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G protein-coupled receptor (GPCR) signaling is crucial for many physiological processes. A signature of such pathways is high amplification, a concept originating from retinal rod phototransduction, whereby one photoactivated rhodopsin molecule (Rho*) was long reported to activate several hundred transducins (G(T)*s), each then activating a cGMP-phosphodiesterase catalytic subunit (G(T)*·PDE*). This high gain at the Rho*-to-G(T)* step has been challenged more recently, but estimates remain dispersed and rely on some nonintact rod measurements. With two independent approaches, one with an extremely inefficient mutant rhodopsin and the other with WT bleached rhodopsin, which has exceedingly weak constitutive activity in darkness, we obtained an estimate for the electrical effect from a single G(T)*·PDE* molecular complex in intact mouse rods. Comparing the single-G(T)*·PDE* effect to the WT single-photon response, both in Gcaps(−/−) background, gives an effective gain of only ∼12–14 G(T)*·PDE*s produced per Rho*. Our findings have finally dispelled the entrenched concept of very high gain at the receptor-to-G protein/effector step in GPCR systems.

The pupil-aligned orientation of retinal photoreceptors is dynamically phototropic, but different between outer and inner segment., Almost a century ago, Stiles and Crawford reported that the human eye is more sensitive to light entering through the pupil center than through its periphery (Stiles-Crawford effect). This psychophysical phenomenon, later found to correlate with photoreceptor orientation toward the pupil, was dynamically phototropic, adjustable within days to an eccentrically displaced pupil. For decades, this phototropism has been speculated to involve coordinated movements of the rectilinear photoreceptor outer and inner segments. We report here that, unexpectedly, the murine photoreceptor outer segment has a seemingly light-independent orientation, but the inner segment’s orientation undergoes light-dependent movement, giving rise to nonrectilinear outer and inner segments in adult mice born and reared in darkness. Light during an early critical period (~P0 to P8), however, largely sets the correct photoreceptor orientation permanently afterward. Unexpectedly, abolishing rod and cone phototransductions did not mimic darkness in early life, suggesting photosignaling extrinsic to rods and cones is involved.

Retinal rod and cone photoreceptors mediate vision in dim and bright light, respectively, by transducing absorbed photons into electrical signals. Their phototransduction mechanisms are essentially identical. However, one distinct difference between them is that, whereas a rod visual pigment remains stable in darkness, a cone pigment is liable to spontaneously dissociate into apo-opsin and retinal (the chromophore) without isomerization. This cone-pigment property has long been reported, but it has often been overlooked in the field. Importantly, because apo-opsin has constitutive activity, it triggers transduction signaling to produce electrical noise even in darkness. Currently, the precise apo-opsin content in dark-adapted cones is unknown, as is its associated dark activity. We report here a systematic study of red (L), green (M) and blue (S) cones, finding different percentages of apo-opsin across them in darkness. Surprisingly, we also found that apo-opsin signaling in L and M cones in darkness is actually much higher than the well-known holo-pigment thermal isomerization activity. As such, given the very low signal amplification at the pigment-to-transducin/phosphodiesterase step in cone transduction, apo-opsin noise may not be easily distinguishable from light-triggered responses and may affect cone vision near threshold.

Olfactory transduction in vertebrate olfactory receptor neurons (ORNs) involves primarily a cAMP-signaling cascade that leads to the opening of cyclic-nucleotide–gated (CNG), nonselective cation channels. The consequent Ca2+ influx triggers adaptation but also signal amplification, the latter by opening a Ca2+-activated Cl channel (ANO2) to elicit, unusually, an inward Cl current. Hence the olfactory response has inward CNG and Cl components that are in rapid succession and not easily separable. We report here success in quantitatively separating these two currents with respect to amplitude and time course over a broad range of odorant strengths. Importantly, we found that the Cl current is the predominant component throughout the olfactory dose–response relation, down to the threshold of signaling to the brain. This observation is very surprising given a recent report by others that the olfactory-signal amplification effected by the Ca2+-activated Cl current does not influence the behavioral olfactory threshold in mice.

It is a deeply engrained notion that the visual pigment rhodopsin signals light as a monomer, even though many G protein-coupled receptors are now known to exist and function as dimers. Nonetheless, recent studies (albeit all in vitro) have suggested that rhodopsin and its chromophore-free apoprotein, R-opsin, may indeed exist as a homodimer in rod disk membranes. Given the overwhelmingly strong historical context, the crucial remaining question, therefore, is whether pigment dimerization truly exists naturally and what function this dimerization may serve. We addressed this question in vivo with a unique mouse line (S-opsin+Lrat−/−) expressing, transgenically, short-wavelength–sensitive cone opsin (S-opsin) in rods and also lacking chromophore to exploit the fact that cone opsins, but not R-opsin, require chromophore for proper folding and trafficking to the photoreceptor’s outer segment. In R-opsin’s absence, S-opsin in these transgenic rods without chromophore was mislocalized; in R-opsin’s presence, however, S-opsin trafficked normally to the rod outer segment and produced functional S-pigment upon subsequent chromophore restoration. Introducing a competing R-opsin transmembrane helix H1 or helix H8 peptide, but not helix H4 or helix H5 peptide, into these transgenic rods caused mislocalization of R-opsin and S-opsin to the perinuclear endoplasmic reticulum. Importantly, a similar peptide-competition effect was observed even in WT rods. Our work provides convincing evidence for visual pigment dimerization in vivo under physiological conditions and for its role in pigment maturation and targeting. Our work raises new questions regarding a potential mechanistic role of dimerization in rhodopsin signaling.

Retinal rod and cone photoreceptors mediate vision in dim and bright light, respectively, by transducing absorbed photons into neural electrical signals. Their phototransduction mechanisms are essentially identical. However, one difference is that, whereas a rod visual pigment remains stable in darkness, a cone pigment has some tendency to dissociate spontaneously into apo-opsin and retinal (the chromophore) without isomerization. This cone-pigment property is long known but has mostly been overlooked. Importantly, because apo-opsin has weak constitutive activity, it triggers transduction to produce electrical noise even in darkness. Currently, the precise dark apo-opsin contents across cone subtypes are mostly unknown, as are their dark activities. We report here a study of goldfish red (L), green (M), and blue (S) cones, finding with microspectrophotometry widely different apo-opsin percentages in darkness, being ∼30% in L cones, ∼3% in M cones, and negligible in S cones. L and M cones also had higher dark apo-opsin noise than holo-pigment thermal isomerization activity. As such, given the most likely low signal amplification at the pigment-to-transducin/phosphodiesterase phototransduction step, especially in L cones, apo-opsin noise may not be easily distinguishable from light responses and thus may affect cone vision near threshold.

