Rod Photoreceptor Outer Segments Research Articles

Generation of a genetically encoded marker of rod photoreceptor outer segment growth and renewal

SummaryVertebrate photoreceptors are specialized light sensing neurons. The photoreceptor outer segment is a highly modified cilium where photons of light are transduced into a chemical and electrical signal. The outer segment has the typical cilary axoneme but, in addition, it has a large number of densely packed, stacked, intramembranous discs. The molecular and cellular mechanisms that contribute to vertebrate photoreceptor outer segment morphogenesis are still largely unknown. Unlike typical cilia, the outer segment is continuously regenerated or renewed throughout the life of the animal through the combined process of distal outer segment shedding and proximal outer segment growth. The process of outer segment renewal was discovered over forty years ago, but we still lack an understanding of how photoreceptors renew their outer segments and few, if any, molecular mechanisms that regulate outer segment growth or shedding have been described. Our lack of progress in understanding how photoreceptors renew their outer segments has been hampered by the difficulty in measuring rates of renewal. We have created a new method that uses heat-shock induction of a fluorescent protein that can be used to rapidly measure outer segment growth rates. We describe this method, the stable transgenic line we created, and the growth rates observed in larval and adult rod photoreceptors using this new method. This new method will allow us to begin to define the genetic and molecular mechanisms that regulate rod outer segment renewal, a crucial aspect of photoreceptor function and, possibly, viability.

The ciliopathy gene cc2d2a controls zebrafish photoreceptor outer segment development through a role in Rab8-dependent vesicle trafficking

Ciliopathies are a genetically and phenotypically heterogeneous group of human developmental disorders whose root cause is the absence or dysfunction of primary cilia. Joubert syndrome is characterized by a distinctive hindbrain malformation variably associated with retinal dystrophy and cystic kidney disease. Mutations in CC2D2A are found in ∼10% of patients with Joubert syndrome. Here we describe the retinal phenotype of cc2d2a mutant zebrafish consisting of disorganized rod and cone photoreceptor outer segments resulting in abnormal visual function as measured by electroretinogram. Our analysis reveals trafficking defects in mutant photoreceptors affecting transmembrane outer segment proteins (opsins) and striking accumulation of vesicles, suggesting a role for Cc2d2a in vesicle trafficking and fusion. This is further supported by mislocalization of Rab8, a key regulator of opsin carrier vesicle trafficking, in cc2d2a mutant photoreceptors and by enhancement of the cc2d2a retinal and kidney phenotypes with partial knockdown of rab8. We demonstrate that Cc2d2a localizes to the connecting cilium in photoreceptors and to the transition zone in other ciliated cell types and that cilia are present in these cells in cc2d2a mutants, arguing against a primary function for Cc2d2a in ciliogenesis. Our data support a model where Cc2d2a, localized at the photoreceptor connecting cilium/transition zone, facilitates protein transport through a role in Rab8-dependent vesicle trafficking and fusion.

Rod Outer Segment Retinol Formation Is Independent of Abca4, Arrestin, Rhodopsin Kinase, and Rhodopsin Palmitylation

The reactive aldehyde all-trans retinal is released in rod photoreceptor outer segments by photoactivated rhodopsin and is eliminated through reduction to all-trans retinol. This study was undertaken to determine whether all-trans retinol formation depends on Abca4, arrestin, rhodopsin kinase, and the palmitylation of rhodopsin, all of which are factors that affect the release and sequestration of all-trans retinal. Experiments were performed in isolated retinas and single living rods derived from 129/sv wild-type mice and Abca4-, arrestin-, and rhodopsin kinase-deficient mice and in genetically modified mice containing unpalmitylated rhodopsin. Formation of all-trans retinol was measured by imaging its fluorescence and by HPLC of retina extracts. The release of all-trans retinal from photoactivated rhodopsin was measured in purified rod outer segment membranes according to the increase in tryptophan fluorescence. All experiments were performed at 37°C. The kinetics of all-trans retinol formation in the different types of genetically modified mice are in reasonable agreement with those in wild-type animals. The kinetics of all-trans retinol formation in 129/sv mice are similar to those in C57BL/6, although the latter are known to regenerate rhodopsin much more slowly. The release of all-trans retinal from rhodopsin in purified membranes is significantly faster than the formation of all-trans retinol in intact cells and is independent of the presence of the palmitate groups. The regeneration of rhodopsin and the recycling of its chromophore are not strongly coupled. Neither the activities of Abca4, rhodopsin kinase, and arrestin, nor the palmitylation of rhodopsin affects the formation of all-trans retinol.

