In the current model of visual transduction, the lifetime of active cGMP phosphodiesterase depends upon the period of its interaction with GTP-bound transducin. If recoverin regulates the lifetime of light-activated cGMP phosphodiesterase through inhibition of rhodopsin phosphorylation, rhodopsin should directly interact with cGMP phosphodiesterase and/or GTP-bound transducin complexed with cGMP phosphodiesterase. Is this true?

The regulatory properties of the neurospecific, type I adenylyl cyclase and its distribution within brain have suggested that this enzyme may be important for neuroplasticity. To address this issue, the murine, Ca2+ -stimulated adenylyl cyclase (type I), was inactivated by targeted mutagenesis. Ca2+ -stimulated adenylyl cyclase activity was reduced 40% to 60% in the hippocampus, neocortex, and cerebellum. Long term potentiation in the CA1 region of the hippocampus from mutants was perturbed relative to controls. Both the initial slope and maxim (...) um extent of changes in synaptic response were reduced. Although mutant mice learned to find a hidden platform normally in the Morris water task, they did not display a preference for the region where the platform had been when it was removed. The behavioral phenotype of these mice is very similar to that exhibited by mice which have been surgically lesioned in the hippocampus. These results indicate that disruption of the gene for the type I adenylyl cyclase produces changes in spatial memory and indicate that the cAMP signal transduction pathway may play an important role for synaptic plasticity. (shrink)

Cyclic nucleotides can regulate the sensitivity of retinal rods to light through phosducin. The phosphorylation state of phosducin determines the amount of G available for activation by Rho*. Phosducin phosphorylation is regulated by cyclic nucleotides through their activation of cAMP-dependent protein kinase. The regulation of phosphodiesterase activity by the noncatalytic cGMP binding sites as well as Ca2+/calmodulin dependent regulation of cGMP binding to the cation channel are also discussed.

Our understanding of the molecular properties and cellular role of cGMP-gated channels in outer segments of vertebrate photo-receptors has come from over a decade of studies which have continuously altered and refined ideas about these channels. Further examination of this current view may lead to future surprises and further refine the understanding of cGMP-gated channels.

Site-specific phenotypic effects of the 73 known alleles in the rhodopsin gene that cause retinal degeneration are difficult to interpret because most alleles are documented in only one case or one family, which means variation in effects could actually arise from interactions with other loci. However, sample sizes necessary to detect epistatic interaction may place an answer to this question beyond our grasp.

Genetic abnormalities of three factors related to the photoreceptor mechanism have been reported in both animal models and humans. Apoptotic mechanism has also been suggested as a final common pathway of photoreceptor cell death. Our findings of increased level of glutamate in photoreceptor cells in rds mice suggest that amino acid might mediate between these two pathological mechanisms.

A number of proteins homologous to recoverin have been identified in the brains of the several vertebrate species. The brainderived members originally contain four EF-hand domains, but NH2- terminal domain is aberrant. Many of these proteins inhibited light-induced rhodopsin phosphorylation at high [Ca2+], suggesting that the brain-derived members may act as a Ca2+-sensitive modulator of receptor phosphorylation, as recoverin does.

Magic angle spinning (MAS) NMR methods provide a means of obtaining high resolution structural data on rhodopsin and its photoin termediates. Current work has focused on the structure of the retinal chromophore and its interactions with surrounding protein charges. The recent development of MAS NMR methods for measuring internuclear distances with a resolution of ∼0.2 will complement diffraction methods for addressing key mechanistic questions.

Inactivation of the Ca2+ extrusion mode of the retinal rod Na- Ca + K exchanger is suggested to be the mechanism that prevents lowering of cytosolic free Ca2+ to < 1 nM when rod cells are saturated for a prolonged time under bright light conditions. Under these conditions, Ca2+ fluxes across disk membranes can contribute significantly to Ca2+ homeostasis in rods.