Visual pigments can be spontaneously activated by internal thermal energy, generating noise that interferes with real-light detection. Recently, we developed a physicochemical theory that successfully predicts the rate of spontaneous activity of representative rod and cone pigments from their peak-absorption wavelength (λmax), with pigments having longer λmax being noisier. Interestingly, cone pigments may generally be ~25 fold noisier than rod pigments of the same λmax, possibly ascribed to an ‘open’ chromophore-binding pocket in cone pigments defined by the capability of chromophore-exchange in darkness. Here, we show in mice that the λmax-dependence of pigment noise could be extended even to a mutant pigment, E122Q-rhodopsin. Moreover, although E122Q-rhodopsin shows some cone-pigment-like characteristics, its noise remained quantitatively predictable by the ‘non-open’ nature of its chromophore-binding pocket as in wild-type rhodopsin. The openness/closedness of the chromophore-binding pocket is potentially a useful indicator of whether a pigment is intended for detecting dim or bright light. DOI: http://dx.doi.org/10.7554/eLife.18492.001, eLife digest At the back of our eyes is a thin layer of cells that contain light-absorbing pigment molecules. These cells convert light energy into electrical signals that the brain then interprets to allow us to see. In this cell layer, the so-called cone cells work in bright light and provide us with the sense of color, whereas rod cells are for vision in dim light. Each visual pigment consists of a protein with a pocket-like space that holds a compound called a chromophore. Light causes the chromophore to change shape inside the pocket, which in turn activates the pigment. However, the pigments can also become activated at random, even in darkness. These false signals, nicknamed “dark light”, are caused by heat instead of light and essentially create a kind of visual noise that can interfere with vision. In 2011, researchers found that pigments that are most sensitive to the longer wavelengths of light (that is, light redder in color) tend to be noisier. The researchers also found that cone pigments are noisier than rod pigments even if they are most sensitive to the same wavelengths of light. To understand what causes this difference between cone and rod pigments, Yue, Frederiksen et al. – who include many of the researchers involved in the 2011 study – made use of mice with a mutated pigment in their rod cells. The mutant pigment was more sensitive to light of shorter wavelengths and, importantly, it behaved like a cone pigment in some ways but kept the closed pocket that is found in rod pigments. Indeed,Yue, Frederiksen et al. showed that the noise level of this mutant pigment could be accurately predicted from the wavelength it was most sensitive to and how closed its pocket was (in other words, the pocket's “closedness”). Further analyses revealed that an open pocket seems to be common to cone pigments from different species. So, it appears that cone pigments are noisier because they have a more open pocket, and the extra space might allow the chromophore to move around and change shape more easily. Going forward, more visual pigments need to be tested to confirm the relationship between the openness of the chromophore-binding pocket and spontaneous activity. If confirmed, it might be possible to one day predict whether a pigment is intended for dim- or bright-light vision simply by knowing whether its chromophore-binding pocket is more open or closed. DOI: http://dx.doi.org/10.7554/eLife.18492.002

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are recently discovered photoreceptors in the mammalian eye. These photoreceptors mediate primarily nonimage visual functions, such as pupillary light reflex and circadian photoentrainment, which are generally expected to respond to the absolute light intensity. The classical rod and cone photoreceptors, on the other hand, mediate image vision by signaling contrast, accomplished by adaptation to light. Experiments by others have indicated that the ipRGCs do, in fact, light-adapt. We found the same but, in addition, have now quantified this light adaptation for the M1 ipRGC subtype. Interestingly, in incremental-flash-on-background experiments, the ipRGC’s receptor current showed a flash sensitivity that adapted in background light according to the Weber–Fechner relation, well known to describe the adaptation behavior of rods and cones. Part of this light adaptation by ipRGCs appeared to be triggered by a Ca 2+ influx, in that the flash response elicited in the absence of extracellular Ca 2+ showed a normal rising phase but a slower decay phase, resulting in longer time to peak and higher sensitivity. There is, additionally, a prominent Ca 2+ -independent component of light adaptation not typically seen in rods and cones or in invertebrate rhabdomeric photoreceptors.

Parietopsin is a nonvisual green light-sensitive opsin closely related to vertebrate visual opsins and was originally identified in lizard parietal eye photoreceptor cells. To obtain insight into the functional diversity of opsins, we investigated by UV-visible absorption spectroscopy the molecular properties of parietopsin and its mutants exogenously expressed in cultured cells and compared the properties to those of vertebrate and invertebrate visual opsins. Our mutational analysis revealed that the counterion in parietopsin is the glutamic acid (Glu) in the second extracellular loop, corresponding to Glu181 in bovine rhodopsin. This arrangement is characteristic of invertebrate rather than vertebrate visual opsins. The photosensitivity and the molar extinction coefficient of parietopsin were also lower than those of vertebrate visual opsins, features likewise characteristic of invertebrate visual opsins. On the other hand, irradiation of parietopsin yielded meta-I, meta-II, and meta-III intermediates after batho and lumi intermediates, similar to vertebrate visual opsins. The pH-dependent equilibrium profile between meta-I and meta-II intermediates was, however, similar to that between acid and alkaline metarhodopsins in invertebrate visual opsins. Thus, parietopsin behaves as an "evolutionary intermediate" between invertebrate and vertebrate visual opsins.