Suppression and Replacement Gene Therapy for Autosomal Dominant Disease in a Murine Model of Dominant Retinitis Pigmentosa

For dominantly inherited disorders development of gene therapies, targeting the primary genetic lesion has been impeded by mutational heterogeneity. An example is rhodopsin-linked autosomal dominant retinitis pigmentosa with over 150 mutations in the rhodopsin gene. Validation of a mutation-independent suppression and replacement gene therapy for this disorder has been undertaken. The therapy provides a means of correcting the genetic defect in a mutation-independent manner thereby circumventing the mutational diversity. Separate adeno-associated virus (AAV) vectors were used to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. Viruses were subretinally coinjected into P347S mice, a model of dominant rhodopsin-linked retinitis pigmentosa. Benefit in retinal function and structure detected by electroretinography (ERG) and histology, respectively, was observed for at least 5 months. Notably, the photoreceptor cell layer, absent in 5-month-old untreated retinas, contained 3–4 layers of nuclei, whereas photoreceptor ultrastructure, assessed by transmission electron microscopy (TEM) improved significantly. The study provides compelling evidence that codelivered suppression and replacement is beneficial, representing a significant step toward the clinic. Additionally, dual-vector delivery of combined therapeutics represents an exciting approach, which is potentially applicable to other inherited disorders.

A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa

The rd1 natural mutant is one of the first and probably the most commonly studied mouse model for retinitis pigmentosa (RP), a severe and frequently blinding human retinal degeneration. In several decades of research, the link between the increase in photoreceptor cGMP levels and the extremely rapid cell death gave rise to a number of hypotheses. Here, we provide clear evidence that the presence of cyclic nucleotide gated (CNG) channels in the outer segment membrane is the key to rod photoreceptor loss. In Cngb1(-/-) × rd1 double mutants devoid of regular CNG channels, cGMP levels are still pathologically high, but rod photoreceptor viability and outer segment morphology are greatly improved. Importantly, cone photoreceptors, the basis for high-resolution daylight and colour vision, survived and remained functional for extended periods of time. These findings strongly support the hypothesis of deleterious calcium (Ca(2+))-influx as the cause of rapid rod cell death and highlight the importance of CNG channels in this process. Furthermore, our findings suggest that targeting rod CNG channels, rather than general Ca(2+)-channel blockade, is a most promising symptomatic approach to treat otherwise incurable forms of cGMP-related RP.

Rapid formation of all-trans retinol after bleaching in frog and mouse rod photoreceptor outer segments

All-trans retinol is formed in the outer segments of vertebrate rod photoreceptors from the reduction of the all-trans retinal released by photoactivated rhodopsin. The reduction requires NADPH and is therefore dependent on metabolic input. In metabolically intact photoreceptors, a large increase in rod outer segment fluorescence, attributed to the fluorescence of all-trans retinol, follows rhodopsin photoactivation. The fluorescence increase is biphasic, including a rapid and a slow component. In metabolically compromised cells, there is a much smaller fluorescence increase following rhodopsin photoactivation, but it too contains a rapid component. We have measured the fluorescence signal in single living frog and mouse rod photoreceptors, and have characterized its dependence on the wavelengths of light selected for excitation and for collecting emission. We find that in metabolically intact cells, the excitation and emission properties of both the rapid and slow components of the fluorescence signal are in close agreement with those of all-trans retinol fluorescence. In metabolically compromised cells, however, the signal can only partially be due to all-trans retinol, and most of it is consistent with all-trans retinal. The results suggest that in the outer segments of living rod photoreceptors there is rapid release of all-trans retinal, which in metabolically intact cells is accompanied by rapid conversion to all-trans retinol.