Amino-terminal heteroacylation has been identified in retinal proteins including recoverin and α subunit of G-protein, transducin. The tissue-specific modification seems to mediate not only a proteinmembrane interaction but also a specific protein-protein interaction. The mechanism generating the heterogeneity and its physiological role are still unclear, but an interesting idea for the latter postulates a fine regulation of the signal transduction pathway by distinct N-acyl groups.

Calcium acting through calmodulin has been shown to regulate the affinity of cyclic nucleotide-gated channels expressed in cell lines. But is calmodulin the Ca-sensor that normally regulates these channels?

Our results on hippocampal long-term potentiation are considered in the context of Xia et al.'s hypothesis. Whereas the target article proposes presynaptic PKC involvement in adenylyl cyclase activation by phosphorylation of nenromodulin, we suggest an additional postsynaptic role involving RC3/nenrogranin. Finally, we examine the possibility that the adenylyl cyclase mutant mouse may display normal learning with a selective impairment of memory.

It has been suggested that type I adenylyl cyclase may play a unique role in long-term potentiation, due to both unique regulatory properties as well as a specialized distribution within the mammalian brain. This would allow an integration of the signals wrought by increased intracellular calcium with those conveyed into the cellular milieu via increased cAMP. These results are discussed in the context of changes in cellular structure, because of changing interactions between G proteins and cytoskeletal components, which might be (...) expected to accompany chronic synaptic activation. (shrink)

Considerable progress has been made toward understanding the involvement of recoverin in a cancer-associated retinopathy (CAR) that results in blindness. We describe the expression of recoverin in tumors of individuals afflicted with CAR, characterize the immunological response towards recoverin in these patients, and demonstrate how the disease can be induced in rodents using recoverin as an immunogen.

The subunit structure of the cGMP-gated cation channel of rod photoreceptors is rapidly being defined, and in the process the mode of regulation by Ca2+-calmodulin unraveled. Intriguingly, early results suggest that additional subunits of unknown function are associated with the channel and remain to be identified.

Recent studies from several different laboratories have provided further insight into structure-function relationships of cyclic nucleotide-gated channel and in particular the cCMPgated channel of rod photoreceptors. Site-directed mutagenesis and rod-olfactory chimeria constructs have defined important amino acids and peptide segments of the channel that are important in ion blockage, ligand specificity, and gating properties. Molecular cloning studies have indicated that cyclic nucleotide-gated channels consist of two subunits that are required to reproduce the properties of the native channels. Biochemical analysis of (...) the cGMP-gated channel of rodcells have indicated that the 240 kDa protein that co-purifies with the 63 kDa channel subunit contains both the previously cloned second subunit of the channel and a glutamic acid-rich protein. The regulatory properties of the cGMP-gated channel from rod cells has also been studied in more detail. Studies indicate that the beta subunit of the cGMP-gated channel of rod cells contains the binding site for calmodulin. Interaction of calmodulin with the channel alters the apparent affinity of the channel for cGMP in all in vitro systems that have been studied. The significance of these recent studies are discussed in relation to the commentaries on the target article. (shrink)

Hereditary retinal degeneration due to mutations in visual genes may be amenable to therapeutic interventions that modulate, either positively or negatively, the amount of protein product. Some of the proteins involved in phototransduction are rapidly moved by a lightdependent mechanism between the inner segment and the outer segment in rod photoreceptor cells, and this phenomenon is important in phototransduction.

Light adaptation is modulated almost exclusively by changes in intracellular Ca2+ concentration, and other Ca2+-independent mechanisms are likely to play only a minor role. Changes in Ca2+i may be not only necessary for light adaptation to take place but sufficient to cause it.

This commentary discusses the balance of phosphodiesterase and guanylate cyclase activities in vertebrate photoreceptors at moderate light intensities. The rate of cGMP hydrolysis and synthesis seem to equal each other. Ca2+as regulator of both enzyme activities is also effectively buffered in photoreceptor cells by cytoplasmic buffer components.