Rpe65−/−mice are unable to produce 11-cis-retinal, the chromophore of visual pigments. Consequently, the pigment is present as the apoprotein opsin with a minute level of pigment containing 9-cis-retinal as chromophore. Notably, a 10–20% fraction of this opsin is mono-phosphorylated independently of light conditions. To determine the role of rhodopsin kinase (GRK1) in phosphorylating this opsin and to test whether eliminating this phosphorylation would accelerate photoreceptor degeneration, we generated theRpe65−/−Grk1−/−mouse. The retinae ofRpe65−/−Grk1−/−mice had negligible opsin phosphorylation, extensive degeneration with decreased opsin levels, and diminished light-evoked rod responses relative toRpe65−/−mice. These data show that opsin phosphorylation in theRpe65−/−mouse is due to the action of GRK1 and is neuroprotective. However, despite the higher activity of unphosphorylated opsin, the severe loss of opsin in the rapidly degeneratingRpe65−/−Grk1−/−mice resulted in lower overall opsin activity and in higher rod sensitivity compared withRpe65−/−mice. InRpe65−/−Grk1−/−Gnat1−/−mice where transduction activation was blocked, degeneration was only partially prevented. Therefore, increased opsin activity in the absence of phosphorylation was not the only mechanism for the accelerated retinal degeneration. Finally, the deletion of GRK1 triggered retinal degeneration inGrk1−/−mice after 1 month, even in the absence of apo-opsin. This degeneration was independent of light conditions and occurred even in the absence of transducin inGrk1−/−Gnat1−/−mice. Taken together, our results demonstrate a light-independent mechanism for retinal degeneration in the absence of GRK1, suggesting a second, not previously recognized role for that kinase.

A subset of retinal ganglion cells has recently been discovered to be intrinsically photosensitive, with melanopsin as the pigment. These cells project primarily to brain centers for non-image-forming visual functions such as the pupillary light reflex and circadian photoentrainment. How well they signal intrinsic light absorption to drive behavior remains unclear. Here we report fundamental parameters governing their intrinsic light responses and associated spike generation. The membrane density of melanopsin is 104-fold lower than that of rod and cone pigments, resulting in a very low photon-catch and a phototransducing role only in relatively bright light. Nonetheless, each captured photon elicits a large and extraordinarily prolonged response, with a unique shape among known photoreceptors. Remarkably, like rods, these cells are capable of signalling single-photon absorption. A flash causing a few hundred isomerized melanopsin molecules in a retina is sufficient for reaching threshold for the pupillary light reflex.

Retinal rod and cone pigments consist of an apoprotein, opsin, covalently linked to a chromophore, 11-cis retinal. Here we demonstrate that the formation of the covalent bond between opsin and 11-cis retinal is reversible in darkness in amphibian red cones, but essentially irreversible in red rods. This dissociation, apparently a general property of cone pigments, results in a surprisingly large amount of free opsin--about 10% of total opsin--in dark-adapted red cones. We attribute this significant level of free opsin to the low concentration of intracellular free 11-cis retinal, estimated to be only a tiny fraction (approximately 0.1 %) of the pigment content in red cones. With its constitutive transducin-stimulating activity, the free cone opsin produces an approximately 2-fold desensitization in red cones, equivalent to that produced by a steady light causing 500 photoisomerizations s-1. Cone pigment dissociation therefore contributes to the sensitivity difference between rods and cones.

Many ion channels in vivo are heteromeric complexes with well defined subunit compositions. For some channels, domains have been identified that determine whether two or more subunit species are compatible in forming a complex. Nonetheless, an unsolved fundamental question is how the native composition of an ion channel is selected during assembly over functional alternatives, such as heteromeric complexes favored over homomers. Cyclic nucleotide-gated channels are tetramers and, in their native forms, are composed of A and B subunits. Although most A subunits can form functional homomeric channels when expressed alone, A/B heteromeric channels are selectively formed in the presence of a B subunit. Here, we show that this selective assembly of heteromeric channels requires a trimer-forming C-terminal leucine zipper (CLZ) domain recently identified in the distal C terminus of A, but not B, subunits. Thus, a CLZ-defective A subunit no longer forms predominantly A/B heteromeric channels with the B subunit. A mechanism for this specificity involving the trimerization of the CLZ domain is proposed.

We have measured the sensitivity of rod photoreceptors from overnight-dark-adapted Xenopus laevis through developmental stages 46-66 into adulthood by using suction-pipette recording. The dark current increased gradually from approximately 5 pA at stage 46 to approximately 20 pA at stage 57, compared with an adult (metamorphosed) current of approximately 35 pA. This increase in dark current largely paralleled the progressive increase in length and diameter of the rod outer segment (ROS). Throughout stages 46-66, the dark current increased approximately linearly with ROS surface area. At stage 53, there was a steep (approximately 10-fold) increase in the rod flash sensitivity, accompanied by a steep increase in the time-to-peak of the half-saturated flash response. This covariance of sensitivity and time-to-peak suggested a change in the state of adaptation of rods at stage 53 and thereafter. When the isolated retina was preincubated with 11-cis-retinal, the flash sensitivity and the response time-to-peak of rods before stage 53 became similar to those at or after stage 53, suggesting that the presence of free opsin (i.e., visual pigment without chromophore) in rods before stage 53 was responsible for the adapted state (low sensitivity and short time-to-peak). By comparing the response sensitivity before stage 53 to the sensitivity at/after stage 53 measured from rods that had been subjected to various known bleaches, we estimated that 22-28% of rod opsin in stage 50-52 tadpoles (i.e., before stage 53) was devoid of chromophore despite overnight dark-adaptation. When continuously dark adapted for 7 d or longer, however, even tadpoles before stage 53 yielded rods with similar flash sensitivity and response time-to-peak as those of later-stage animals. In conclusion, it appears that chromophore regeneration is very slow in tadpoles before stage 53, but this regeneration becomes much more efficient at stage 53. A similar delay in the maturity of chromophore regeneration may partially underlie the low sensitivity of rods observed in newborn mammals, including human infants.

Photoreceptors of the lizard parietal eye, unlike rods and cones but like most invertebrate photoreceptors, respond to light under dark-adapted conditions with a depolarization. Using excised-patch recordings, we have nonetheless found a cGMP-gated, non-selective cation channel present at high density at the presumptive light-sensitive part (the outer segment) of these cells. This channel resembles the rod cGMP-gated channel in its activation characteristics, and by showing a relative non-selectivity among alkali monovalent cations, a high permeability to Ca 2+ , a high sensitivity to l - cis -diltiazem, as well as a negative modulation by Ca 2+ –calmodulin. This channel appears to mediate phototransduction by opening in the light to produce the depolarizing response.