2-Hydroxypropyl-β-Cyclodextrin Removes All-TransRetinol from Frog Rod Photoreceptors in a Concentration-Dependent Manner

To determine whether a nonprotein lipophilic carrier, 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), can remove all-trans retinol from rod photoreceptor outer segments. All-trans retinol is generated in rod outer segments after light exposure. It is highly insoluble, and its efficient transport across extra- and intracellular aqueous space requires specialized carriers. Experiments were carried out with isolated frog rod photoreceptor cells. The removal of all-trans retinol by different concentrations of this carrier was measured by imaging its fluorescence in single-rod photoreceptors. HP-beta-CD concentrations >0.3 mM significantly increased the rate of all-trans retinol removal. The rate of removal increased linearly with carrier concentration, with a slope of 0.0058 min(-1)/mM. The effectiveness of HP-beta-CD shows that a specialized interaction with the cell membrane is not necessary for the efficient transfer of all-trans retinol between the cell membrane and the carrier. The transfer occurs through a collision-based mechanism, as indicated by the linear increase of the rate of removal with the carrier concentration.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 1: Identification of its inhibitory subunit complexes and their roles

Cyclic GMP phosphodiesterase (PDE) in bovine rod photoreceptor outer segments (OS) comprises a catalytic subunit complex (Palphabeta) and two inhibitory subunits (Pgamma) and is regulated by the alpha subunit of transducin (Talpha). Here, we show an overall mechanism for PDE regulation by identifying Pgamma complexes in OS homogenates prepared with an isotonic buffer. Before Talpha activation, three Pgamma complexes exist in the soluble fraction. Complex a, a minor complex, contains Palphabeta, Talpha, and a protein named Pdelta. Complex b, Palphabetagammagamma( b ), has a PDE activity similar to that of membranous Palphabetagammagamma, Palphabetagammagamma( M ), and its level, although its large portion is Pdelta-free, is estimated to be 20-30% of the total Palphabetagammagamma. Complex c, (Pgamma.GDP-Talpha) (2) ( c ) , appears to be a dimer of Pgamma.GDP-Talpha. Upon Talpha activation, (1) complex a stays unchanged, (2) Palphabetagammagamma( b ) binds to membranes, (3) the level of (Pgamma.GDP-Talpha) (2) ( c ) is reduced as its GTP-form is produced, (4) complex d, Pgamma.GTP-Talpha( d ), is formed on membranes and its substantial amount is released to the soluble fraction, and (5) membranous Palphabetagammagamma, Palphabetagammagamma( M ) and/or Palphabetagammagamma( b ), becomes Pgamma-depleted. These observations indicate that Pgamma as a complex with GTP-Talpha dissociates from Palphabetagammagamma on membranes and is released to the soluble fraction and that Pgamma-depleted PDE is the GTP-Talpha-activated PDE. After GTP hydrolysis, both (Pgamma.GDP-Talpha) (2) ( c ) and Pgamma.GDP-Talpha( d ), without liberating Pgamma, deactivate Pgamma-depleted PDE. The preferential order to be used for the deactivation is membranous Pgamma.GDP-Talpha( d ), solubilized Pgamma.GDP-Talpha( d ) and (Pgamma.GDP-Talpha) (2) ( c ) . Release of Pgamma.GTP-Talpha complexes to the soluble fraction is relevant to light adaptation.

The Retinitis Pigmentosa Mutation c.3444+1G>A in CNGB1 Results in Skipping of Exon 32

Retinitis pigmentosa (RP) is a severe hereditary eye disorder characterized by progressive degeneration of photoreceptors and subsequent loss of vision. Two of the RP associated mutations were found in the CNGB1 gene that encodes the B subunit of the rod cyclic nucleotide-gated channel (CNGB1a). One of them (c.3444+1G>A) is located at the donor site of exon 32 and has been proposed to result in a frameshift and truncation of the last 28 aa of the corresponding protein. However, this ambiguous conclusion was not verified by experimental data. Recently, another study reported that the last 28 aa of CNGB1a harbor a motif required for the proper targeting of this subunit to rod photoreceptor outer segments. This suggests that defective targeting is the major cause for the RP phenotype in affected patients. Here, we investigated the splicing of c.3444+1G>A by exon trapping experiments and could demonstrate that instead of the proposed truncation of the last 28 aa this mutation leads to replacement of the last 170 aa of CNGB1a by 68 unrelated amino acids. The 170 aa deletion covers the complete distal C-terminus including the last 10 aa of an important alpha (αC) helix within the ligand-binding domain of CNGB1a. When expressed in a heterologous expression system the corresponding mutant full-length CNGB1a subunit was more susceptible to proteosomal degradation compared to the wild-type counterpart. In conclusion, our experimental data do not support the hypothesis proposed by the original study on the c.3444+1G>A mutation. Based on this, we suggest that apart from the defective targeting other mechanisms may be responsible for the RP phenotype in affected individuals.

