the noncatalytic binding sites in intact rods with kinetics approximating that for the slow adaptation phase, this hypothesis was ruled out because the intensity of light required for cGMP dissociation far exceeded that required to evoke the slow phase. Other possible mechanisms are discussed.

Dependence of RGS9–1 Membrane Attachment on Its C-terminal Tail

RGS9-1 is a GTPase-accelerating protein (GAP) required for rapid recovery of the light response in vertebrate rod and cone photoreceptors. Similar to its phototransduction partners transducin (G(t)) and cGMP phosphodiesterase, it is a peripheral protein of the disc membranes, but it binds membranes much more tightly. It lacks the lipid modifications found on G(t) and cGMP phosphodiesterase, and the mechanism for membrane attachment is unknown. We have used limited proteolysis to generate a fragment of RGS9-1 that is readily removed from membranes under moderate salt conditions. Immunoblots reveal that this soluble fragment lacks a 3-kDa fragment from the C-terminal domain, the only domain within RGS9-1 that differs in sequence from the brain-specific isoform RGS9-2. Recombinant fragments of RGS9-1 with or without the partner subunit G beta(5L) were constructed with or without the C-terminal domain. Those lacking the C-terminal domain bound to photoreceptor membranes much less tightly than those containing it. Removal by urea of G beta(5L) from endogenous or recombinant RGS9-1 bound to rod outer segment membranes left RGS9-1 tightly membrane-bound, and recombinant RGS9-1 was urea-soluble in the absence of membranes. Thus the C-terminal domain of RGS9-1 is critical for membrane binding, whereas G beta(5L) does not play an important role in membrane attachment.

Folding and subunit assembly of photoreceptor peripherin/rds is mediated by determinants within the extracellular/intradiskal EC2 domain: implications for heterogeneous molecular pathologies.

Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes.

Growth factors regulate phototransduction in retinal rods by modulating cyclic nucleotide-gated channels through dephosphorylation of a specific tyrosine residue.

Illumination of vertebrate rod photoreceptors leads to a decrease in the cytoplasmic cGMP concentration and closure of cyclic nucleotide-gated (CNG) channels. Except for Ca(2+), which plays a negative feedback role in adaptation, and 11-cis-retinal, supplied by the retinal pigment epithelium, all of the biochemical machinery of phototransduction is thought to be contained within rod outer segments without involvement of extrinsic regulatory molecules. Here we show that insulin-like growth factor-I (IGF-I), a paracrine factor released from the retinal pigment epithelium, alters phototransduction by rapidly increasing the cGMP sensitivity of CNG channels. The IGF-I-signaling pathway ultimately involves a protein tyrosine phosphatase that catalyzes dephosphorylation of a specific residue in the alpha-subunit of the rod CNG channel protein. IGF-I conjointly accelerates the kinetics and increases the amplitude of the light response, distinct from events that accompany adaptation. These effects of IGF-I could result from the enhancement of the cGMP sensitivity of CNG channels. Hence, in addition to long-term control of development and survival of rods, growth factors regulate phototransduction in the short term by modulating CNG channels.

Role of Noncovalent Binding of 11- cis-Retinal to Opsin in Dark Adaptation of Rod and Cone Photoreceptors

Regeneration of visual pigments of vertebrate rod and cone photoreceptors occurs by the initial noncovalent binding of 11- cis-retinal to opsin, followed by the formation of a covalent bond between the ligand and the protein. Here, we show that the noncovalent interaction between 11- cis-retinal and opsin affects the rate of dark adaptation. In rods, 11- cis-retinal produces a transient activation of the phototransduction cascade that precedes sensitivity recovery, thus slowing dark adaptation. In cones, 11- cis-retinal immediately deactivates phototransduction. Thus, the initial binding of the same ligand to two very similar G protein receptors, the rod and cone opsins, activates one and deactivates the other, contributing to the remarkable difference in the rates of rod and cone dark adaptation.

Looking slim: peripherin/rds and photoreceptor cell morphology

The outer segments of vertebrate rod photoreceptor cells are specialized organelles that function in visual excitation by transducing light into electrical signals. These organelles contain thousands of closely stacked disc membranes, with distinctive protein compositions in their lamellar and rim regions: the lamellae host the visual receptors (opsins), and peripherin/rds (rds, retinal degeneration slow) and a similar transmembrane protein, Rom1, inhabit the rims. The precise structural organization of photoreceptor cells is critical to the visual process; disc flattening maximizes the area available for photoreception and allows efficient renewal of the rod outer segments. Thus, visual processing depends on the correct formation and maintenance of this structure.

Soluble factors and the development of rod photoreceptors.