Cyclic-nucleotide-activated, nonselective cation channels have a central role in sensory transduction. They are most likely tetramers, composed of two subunits ( α and β or 1 and 2), with the former, but not the latter, being able to form homomeric cyclic-nucleotide-activated channels. Identified members of this channel family now include, in vertebrates, the rod and cone channels mediating visual transduction and the channel mediating olfactory transduction, each apparently with distinct α - and β -subunits. Homologous channels have also been identified in Drosophila melanogaster and Caenorhabditis elegans . By co-expressing any combination of two α -subunits, or α - and β -subunits, of this channel family in HEK 293 cells, we have found that they can all co-assemble functionally with each other, including those from fly and nematode. This finding suggests that the subunit members so far identified form a remarkably homogeneous and conserved group, functionally and evolutionarily, with no subfamilies yet identified. The ability to cross-assemble allows these subunits to potentially generate a diversity of heteromeric channels, each with properties specifically suited to a particular cellular function.

We have cloned from human retina a cyclic nucleotide-gated (CNG) ion channel that is distinct from the one found in rod photoreceptors. This channel protein is highly homologous to the CNG channel that recently has been cloned from bovine testis and kidney and has been shown to be present i retinal cone photoreceptors. When expressed in human embryonic kidney cells, the protein forms functional ion channels with properties broadly similar to those described for the cloned bovine channel. The gene for this channel resides on chromosome 2.

Cyclic GMP (cGMP) is the intracellular messenger that mediates phototransduction in retinal rods. As photoisomerizations of rhodopsin molecules are local events, the longitudinal diffusion of cGMP in the rod outer segment should be a contributing factor to the response of the cell to light. We have employed the truncated rod outer segment preparation from bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) to measure the cGMP diffusion coefficient. In this preparation, the distal portion of a rod outer segment was drawn into a suction pipette for measuring membrane current, and the rest of the cell was then sheared off with a glass probe, allowing bath cGMP to diffuse into the outer segment and activate the cGMP-gated channels on the surface membrane. Addition and removal of bath cGMP were fast enough to produce effectively step changes in cGMP concentration at the open end of the outer segment. When cGMP hydrolysis is inhibited by isobutylmethylxanthine (IBMX), the equation for the diffusion of cGMP inside the truncated rod outer segment has a simple analytical solution, which we have used to analyze the rise and decay kinetics of the cGMP-elicited currents. From these measurements we have obtained a cGMP diffusion coefficient of approximately 70 x 10(-8) cm2 s-1 for bullfrog rods and approximately 60 x 10(-8) cm2 s-1 for tiger salamander rods. These values are six to seven times lower than the expected value in aqueous solution. The estimated diffusion coefficient is the same at high (20–1000 microM) and low (5–10 microM) concentrations of cGMP, suggesting no significant effect from buffering over these concentration ranges.

We have used the truncated outer segment preparation to measure rod cGMP-phosphodiesterase activity, as well as its modulation by Ca2+, in darkness and in light. The basal enzyme activity in darkness was approximately 0-3 s-1, and was largely independent of Ca2+ concentration from 10 nM to 10 microM. The steady state activity elicited by a step of light (lambda = 520 nm) was strongly enhanced by Ca2+, increasing from approximately 0.005 s-1/(h nu micron-2 s-1) at 10 nM Ca2+ to approximately 0.16 s-1/h nu micron-2 s-1) at 10 microM Ca2+. Based on these measurements, as well as previous measurements on the effects of Ca2+ on rod guanylate cyclase and the cGMP-gated channel, we have calculated the step response-intensity relation for the rod cell in steady state. This relation agrees reasonably well with the relation directly measured from intact rods. We have also evaluated the relative contributions from the three Ca2+ effects to rod sensitivity. At low background light intensities, the Ca2+ modulation of the guanylate cyclase appears to be the most important for sensitivity regulation. At higher light intensities, especially above half-saturation of the response, the Ca2+ modulation of the light-stimulated phosphodiesterase shows a progressively important influence on the light response; it also extends the Weber-Fechner behavior of the cell to higher intensities. The contribution of the Ca2+ modulation of the cGMP-gated channel is slight throughout.

The cGMP-gated cation channel mediating visual transduction in retinal rods was recently found to comprise at least two subunits, 1 and 2 (or alpha and beta). SDS gels of the purified channel show, in addition to a 63-kDa protein band (subunit 1), a 240-kDa protein band that binds Ca(2+)-calmodulin, a modulator of the channel. To examine any connection between subunit 2 and the 240-kDa protein, cGMP-gated channels formed from the expressed cloned subunits in human embryonic kidney (HEK) 293 cells were tested for Ca(2+)-calmodulin effect. Homooligomeric channels formed by subunit 1 alone showed no sensitivity to Ca(2+)-calmodulin, and neither did heterooligomeric channels formed by subunit 1 and the short alternatively spliced form of subunit 2 (2a). By contrast, the cGMP half-activation constant (K1/2) for heterooligomeric channels formed from subunit 1 and the long form of subunit 2 (2b) was increased 1.5- to 2-fold by Ca(2+)-calmodulin, similar to the increase observed for the native channel. In Western blots of rod outer segment membranes, a subunit 2-specific antibody also recognized the 240-kDa protein. Finally, amino acid sequences derived from peptide fragments of the bovine 240-kDa protein showed approximately 80% identity to regions of subunit 2b of the human channel. These results together suggest that subunit 2b of the rod channel is a component of the 240-kDa protein and that it mediates the Ca(2+)-calmodulin modulation of the channel.

Olfactory receptor neurons depolarize in response to odorant stimulation of their sensory cilia. One transduction mechanism involves a G-protein-mediated increase in adenylate cyclase activity, raising the internal cyclic AMP concentration to open a cyclic nucleotide-activated cation channel on the plasma membrane. An influx of Ca2+ through this channel, which is permeable to both monovalent and divalent cations, triggers olfactory adaptation. Previous work has indicated that at least part of this Ca(2+)-mediated adaptation resides in the channel itself, but the mechanism remains unclear and controversial. Here we use the cloned channel from rat expressed in a cell line and the native channel from rat olfactory receptor cells to show that Ca2+ reduces the apparent affinity of the channel for cAMP by up to 20-fold in the presence of calmodulin, an abundant protein in olfactory cilia. This decrease in apparent affinity appears to involve a direct interaction between Ca(2+)-calmodulin and the channel, and it can reduce the activation of the channel by cAMP by up to a few hundred-fold, suggesting that it may be a key component of the Ca(2+)-triggered olfactory adaptation.