Proteomic Changes in the Photoreceptor Outer Segment upon Intense Light Exposure

Acute light-induced photoreceptor degeneration has been studied in experimental animals as a model for photoreceptor cell loss in human retinal degenerative diseases. Light absorption by rhodopsin in rod photoreceptor outer segments (OS) induces oxidative stress and initiates apoptotic cell death. However, the molecular events that induce oxidative stress and initiate the apoptotic cascade remain poorly understood. To better understand the molecular mechanisms of light-induced photoreceptor cell death, we studied the proteomic changes in OS upon intense light exposure by using a proteolytic (18)O labeling method. Of 171 proteins identified, the relative abundance of 98 proteins in light-exposed and unexposed OS was determined. The quantities of 11 proteins were found to differ by more than 2-fold between light-exposed OS and those remaining in darkness. Among the 11 proteins, 8 were phototransduction proteins and 7 of these were altered such that the efficiency of phototransduction would be reduced or quenched during light exposure. In contrast, the amount of OS rhodopsin kinase was reduced by 2-fold after light exposure, suggesting attenuation in the mechanism of quenching phototransduction. Liquid chromatography multiple reaction monitoring (LC-MRM) was performed to confirm this reduction in the quantity of rhodopsin kinase. As revealed by immunofluorescence microscopy, this reduction of rhodopsin kinase is not a result of protein translocation from the outer to the inner segment. Collectively, our findings suggest that the absolute quantity of rhodopsin kinase in rod photoreceptors is reduced upon light stimulation and that this reduction may be a contributing factor to light-induced photoreceptor cell death. This report provides new insights into the proteomic changes in the OS upon intense light exposure and creates a foundation for understanding the mechanisms of light-induced photoreceptor cell death.

Pore Forming Properties of Cecropin-Melittin Hybrid Peptide in a Natural Membrane

The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH2; CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in ~50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was ≥1 GΩ, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15.

RPGRIP1 is essential for normal rod photoreceptor outer segment elaboration and morphogenesis

The function of the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) gene is currently not known. However, mutations within the gene lead to Leber Congenital Amaurosis and autosomal recessive retinitis pigmentosa in human patients. In a previously described knockout mouse model of the long splice variant of Rpgrip1, herein referred to as Rpgrip1(tm1Tili) mice, mislocalization of key outer segment proteins and dysmorphogenesis of outer segment discs preceded subsequent photoreceptor degeneration. In this report, we describe a new mouse model carrying a splice acceptor site mutation in Rpgrip1, herein referred to as Rpgrip1(nmf247) that is phenotypically distinct from Rpgrip1(tm1Tili) mice. Photoreceptor degeneration in homozygous Rpgrip1(nmf247) mice is earlier in onset and more severe when compared with Rpgrip1(tm1Tili) mice. Also, ultrastructural studies reveal that whereas Rpgrip1(nmf247) mutants have a normal structure and number of connecting cilia, unlike Rpgrip1(tm1Tili) mice, they do not elaborate rod outer segments (OS). Therefore, in addition to its role in OS disc morphogenesis, RPGRIP1 is essential for rod OS formation. Our study indicates the absence of multiple Rpgrip1 isoforms in Rpgrip1(nmf247) mice, suggesting different isoforms may play different roles in photoreceptors and underscores the importance of considering splice variants when generating targeted null mutations.