Photoreceptors are the most abundant cell type in the vertebrate neural retina. Like the other retinal neurons and the Müller glia, they arise from a population of precursor cells that are multipotent and intrinsic to the retina. Approximately 10 years ago, several studies demonstrated that retinal precursor cells (RPCs) are competent to respond to environmental factors that promote cell type determination and differentiation. Since those studies, significant effort has been directed at identifying the molecular nature of these environmental signals and understanding the precise mechanisms they employ to drive RPCs towards the different retinal fates. In this review, we describe the recent progress toward understanding how environmental factors influence the development of vertebrate rod photoreceptors.

Regulation of Photoreceptor Membrane Guanylyl Cyclases by Guanylyl Cyclase Activator Proteins

Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca2+ and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca2+ to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca2+-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca2+ at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca2+ GCAP stimulates and in the presence of Ca2+ it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.

Activity-Dependent Modulation of Rod Photoreceptor Cyclic Nucleotide-Gated Channels Mediated by Phosphorylation of a Specific Tyrosine Residue

Cyclic nucleotide-gated (CNG) channels are crucial for phototransduction in vertebrate rod photoreceptors. The cGMP sensitivity of these channels is modulated by diffusible intracellular messengers, including Ca2+/calmodulin, contributing to negative feedback during sensory adaptation. Membrane-associated protein tyrosine kinases and phosphatases also modulate rod CNG channels, but whether this results from direct changes in the phosphorylation state of the channel protein has been unclear. Here, we show that bovine rod CNG channel alpha-subunits (bRET) contain a tyrosine phosphorylation site crucial for modulation. bRET channels expressed in Xenopus oocytes exhibit modulation, whereas rat olfactory CNG channels (rOLF) do not. Chimeric channels reveal that differences in the C terminus, containing the cyclic nucleotide-binding domain, account for this difference. One specific tyrosine in bRET (Y498) appears to be crucial; replacement of this tyrosine in bRET curtails modulation, whereas installation into rOLF confers modulability. As the channel becomes dephosphorylated, there is an increase in the rate of spontaneous openings in the absence of ligand, indicating that changes in the phosphorylation state affect the allosteric gating equilibrium. Moreover, we find that dephosphorylation, which favors channel opening, requires open channels, whereas phosphorylation, which promotes channel closing, requires closed channels. Hence, modulation by changes in tyrosine phosphorylation is activity-dependent and may constitute a positive feedback mechanism, contrasting with negative feedback systems underlying adaptation.

12] Purification, characterization, and reconstitution of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels comprise a family of cation-selective channels that are cooperatively activated or inhibited by the binding of cGMP or cAMP. In vertebrate rod and cone photoreceptor cells and olfactory receptor neurons, these channels play a central role in sensory transduction pathways by controlling the flow of Na + and Ca 2+ into the cell in response to signal-mediated changes in intracellular cyclic nucleotide levels. The physiologic role of CNG channels in nonsensory cells, however, has yet to be determined. The CNG channels of rod photoreceptors and olfactory neurons have been most extensively studied at a molecular level. This chapter describes several procedures that have been used to purify the cGMP-gated channel from ROS membranes and reconstitute the channel into lipid vesicles for functional analysis. The application of these methods to the isolation of CNG channels from other cells systems is discussed.

Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins.

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.

Cysteine residues of photoreceptor peripherin/rds: role in subunit assembly and autosomal dominant retinitis pigmentosa.

Peripherin/rds is a tetraspanning membrane glycoprotein that is essential for the morphogenesis and stabilization of outer segments of vertebrate rod and cone photoreceptor cells. Mutations in the gene for peripherin/rds are responsible for retinal degeneration in the rds mouse and a variety of progressive human retinal degenerative diseases including autosomal dominant retinitis pigmentosa and macular dystrophy. Peripherin/rds associates with rom-1, a homologous subunit, to form a heterotetrameric complex. This study examines the importance of cysteine residues for the structure of peripherin/rds and its assembly with rom-1. Each of the 13 cysteine residues in bovine peripherin/rds was individually replaced with a serine residue by site-directed mutagenesis, and the resulting mutants were expressed individually or together with rom-1 in COS-1 cells. SDS-polyacrylamide gel electrophoresis, immunoprecipitation, and velocity sedimentation were carried out to evaluate the ability of these mutants to form disulfide-linked homodimers, associate with rom-1, and assemble into tetramers characteristic of wild-type peripherin/rds. Substitution of each of the six nonconserved cysteines had no apparent effect on dimer formation, folding, or subunit assembly. In contrast, replacement of any of the seven conserved cysteine residues predicted to lie within a 150 amino acid intradiscal loop significantly altered these properties. Six of these mutants, including a C214S mutant linked to autosomal dominant retinitis pigmentosa, were unable to fold normally, interact with rom-1, or self-assemble into tetramers but instead formed a mixture of large aggregates and a smaller component, most likely a dimer. The C150S mutant, on the other hand, was incapable of forming intermolecular disulfide bonds but did associate with rom-1 into a heterotetramer. These results suggest that (1) the conserved C150 residue is required for intermolecular disulfide bonding but not subunit assembly; (2) the six other conserved cysteine residues are crucial for proper folding and subunit assembly, possibly through formation of intramolecular disulfide bonds; and (3) the misfolding and defective subunit assembly of the C214S mutant is responsible for a form of monogenic autosomal dominant retinitis pigmentosa.