Although visual pigments play key structural and functional roles in photoreceptors, the relationship between the properties of mammalian cone pigments and those of mammalian cones is not well understood. We generated transgenic mice with rods expressing mouse short-wave cone opsin (S-opsin) to test whether cone pigment can substitute for the structural and functional roles of rhodopsin and to investigate how the biophysical and signaling properties of the short-wave cone pigment (S-pigment) contribute to the specialized function of cones. The transgenic S-opsin was targeted to rod outer segments, and formed a pigment with peak absorption at 360 nm. Expression of S-opsin in rods lacking rhodopsin (rho−/−) promoted outer segment growth and cell survival and restored their ability to respond to light while shifting their action spectrum to 355 nm. Using the spectral separation between S-pigment and rhodopsin, we found that the two pigments produced similar photoresponses. Dark noise did not increase in transgenic rods, indicating that thermal activation of S-pigment might not contribute to the low sensitivity of mouse S-cones. Using rod arrestin knock-out animals (arr1−/−), we found that the physiologically active (meta II) state of S-pigment decays 40 times faster than that of rhodopsin. Interestingly, rod arrestin was efficient in deactivating S-pigment in rods, but its deletion did not have any obvious effect on dim-flash response shutoff in cones. Furthermore, transgenic cone arrestin was not able to rescue the slow shutoff of S-pigment dim-flash response inarr1−/− rods. Thus, the connection between rod/cone arrestins and S-pigment shutoff remains unclear.

Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin alpha-subunit were mostly unaffected. Outer segment membranes of GC1-/- and GC double knock-out cones were destabilized and devoid of cone transducin (alpha- and gamma-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the down-regulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated phototransduction proteins.

Signal transduction in rod cells begins with photon absorption by rhodopsin and leads to the generation of an electrical response. The response profile is determined by the molecular properties of the phototransduction components. To examine how the molecular properties of rhodopsin correlate with the rod-response profile, we have generated a knock-in mouse with rhodopsin replaced by its E122Q mutant, which exhibits properties different from those of wild-type (WT) rhodopsin. Knock-in mouse rods with E122Q rhodopsin exhibited a photosensitivity about 70% of WT. Correspondingly, their single-photon response had an amplitude about 80% of WT, and a rate of decline from peak about 1.3 times of WT. The overall 30% lower photosensitivity of mutant rods can be explained by a lower pigment photosensitivity (0.9) and the smaller single-photon response (0.8). The slower decline of the response, however, did not correlate with the 10-fold shorter lifetime of the meta-II state of E122Q rhodopsin. This shorter lifetime became evident in the recovery phase of rod cells only when arrestin was absent. Simulation analysis of the photoresponse profile indicated that the slower decline and the smaller amplitude of the single-photon response can both be explained by the shift in the meta-I/meta-II equilibrium of E122Q rhodopsin toward meta-I. The difference in meta-III lifetime between WT and E122Q mutant became obvious in the recovery phase of the dark current after moderate photobleaching of rod cells. Thus, the present study clearly reveals how the molecular properties of rhodopsin affect the amplitude, shape, and kinetics of the rod response.

Retinal rods and cones share a phototransduction pathway involving cyclic GMP1. Cones are typically 100 times less photosensitive than rods and their response kinetics are several times faster2, but the underlying mechanisms remain largely unknown. Almost all proteins involved in phototransduction have distinct rod and cone variants. Differences in properties between rod and cone pigments have been described, such as a 10-fold shorter lifetime of the meta-II state (active conformation) of cone pigment3, 4, 5, 6 and its higher rate of spontaneous isomerization7, 8, but their contributions to the functional differences between rods and cones remain speculative. We have addressed this question by expressing human or salamander red cone pigment in Xenopus rods, and human rod pigment in Xenopus cones. Here we show that rod and cone pigments when present in the same cell produce light responses with identical amplification and kinetics, thereby ruling out any difference in their signalling properties. However, red cone pigment isomerizes spontaneously 10,000 times more frequently than rod pigment. This high spontaneous activity adapts the native cones even in darkness, making them less sensitive and kinetically faster than rods. Nevertheless, additional factors are probably involved in these differences.

Odorants activate sensory transduction in olfactory receptor neurons (ORNs) via a cAMP-signaling cascade, which results in the opening of nonselective, cyclic nucleotide-gated (CNG) channels. The consequent Ca2+ influx through CNG channels activates Cl channels, which serve to amplify the transduction signal. We investigate here some general properties of this Ca-activated Cl channel in rat, as well as its functional interplay with the CNG channel, by using inside-out membrane patches excised from ORN dendritic knobs/cilia. At physiological concentrations of external divalent cations, the maximally activated Cl current was approximately 30 times as large as the CNG current. The Cl channels on an excised patch could be activated by Ca2+ flux through the CNG channels opened by cAMP. The magnitude of the Cl current depended on the strength of Ca buffering in the bath solution, suggesting that the CNG and Cl channels were probably not organized as constituents of a local transducisome complex. Likewise, Cl channels and the Na/Ca exchanger, which extrudes Ca2+, appear to be spatially segregated. Based on the theory of buffered Ca2+ diffusion, we determined the Ca2+ diffusion coefficient and calculated that the CNG and Cl channel densities on the membrane were approximately 8 and 62 micro m-2, respectively. These densities, together with the Ca2+ diffusion coefficient, demonstrate that a given Cl channel is activated by Ca2+ originating from multiple CNG channels, thus allowing low-noise amplification of the olfactory receptor current.