The novel distribution of phosphodiesterase-4 subtypes within the rat retina

Phosphodiesterases (PDEs) are important regulators of signal transduction processes. While much is known about the function of cyclic GMP-specific PDEs in the retina, much less is known about the closely related, cyclic AMP-specific PDEs. The purpose of the present study is to characterize and localize PDE4 within the adult rat retina. We have used Western blotting, RT-PCR, and immunohistochemistry together with retrograde labeling to determine the presence and location of each PDE4 subtype. Western blot analysis revealed that multiple isoforms of PDE4A, B, and D subtypes are present within the retina, whereas the PDE4C subtype was absent. These data were confirmed by RT-PCR. Using immunohistochemistry we show that all three PDE4s are abundantly expressed within the retina where they all colocalize with retrograde-labeled retinal ganglion cells, as well as bipolar cells, horizontal cells, and cholinergic amacrine cells, whereas Müller cells lack PDE4 expression. Uniquely, PDE4B was expressed by the inner and outer segments of rod photoreceptors as well as their terminals within the outer plexiform layer. Collectively, our results demonstrate that PDE4s are abundantly expressed throughout the rodent retina and this study provides the framework for further functional studies.

The neurovascular retina in retinopathy of prematurity.

The continuing worldwide epidemic of retinopathy of prematurity (ROP), a leading cause of childhood visual impairment, strongly motivates further research into mechanisms of the disease. Although the hallmark of ROP is abnormal retinal vasculature, a growing body of evidence supports a critical role for the neural retina in the ROP disease process. The age of onset of ROP coincides with the rapid developmental increase in rod photoreceptor outer segment length and rhodopsin content of the retina with escalation of energy demands. Using a combination of non-invasive electroretinographic (ERG), psychophysical, and image analysis procedures, the neural retina and its vasculature have been studied in prematurely born human subjects, both with and without ROP, and in rats that model the key vascular and neural parameters found in human ROP subjects. These data are compared to comprehensive numeric summaries of the neural and vascular features in normally developing human and rat retina. In rats, biochemical, anatomical, and molecular biological investigations are paired with the non-invasive assessments. ROP, even if mild, primarily and persistently alters the structure and function of photoreceptors. Post-receptor neurons and retinal vasculature, which are intimately related, are also affected by ROP; conspicuous neurovascular abnormalities disappear, but subtle structural anomalies and functional deficits may persist years after clinical ROP resolves. The data from human subjects and rat models identify photoreceptor and post-receptor targets for interventions that promise improved outcomes for children at risk for ROP.

Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid, controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments

The biogenesis of cilia-derived sensory organelles, the photoreceptor rod outer segments (ROS), is mediated by rhodopsin transport carriers (RTCs). The small GTPase Rab8 regulates ciliary targeting of RTCs, but their specific fusion sites have not been characterized. Here, we report that the Sec6/8 complex, or exocyst, is a candidate effector for Rab8. We also show that the Qa-SNARE syntaxin 3 is present in the rod inner segment (RIS) plasma membrane at the base of the cilium and displays a microtubule-dependent concentration gradient, whereas the Qbc-SNARE SNAP-25 is uniformly distributed in the RIS plasma membrane and the synapse. Treatment with omega-3 docosahexaenoic acid [DHA, 22:6(n-3)] causes increased co-immunoprecipitation and colocalization of SNAP-25 and syntaxin 3 at the base of the cilium, which results in the increased delivery of membrane to the ROS. This is particularly evident in propranolol-treated retinas, in which the DHA-mediated increase in SNARE pairing overcomes the tethering block, including dissociation of Sec8 into the cytosol. Together, our data indicate that the Sec6/8 complex, syntaxin 3 and SNAP-25 regulate rhodopsin delivery, probably by mediating docking and fusion of RTCs. We show further that DHA, an essential polyunsaturated fatty acid of the ROS, increases pairing of syntaxin 3 and SNAP-25 to regulate expansion of the ciliary membrane and ROS biogenesis.

Knockout of GARPs and the β-subunit of the rod cGMP-gated channel disrupts disk morphogenesis and rod outer segment structural integrity