Gene Structure and Chromosome Localization of the GγcSubunit of Human Cone G-Protein (GNGT2)

Phototransduction in the vertebrate rod and cone photoreceptors is regulated by structurally homologous and yet distinct groups of signaling proteins. We have previously identified in bovine retinas a cone-specific G-protein γ subunit (Gγc, previously named Gγ8), which may play a key role in coupling the cone visual pigment to phosphodiesterase (O. C. Onget al.,1995,J. Biol. Chem.270: 8495–8500). We report here the characterization of human Gγcand its gene structure. Human Gγcsubunit shares a high degree of sequence identity with the corresponding bovine Gγcisoform (85%) and human rod Gγ1(63%). The protein is specifically localized in cones, as indicated by immunohistochemical staining using anti-Gγcantibodies. Nucleotide sequence analysis of the Gγcgene (GNGT2) reveals a structure consisting of three exons and two introns, with the intron splice sites similar to that of the rod Gγ1gene (GNGT1). By using fluorescencein situhybridization, we have further localized the human GNGT2 gene to chromosome 17q21. The elucidation of the Gγcgene structure would facilitate the identification of genetic defects associated with cone degeneration.

Photo-excitation by a half-carotenoid: symbiosis between retinal and the visual protein opsin

Abstract

Ionic current model of the vertebrate rod photoreceptor

We describe voltage- and calcium-dependent ionic currents in the photoreceptor inner segments similar to the Hodgkin and Huxley ( Journal of Physiology, 117, 500–544, 1952) equations. The model is used to describe both rods and cones by adjusting parameters. To simulate the light response, the inner segment model was connected with the phototransduction model proposed by Torre et al. ( Cola Spring Harbor Symposia on Quantitative Biology, 55, 563–573, 1990). The role of individual ionic currents in the inner segment in shaping the light response was analyzed through computer simulations. The results suggest that: (1) the transient hyperpolarization to a bright flash is generated by I h; (2) the oscillation after prolonged hyperpolarization in rods results from the interaction among I Ca, I K(Ca), and I Cl(Ca). Since the present model describes the biophysical processes from phototransduction to voltage response, the model can be used for analyzing the light response properties of the photoreceptors quantitatively.

Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors

Kinesin superfamily proteins (KIFs) are probable motors in vesicular and non-vesicular transport along microtubular tracks. Since a variety of KIFs have been recently identified in the motile flagella of Chlamydomonas, we sought to ascertain whether KIFs are also associated with the connecting cilia of vertebrate rod photoreceptors. As the only structural link between the rod inner segment and the photosensitive rod outer segment, the connecting cilium is thought to be the channel through which all material passes into and out of the outer segment from the rod cell body. We have performed immunological tests on isolated sunfish rod inner-outer segments (RIS-ROS) using two antibodies that recognize the conserved motor domain of numerous KIFs (anti-LAGSE, a peptide antibody, and anti-Klp1 head, generated against the N terminus of Chlamydomonas Klp1) as well as an antibody specific to a neuronal KIF, KIF3A. On immunoblots of RIS-ROS, LAGSE antibody detected a prominent band at approximately 117 kDa, which is likely to be kinesin heavy chain, and Klp1 head antibody detected a single band at approximately 170 kDa; KIF3A antibody detected a polypeptide at approximately 85 kDa which co-migrated with mammalian KIF3A and displayed ATP-dependent release from rod cytoskeletons. Immunofluorescence localizations with anti-LAGSE and anti-Klp1 head antibodies detected epitopes in the axoneme and ellipsoid, and immunoelectron microscopy with the LAGSE antibody showed that the connecting cilium region was particularly antigenic. Immunofluorescence with anti-KIF3A showed prominent labelling of the connecting cilium and the area surrounding its basal body; the outer segment axoneme and parts of the inner segment coincident with microtubules were also labelled. We propose that these putative kinesin superfamily proteins may be involved in the translocation of material between the rod inner and outer segments.

Regulation of sensitivity in vertebrate rod photoreceptors by calcium

Over the past decade and a half, there have been great advances in our understanding of how light is transduced into electrical signals by the retina; rod and cone photoreceptors in vertebrates. One essential feature of these sensory neurons is their ability to adapt to background illumination, which allows them to function over a broad range of light intensities. This adaptatio appears to arise mostly from negative feedback on phototransduction that is mediated by calcium ions. Recent work has suggested that this feedback is fairly complex, and involves several pathways directed at different components of phototransduction. From direct measurements of these feedback pathways in rods, it is possible to evaluate their relative contributions to the overall sensitivity of the cell. At the same time, these feedback mechanisms, as currently known, appear to be sufficient for explaining the change in sensitivity of rods during adaptation to light.