The bestrophins are a newly described family of anion channels unrelated in primary sequence to any previously characterized channel proteins. The human genome codes for four bestrophins, each of which confers a distinctive plasma membrane conductance on transfected 293 cells. Extracellular treatment with methanethiosulfonate ethyltrimethylammonium (MTSET) of a series of substitution mutants that eliminate one or more cysteines from human bestrophin1 demonstrates that cysteine 69 is the single endogenous cysteine responsible for MTSET inhibition of whole-cell current. Cysteines introduced between positions 78–99 and 223–226 are also accessible to external MTSET, with MTSET modification at positions 79, 80, 83, and 90 producing a 2–6-fold increase in whole-cell current. The latter set of four cysteine-substitution mutants define a region that appears to mediate allosteric control of channel activity. Mapping of transmembrane topography by insertion of N-linked glycosylation sites and tobacco etch virus protease cleavage sites provides evidence for cytosolic N and C termini and an unexpected transmembrane topography with at least three extracellular loops that include positions 60–63, 212–227, and 261–267. These experiments provide the first structural analysis of the bestrophin channel family.

Cyclic nucleotide-gated cation channels are essential in visual and olfactory signal transduction. An additional member of the cGMP-gated channel family, termed CNG-3, has been cloned from bovine kidney. Its deduced amino acid sequence is 60% and 62% identical with the CNG-channel proteins from bovine rod outer segment and bovine olfactory epithelium, respectively. Northern analysis and sequences amplified by the PCR showed that the CNG-3 mRNA is present in testis, kidney, and heart. Calcium permeated the expressed channel in the presence of extracellular Mg2+ and Na+ at membrane potentials from -100 to +45 mV. It is likely that CNG-3 protein is responsible for cGMP-induced Ca2+ entry in cells other than sensory cells.

Visual phototransduction, the conversion of incoming light to an electrical signal, takes place in the outer segments of the rod and cone photoreceptor cells. Light reduces the concentration of cGMP, which, in darkness, keeps open cationic channels present in the plasma membrane of the outer segment. Ca2+plays an important role in phototransduction by modulating the cGMP-gated channels as well as cGMP synthesis and breakdown. Ca2+is involved in a negative feedback that is essential for photoreceptor adaptation to background illumination. The effects of Ca2+on the different components of rod phototransduction have been characterized and can quantitatively account for the steady state responses of the rod cell to background illumination. The propagation of the Ca2+feedback signal from the periphery toward the center of the outer segment depends on the Ca2+diffusion coefficient, which has a value of 15±1 μm2s-1. This value shows that diffusion of Ca2+in the radial direction is quite slow providing a significant barrier in the propagation of the feedback signal. Also, because the diffusion coefficient of Ca2+is much smaller than that of cGMP, the decline of Ca2+in the longitudinal direction lags behind the propagation of excitation by the decline of cGMP.

Cyclic nucleotide-gated channels are ion channels the gating of which is directly controlled by cyclic nucleotides. The most well-known of these are non-selective cation channels that require the binding of cyclic nucleotide in order to open (cyclic nucleotide-activated channels). These channels pass not only monovalent cations, but divalent cations even better. They are prominently present in retinal photoreceptors and olfactory receptor neurons, where they play crucial roles in visual and olfactory transductions (for reviews, see

The action of calmodulin (CaM) on target proteins is important for a variety of cellular functions. We demonstrate here, however, that the presence of a CaM-binding site on a protein does not necessarily imply a functional effect. The α-subunit of the cGMP-gated cation channel of human retinal cones has a CaM-binding site on its cytoplasmic N-terminal region, but the homomeric channel that it forms is not functionally modulated by CaM. Mutational analysis based on comparison to the highly homologous olfactory cyclic nucleotide-gated channel α-subunit, which does form a CaM-modulated channel, indicates that residues downstream of the CaM-binding domain on these channels are also important for CaM to have an effect. These findings suggest that a CaM-binding site and complementary structural features in a protein probably evolve independently, and an effect caused by CaM occurs only in the presence of both elements. More generally, the same may be true for other recognized binding sites on proteins for modulators or activators, so that a demonstrated physical interaction does not necessarily imply functional consequence.

Biochemical experiments by others have indicated that protein kinase C activity is present in the rod outer segment, with potential or demonstrated targets including rhodopsin, transducin, cGMP-phosphodiesterase (PDE), guanylate cyclase, and arrestin, all of which are components of the phototransduction cascade. In particular, PKC phosphorylations of rhodopsin and the inhibitory subunit of PDE (PDE γ) have been studied in some detail, and suggested to have roles in downregulating the sensitivity of rod photoreceptors to light during illumination. We have examined this question under physiological conditions by recording from a single, dissociated salamander rod with a suction pipette while exposing its outer segment to the PKC activators phorbol-12-myristate,13-acetate (PMA) or phorbol-12,13-dibutyrate (PDBu), or to the PKC-inhibitor GF109203X. No significant effect of any of these agents on rod sensitivity was detected, whether in the absence or presence of a background light, or after a low bleach. These results suggest that PKC probably does not produce any acute downregulation of rod sensitivity as a mechanism of light adaptation, at least for isolated amphibian rods.

Rods and cones of the two vertebrate lateral eyes hyperpolarize when illuminated, a response generated by a cyclic GMP cascade leading to cGMP hydrolysis and consequently the closure of cGMP-gated, non-selective cation channels that are open in darkness. Lizards and other lower vertebrates also have a parietal (third) eye, which contains ciliary photoreceptors that under dark-adapted conditions depolarize to light instead. Depolarizing light responses are characteristic of most invertebrate rhabdomeric photoreceptors, and are thought to involve a phosphoinositide signalling pathway (see, for example, refs 7-9). Surprisingly, we have found in excised membrane patches a cGMP-gated channel that is selectively present at high density on the outer segment (the presumptive light-sensitive part) of the parietal eye photoreceptor. Like the light-activated channel of the cell, it is non-selective among cations. Inositol trisphosphate (InsP3) had no effect on the same membrane patches. These findings suggest that the photoreceptors of the parietal eye, like rods and cones, use a cGMP cascade and not an InsP3-mediated pathway for phototransduction, but in this case light increases cGMP. A unifying principle of evolutionary significance emerges: that phototransductions in various ciliary photoreceptors, whether hyperpolarizing or depolarizing, uniformly use a cGMP cascade and a cGMP-gated channel to generate the light response, although there are rich variations in the details.