Ion flow into the rod photoreceptor outer segment (ROS) is regulated by a member of the cyclic-nucleotide-gated cation-channel family; this channel consists of two subunit types, alpha and beta. In the rod cells, the Cngb1 locus encodes the channel beta-subunit and two related glutamic-acid-rich proteins (GARPs). Despite intensive research, it is still unclear why the beta-subunit and GARPs are coexpressed and what function these proteins serve. We hypothesized a role for the proteins in the maintenance of ROS structural integrity. To test this hypothesis, we created a Cngb1 5'-knockout photoreceptor null (Cngb1-X1). Morphologically, ROSs were shorter and, in most rods that were examined, some disks were misaligned, misshapen and abnormally elongated at periods when stratification was still apparent and degeneration was limited. Additionally, a marked reduction in the level of channel alpha-subunit, guanylate cyclase I (GC1) and ATP-binding cassette transporter (ABCA4) was observed without affecting levels of other ROS proteins, consistent with a requirement for the beta-subunit in channel assembly or targeting of select proteins to ROS. Remarkably, phototransduction still occurred when only trace levels of homomeric alpha-subunit channels were present, although rod sensitivity and response amplitude were both substantially reduced. Our results demonstrate that the beta-subunit and GARPs are necessary not only to maintain ROS structural integrity but also for normal disk morphogenesis, and that the beta-subunit is required for normal light sensitivity of the rods.

Ankyrin-G promotes cyclic nucleotide-gated channel transport to rod photoreceptor sensory cilia.

Cyclic nucleotide-gated (CNG) channels localize exclusively to the plasma membrane of photosensitive outer segments of rod photoreceptors where they generate the electrical response to light. Here, we report the finding that targeting of CNG channels to the rod outer segment required their interaction with ankyrin-G. Ankyrin-G localized exclusively to rod outer segments, coimmunoprecipitated with the CNG channel, and bound to the C-terminal domain of the channel beta1 subunit. Ankyrin-G depletion in neonatal mouse retinas markedly reduced CNG channel expression. Transgenic expression of CNG channel beta-subunit mutants in Xenopus rods showed that ankyrin-G binding was necessary and sufficient for targeting of the beta1 subunit to outer segments. Thus, ankyrin-G is required for transport of CNG channels to the plasma membrane of rod outer segments.

11-cis Retinol as a Substrate for Cone Dark Adaptation

We have determined the effectiveness of 11-cis retinol as a substrate for visual pigment formation in intact vertebrate cone and rod photoreceptors and measured opsin-mediated transducin activation by 11-cis retinol. Methods were of two types. Firstly, visual pigment absorbance spectra were measured microspectrophotometrically in single cone and rod photoreceptor outer segments before and after bleaching of the native visual pigment and following subsequent treatment with 11-cis retinal and 11-cis retinol. Secondly, we expressed human and salamander cone and rod opsins in COS cells and then tested in a cell free assay the effects of these retinoids on the activation of transducin by opsin. We show that 11-cis retinol promotes pigment formation in bleached red and blue salamander cones but not in bleached salamander red or green rods. Transducin activation experiments show that 11-cis retinol acts as an inverse agonist of red and green cone opsins, but has no effect on the activity of blue cone opsins. In contrast, 11-cis retinol acts as an agonist of rod opsin. We conclude that cones have a mechanism for handling retinoids and regenerating visual pigment that is different from rods. 11-cis Retinal and 11-cis retinol are usable substrates for cone pigment regeneration and dark adaptation as both retinoids promote pigment regeneration and neither elicits activation of the transduction cascade by opsin. On the other hand, 11-cis retinol is not useful for rod function since it does not promote pigment regeneration and its opsin-mediated activation of rod transducin may slow the rate of rod dark adaptation.

NCKX Reaction Cycle: ATP, Voltage And Ion Regulation

Ca2+ concentration in photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce voltage changes exceeding 40 mV: since the Ca2+ extrusion in the OS is entirely controlled by the Na+:Ca2+, K+ exchanger (NCKX), it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors (like Mg-ATP, known to regulate the Na+:Ca2+ exchanger). The NKCX regulation was therefore investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarization increases Ca2+ extrusion rate, the recovery of the dark level of Ca2+ (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of ∼2.3 and ∼2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the NCKX turnover number, although through an unknown mechanism. The ion transport mechanism was further investigated by using voltage and Ca2+ jumps (achieved via photolysis of caged-Ca2+ or fast solution changes) and by studying the NCKX selectivity in different ionic conditions. Ca2+ jumps, but not voltage jumps, produced current transients, possibly originating from electrogenic partial reactions. No monovalent cation substituted for Na+ at the NCKX binding sites, but Rb+ substituted for K+, while Sr2+, Ba2+, Mg2+ substituted for Ca2+ with an apparent permeability ratio of 0.78, 0.20, <0.05:1, respectively.