S-Modulin is a frog homolog of recoverin. The function and the underlying mechanism of the action of these proteins are now understood in general. However, there remain some unsolved issues including; two distinct effects of S-modulin; Ca2+-dependent binding of S-modulin to membranes and a possible target protein; S-modulin-like proteins in other neurons. These issues are considered in this commentary.

Although undoubtedly it will be incomplete by the time it is published, the target article by Daiger et al. organizes mutations in genes that produce retinal degenerations in humans into categories of clinically relevant phenotypes. Such classifications should help us understand the link between altered photoreceptor cell proteins and subsequent cell death, and they may yield insight into methods for preventing consequent blindness.

Recoverin is a Ca2+-binding protein found primarily in vertebrate photoreceptors. The proposed physiological function of recoverin is based on the finding that recoverin inhibits light-stimulated phosphorylation of rhodopsin. Recoverin interacts with rod outer segment membranes in a Ca2+-dependent manner. This interaction requires N-terminal acylation of recoverin. Four types of fatty acids have been detected on the N-terminus of recoverin, but the functional significance of this heterogeneous acylation is not yet clear.

The identification of additional subunits of the cGMP-gated cation channel suggests exciting questions about their regulatory roles and about structure/functional relationships. How do the different subunits interact? How is the complex assembled into the plasma membrane? Divalent cations have been implicated in the regulation of adaptation. One often overlooked cation is magnesium. Could this ion play a role in phototransduction?

The type I CaM-sensitive adenylyl cyclase is in a position to integrate signals from multiple inputs, consistent with the requirements for mediating long term potentiation (LTP). Biochemical and genetic evidence supports the idea that this enzyme plays an important role inc LTP. However, more work is needed before we will be certain of the role that CaM-sensitive adenylyl cyclases play in LTP.

The primary sequence of two subunits of the rod and one subunit of the cone cGMP-gated channel have been described, but describing how structure determines function is only just beginning. The discovery that the affinity of the rod channel for its agonist can be modulated indicates that the relationship between intracellular cGMP and the channel's open probability (current) during the course of the photoresponse may be more complex than previously thought.

To understand how the photoreceptor protein rhodopsin performs in its role as a receptor, its structure needs to be determined at the atomic level. Upon receiving a photon of light, rhodopsin undergoes a change in conformation that allows it to bind and activate the C-protein, transducin. An important future goal should be to determine the structure of both the inactive and the photoactivated state of rhodopsin, R*. This should provide the groundwork necessary for experiments on how rhodopsin achieves its signaling (...) state R*, and how R* functions to activate transducin. To do this, the crystal structure of both rhodopsin and R* must be determined. Few membrane proteins have been successfully crystallized, so this is not a trivial undertaking. Two- or three dimensional crystals of rhodopsin must be prepared that are well ordered, to produce a high-resolution structure. Rhodopsin must be purified to homogeneity and the appropriate detergent(s) selected for crystallization experiments. Long-term thermal stability of the rhodopsin-detergent complex must be achieved in the presence of a precipitant. Two-dimensional crystals may offer advantages in investigating the structure of R*, but the structure obtained may be limited in resolution. It is necessary to work with rhodopsin in the dark, unless suitable light-stable retinal derivatives are developed. Protein engineering of rhodopsin offers attractive opportunities to improve its ability to crystallize, but is presently hindered by the absence of a high-yielding expression system. Knowledge of the structure of rhodopsin will have general importance. Because rhodopsin is a member of the family of C-protein-coupled receptors, knowledge of the structure and the mechanism of action of rhodopsin suggests by analogy how other members of the receptor family may function. (shrink)

NMR (nuclear magnetic resonance) may be useful for determining the structure of retinal and its environment in rhodopsin, but not for determining the complete protein structure. Aggregation and low yield of fragments of rhodopsin may make them difficult to study by NMR. A long-term multidisciplinary attack on rhodopsin structure is required.