An ion channel directly activated by cGMP was first discovered about ten years ago. Since then, a number of ion channels with the same property (cyclic nucleotide-activated channels) have been reported that are involved in a variety of cell functions. In addition, other channels have been found that are not primarily controlled by cyclic nucleotides but are modulated by them (cyclic nucleotide-modulated channels). These channels likewise have diverse functions and tissue distributions. Both channel classes are reviewed here. Coverage includes the cyclic-nucleotide binding site on these channels, ion permeation, pharmacological blockers, channel gating and modulation, and physiological functions of the channels.

cGMP mediates vertebrate phototransduction by directly gating cationic channels on the plasma membrane of the photoreceptor outer segment. This second messenger is produced by a guanylate cyclase and hydrolyzed by a light-activated cGMP-phosphodiesterase. Both of these enzyme activities are Ca2+ sensitive, the guanylate cyclase activity being inhibited and the light-activated phosphodiesterase being enhanced by Ca2+. Changes in these activities due to a light-induced decrease in intracellular Ca2+ are involved in the adaptation of photoreceptors to background light. We describe here experiments to characterize the guanylate cyclase activity and its modulation by Ca2+ using a truncated rod outer segment preparation, in order to evaluate the enzyme's role in light adaptation. The outer segment of a tiger salamander rod was drawn into a suction pipette to allow recording of membrane current, and the remainder of the cell was sheared off with a probe to allow internal dialysis. The cGMP-gated channels on the surface membrane were used to monitor conversion of GTP, supplied from the bath, into cGMP by the guanylate cyclase in the outer segment. At nominal 0 Ca2+, the cyclase activity had a Km of 250 microM MgGTP and a Vmax of 25 microM cGMP s-1 in the presence of 1.6 mM free Mg2+; in the presence of 0.5 mM free Mg2+, the Km was 310 microM MgGTP and the Vmax was 17 microM cGMP s-1. The stimulation by Mg2+ had an EC50 of 0.2 mM Mg2+ for MgGTP at 0.5 mM. Ca2+ inhibited the cyclase activity. In a K+ intracellular solution, with 0.5 mM free Mg2+ and 2.0 mM GTP, the cyclase activity was 13 microM cGMP s-1 at nominal 0 Ca2+; Ca2+ decreased this activity with a IC50 of approximately 90 nM and a Hill coefficient of approximately 2.0.

1. The outer segment of an isolated rod photoreceptor from the bullfrog retina was drawn into a pipette containing choline solution for recording membrane current. The rest of the cell was sheared off with a glass probe to allow internal dialysis of the outer segment with a bath potassium solution ('truncated rod outer segment' preparation). The potential between the inside and the outside of the pipette was held at 0 mV. 2. Application of bath cGMP, in the presence of 3-isobutyl-1-methylxanthine (IBMX), gave rise to an outward membrane current. At saturating cGMP concentrations, this current was insensitive to intracellular Ca2+ at concentrations between 0 and 10 microM. At subsaturating cGMP concentrations, however, this current was inhibited by intracellular Ca2+. This sensitivity to Ca2+ declined after dialysis with a low-Ca2+ solution, suggesting the involvement of a soluble factor. 3. At low (nominally 0) Ca2+, the half-maximal activation constant and Hill coefficient for the activation of the cGMP-gated current by cGMP were 27 microM and 2.0, respectively. At high (ca 10 microM) Ca2+, the corresponding values were 40 microM cGMP and 2.4. 4. The inhibition of the current by Ca2+ was characterized at 20 microM cGMP. Ca2+ inhibited the current by up to 60%, with half-maximal inhibition at 48 nM Ca2+ and a Hill coefficient of 1.6.

Although several ion channels have been reported to be directly modulated by calcium-calmodulin, they have not been conclusively shown to bind calmodulin, nor are the modulatory mechanisms understood. Study of the olfactory cyclic nucleotide-activated cation channel, which is modulated by calcium-calmodulin, indicates that calcium-calmodulin directly binds to a specific domain on the amino terminus of the channel. This binding reduces the effective affinity of the channel for cyclic nucleotides, apparently by acting on channel gating, which is tightly coupled to ligand binding. The data reveal a control mechanism that resembles those underlying the regulation of enzymes by calmodulin. The results also point to the amino-terminal part of the olfactory channel as an element for gating, which may have general significance in the operation of ion channels with similar overall structures.

Odor stimulation induces a depolarization of olfactory receptor cells. It is now generally accepted that this signal transduction process is mediated by second messenger cascades, cAMP and/or IP3 pathway [1–6]. Because both cAMP- and IP3-gated channels are known to be cation selective, it is natural to think that odorant-induced current is carried solely by cations. In this study, however, we show that the odorant-induced current also contains an inward chloride component triggered by calcium influx through the cation-selective channel. The cationic component has characteristics very similar to the current induced by an injection of cyclic AMP (cAMP), suggesting that the cationic conductance is identical to cAMP-activated channels found in olfactory cilia [1]. Because the Cl component is dependent on the presence of external Ca, it seems likely that the anionic channel is identical to the Ca-activated Cl channel also found in cilia [7].

The effect of Zn2+ on a voltage-dependent, transient potassium current (IA) in acutely dissociated neurons from the suprachiasmatic nucleus was studied with the whole-cell patch-clamp technique. At micromolar concentrations, Zn2+ markedly potentiated IA activated from a holding potential of -60 mV, which is the resting potential of these neurons. This potentiation occurred at a Zn2+ concentration as low as 2 microM and increased with higher Zn2+ concentrations. The Zn2+ action appears to arise from a shift in the steady-state inactivation of IA to more positive voltages. At 30 microM, Zn2+ shifted the half-inactivation voltage by +20 mV (from -80 mV to -60 mV), and 200 microM Zn2+ shifted this voltage by +45 mV (from -80 mV to -35 mV). Histochemically, we have also observed Zn2+ staining throughout the suprachiasmatic nucleus; the staining is particularly intense in the ventrolateral region of the nucleus, which receives the major fiber inputs. Our findings suggest that Zn2+, presumably synaptically released, may modulate the electrical activity of suprachiasmatic nucleus neurons through IA. Because vesicular Zn2+ is fairly widespread in the central nervous system, it is conceivable that this kind of Zn2+ modulation on IA, and possibly on other voltage-activated currents, exists elsewhere in the brain.

Odorant stimulation leads to a depolarization of olfactory receptor neurons. A mechanism underlying this transduction, which occurs in the sensory cilia, involves a G-protein-mediated increase in adenylyl cyclase activity, and therefore a rise in internal cyclic AMP and consequent opening of a cAMP-gated cation channel on the plasma membrane. Another mechanism, not as well established, involves the opening of an inositol trisphosphate-activated cation channel on the plasma membrane as a result of phospholipase C activity. In both cases, an influx of cations is thought to generate the depolarizing receptor potential. We now report, however, that the mechanism is actually more complex. The odorant-induced current appears to contain an inward chloride component also, which is triggered by calcium influx through the cation-selective channel. This newly found chloride component can be as large as the cationic component. The co-existence of cationic and chloride components in the odorant response, possibly unique among sensory transduction mechanisms, may serve to reduce variations in the transduction current resulting from changes in external ionic concentrations around the olfactory cilia. Our finding can explain the long-standing puzzle of why removal of most mucosal cations still does not diminish the amplitude of the olfactory receptor cell response.

RETINAL rods respond to light with a membrane hyperpolarization produced by a G-protein-mediated signalling cascade that leads to cyclic GMP hydrolysis and the consequent closure of a cGMP-gated channel that is open in darkness1–7. A protein that forms this channel has recently been purified from bovine retina8 and molecularly cloned9, suggesting that the native cGMP-gated channel might be a homo-oligomer10. Here we report the cloning of another protein from human retina which has only about 30% overall identity to the rod channel subunit. This protein, immunocytochemically localized to rod outer segments, does not form functional channels by itself. However, when co-expressed with the cloned human rod channel protein11, it introduces rapid flickers to the channel openings that are characteristic of the native channel12–16. The hetero-oligomeric channel is also highly sensitive to the blocker L-cis-diltiazem, like the native channel15,17,18. This new protein thus seems to be another subunit of the native rod channel. The hetero-oligomeric nature of the rod channel means that it is no exception to a common motif shared by other ligand-gated channels.

We briefy summarize here a study of the cyclic GMP-activated conductance in retinal cones. With its lack of desensitization and fast gating kinetics, this conductance is very similar to the homologous conductance in rods. The lack of channel desensitization permits a steady dark current to be maintained, while the fast gating kinetics allow the channel to act as a rapid sensor for the decrease in free cGMP concentration in the photoreceptor outer segment during illumination.

The responses of rabbit rods to light were studied by drawing a single rod outer segment projecting from a small piece of retina into a glass pipette to record membrane current. The bath solution around the cells was maintained at near 40 degrees C. Light flashes evoked transient outward currents that saturated at up to approximately 20 pA. One absorbed photon produced a response of approximately 0.8 pA at peak. At the rising phase of the flash response, the relation between response amplitude and flash intensity (IF) had the exponential form 1-e-kappa FIF (where kappa F is a constant denoting sensitivity) expected from the absence of light adaptation. At the response peak, however, the amplitude-intensity relation fell slightly below the exponential form. At times after the response peak, the deviation was progressively more substantial. Light steps evoked responses that rose to a transient peak and rapidly relaxed to a lower plateau level. The response-intensity relation again indicated that light adaptation was insignificant at the early rising phase of the response, but became progressively more prominent at the transient peak and the steady plateau of the response. Incremental flashes superposed on a steady light of increasing intensity evoked responses that had a progressively shorter time-to-peak and faster relaxation, another sign of light adaptation. The flash sensitivity changed according to the Weber-Fechner relation (i.e., inversely) with background light intensity. We conclude that rabbit rods adapt to light in a manner similar to rods in cold-blooded vertebrates. Similar observations were made on cattle and rat rods.

Light generates a hyperpolarizing response in retinal photoreceptors by stopping a steady inward current (the dark current) that is present at their outer segments in darkness.(1–3) The mechanism underlying this phototransduction process has been a subject of intense interest and controversy for many years, and is only now becoming fairly clear. The present picture for rods(4–6) is that upon absorbing a photon a visual pigment molecule isomerizes and catalytically activates a GTP-binding protein (also called G-protein) that is bound peripherally to disk membranes within the outer segment. The activated G-protein in turn stimulates a phosphodiesterase that hydrolyzes cGMP, a cyclic nucleotide known to be present at a high concentration in the outer segment. In darkness, this high cGMP level maintains a plasma membrane cationic conductance (the light-regulated conductance) in the open state, thus sustaining the dark current. In the light, the fall in the cGMP level leads to the closure of this conductance, and therefore the cessation of the dark current. This picture for phototransduction in rods now seems to apply to cones as well,(7) despite their different surface membrane geometries.

The plasma membrane of retinal rod outer segments contains a cyclic GMP-activated conductance1–6 which appears to be the light-sensitive conductance involved in phototransduction7. Recently, it has been found that this conductance is partially blocked by Mg2+ (refs 3, 8, 9) and Ca2+ (refs 3, 4, 8–11) at physiological concentrations, thus possibly accounting for the absence of observable single-channel activity in excised membrane patches1,3,8 and for the unusually small apparent unit conductance deduced from noise measurements on intact cells4,10,12,13. We now report that, as expected from this idea, single cGMP-activated channel activity can be detected from an excised rod membrane patch in the absence of divalent cations. The most prominent unitary current had a mean conductance of ∼25 pS. Both individual channel openings (mean open time ∼1 ms) and short bursts of openings (mean burst duration of about a few milliseconds) were observed. In addition, there were smaller events which probably represented other states of the conductance. The mean current increased with the third power of cGMP concentration, suggesting that there are at least three cGMP-binding sites on the channel molecule. With 0.2 mM Mg2+ in the cGMP-containing solution, a flickering block of the open channel was observed; the effect of Ca2+ was similar. The results resolve a puzzle about the light-sensitive conductance by demonstrating that it is an aqueous pore rather than a carrier.

The effect of ions on the light-sensitive current of retinal rods was studied by sucking the inner segment into a tightly fitting capillary with the outer segment projecting into a flowing solution. This new method showed that the light-sensitive pathway, in which Na+ is them normal carrier of current, has an ionic selectivity different from that of other known sodium channels. External calcium has a striking effect on the current, which increased about 20-fold when all calcium was removed. Reducing the sodium concentration gradient greatly prolonged the response to a flash of light, as would be expected if internal calcium blocks sodium channels and if light releases calcium which is subsequently extruded by a sodium–calcium exchange mechanism.