Molday and Hsu review results from in vitro experiments, which indicate that Ca-bound calmodulin reduces the cGMP sensitivity of the cyclic nucleotide-gated channel of photoreceptor cells, and speculate about the role they might play in the recovery of the light response. We discuss results from in vivo experiments that argue against the participation of Ca-calmodulin in photorecovery.

Strong arguments are presented by Hargrave suggesting that the crystallization of visual rhodopsin for high resolution analysis by X-ray crystallography or electron microscopy is feasible. However, the effort needed to achieve this goal will most likely exceed the resources of a single laboratory and a concerted approach to the research is necessary.

Light activated rhodopsin functions by catalyzing the exchange of GTP for GDP on numerous copies of transducin. Peptide mapping has shown that at least six regions, three on rhodopsin and three on the transducin alpha subunit, are involved in the active interface between the two proteins. The most informative structural studies of rhodopsin should include focus on the transducin interaction.

Recent findings emphasize the complexity, both genetic and functional, of the manifold genes and mutations causing inherited retinal degeneration in humans. Knowledge of the genetic bases of these diseases can contribute to design of rational therapy, as well as elucidating the function of each gene product in normal visual processes.

Rapidly advancing studies on rhodopsin have focused on new strategies for crystallization of this integral membrane protein for x-ray analysis and on alternative methods for structural determination from nuclear magnetic resonance data. Functional studies of the interactions between the apoprotein and its chromophore have clarified the role of the chromophore in deactivation of opsin and in photoactivation of the pigment.

A photoaffinity analog of cGMP has been used to biochemically identify a new ligand-binding subunit of the retinal rod cGMP-activated ion channel, as well as amino acids in contact with cGMP in the original subunit. Covalent tethering of this probe to channels in excised menbrane patches has revealed a functional heteogeneity in the ligand-binding sites that may arise from the two biochemically identified subunits.

The generally positive response to our target article indicates that most of the commentators accept our contention that light adaptation consists of multiple and possibly redundant mechanisms. The commentaries fall into three general categories. The first deals with putative mechanisms that we chose not to emphasize. The second is a more extended discussion of the role of calcium in adaptation. Finally, additional aspects of cGMP involvement in adaptation are considered. We discuss each of these points in turn.

Large scale DNA-mutation screening in patients with hereditary retinal diseases greatly enhances our knowledge about retinal function and diseases. Scientists, clinicians, patients, and families involved with retinal disorders may directly benefit from these developments. However, certain aspects of this expanding knowledge, such as the correlation between genotype and phenotype, may be much more complicated than we expect at present.

The candidate gene approach has identified many causes of photoreceptor rod cell death in retinitis pigmentosa. Some mutations lead to increased cyclicGMP concentrations in rods. Rod photoreceptors are also particularly susceptible to some mutations in housekeeping genes. Although many more cases of macular degeneration than retinitis pigmentosa occur each year, there is much less known about both genetic and sporadic forms of this disease.

The structure of rhodopsin is a subject of intense interest. Solving the structure by traditional methods has proved exceedingly challenging. It may therefore be useful to confront the problem by a combination of alternate techniques. These include FTIR (Fourier transform infrared spectroscopy) and AFM (atomic force microscopy) on the intact protein. Furthermore, additional insights may be gained through structural investigations of discrete rhodopsin domains.

Evidence suggests that the Ca2+/calmodulin-sensitive adenylyl cyclase may play a key role in neural plasticity and learning in Aplysia, Drosophila, and mammals. This dually-regulated enzyme has been proposed as a possible site of stimulus convergence during associative learning. This commentary discusses the evidence that is required to demonstrate that a protein in a second messenger cascade actually functions as a molecular site of associative integration. It also addresses the issue of how a dually-regulated protein could contribute to the temporal pairing (...) requirements of classical conditioning: that relationship between stimuli display both temporal contiguity and predictability. (shrink)