Rod Photoreceptor cGMP-gated Cation Channel Research Articles

Overexpression of rod photoreceptor glutamic acid rich protein 2 (GARP2) increases gain and slows recovery in mouse retina

BackgroundThe rod photoreceptor cGMP-gated cation channel, consisting of three α- and one β subunit, controls ion flow into the rod outer segment (ROS). In addition to the β-subunit, the Cngb1 locus encodes an abundant soluble protein, GARP2 that binds stoichiometrically to rod photoreceptor cGMP phosphodiesterase type 6 (PDE6). To examine the in vivo functional role of GARP2 we generated opsin promoter-driven transgenic mice overexpressing GARP2 three-fold specifically in rod photoreceptors.ResultsIn the GARP2 overexpressing transgenic mice (tg), the endogenous channel β-subunit, cGMP phosphodiesterase α-subunit, peripherin2/RDS and guanylate cyclase I were present at WT levels and were properly localized within the ROS. While localized properly within ROS, two proteins cGMP phosphodiesterase α-subunit (1.4-fold) and cGMP-gated cation channel α-subunit (1.2-fold) were moderately, but significantly elevated. Normal stratification of all retinal layers was observed, and ROS were stable in numbers but were 19% shorter than WT. Analysis of the photoresponse using electroretinography (ERG) showed that tg mice exhibit no change in sensitivity indicating overall normal rod function, however two parameters of the photoresponse significantly differed from WT responses. Fitting of the rising phase of the ERG a-wave to an accepted model of phototransduction showed a two-fold increase in phototransduction gain in the tg mice. The increase in gain was confirmed in isolated retinal tissue and by suction electrode recordings of individual rod photoreceptor cells. A measure of response recovery, the dominant time constant (τD) was elevated 69% in isolated retina compared to WT, indicating slower shutoff of the photoresponse.ConclusionsGARP2 may participate in regulating visual signal transduction through a previously unappreciated role in regulating phototransduction gain and recovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0067-5) contains supplementary material, which is available to authorized users.

Three-Dimensional Architecture of the Rod Sensory Cilium and Its Disruption in Retinal Neurodegeneration

Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Age-related changes in Cngb1-X1 knockout mice: prolonged cone survival

The rod photoreceptor cGMP-gated cation channel has an essential role in phototransduction functioning as the primary point for calcium and sodium entry into the rod outer segment. The channel consists of two subunits, α and β. The α-subunit can function in isolation as an ion channel, and the β-subunit modulates channel activity and has a structural role. We previously reported that a mouse knockout (KO) of the β-subunit and related glutamic acid-rich proteins (GARPs) attenuates rod function and causes structural alterations and slowly progressive retinal degeneration. Here, we have extended our functional analyses of the KO mice evaluating rod and cone function using the electroretinogram in mice up to 4 months of age. Retinal stratification is preserved in the knockout mice at 3 months, and a significant number of cones remain up to 7 months based on PNA staining of cone sheaths. Electroretinography of KO mice at 1 month old revealed a diminished dark-adapted b-wave and normal light-adapted b-wave compared to wild-type mice. Over the next 3 months, both dark- and light-adapted b-wave amplitudes declined, but the reduction was greater for dark-adapted b-wave amplitudes. In one-month-old mice, the critical flicker frequency (CFF) was substantially lower for the KO mice at scotopic intensities, but normal at photopic intensities. CFF values remained stable in the KO mice as the b-wave amplitudes decreased with age. Declining b-wave amplitudes confirm an RP phenotype of rod followed by cone degeneration. Flicker responses show that the cone circuits function normally at threshold despite significant losses in the maximum light-adapted b-wave amplitude. These results confirm that rods are marginally functional in the absence of the β-subunit and in addition show that CFF may be a more sensitive measure of remaining functional cone vision in animal models of RP undergoing progressive rod-cone degeneration.

Focus on Molecules: Rod photoreceptor cGMP-gated cation channel

The rod photoreceptor cGMP-gated cation (rod CNG) channel is an essential component of phototransduction and also may play a critical role in maintenance of photoreceptor outer segment structural integrity. It is a non-selective cation channel that belongs to the superfamily of voltage-gated channels, and the subfamily of cyclic nucleotide gated channels (Kaupp and Seifert, 2002). This channel is composed of a heterotetramer consisting of three α-subunits (encoded by the CNGA1 locus; also called A subunit) and one β-subunit (encoded by the CNGB1 locus; also called the B subunit; Genbank IDs for Murine and Human mRNA: α-subunit 34328042, 71143140; β-subunit 28386104, 4502918; uniprot IDs for Murine and Human proteins are: α-subunit P29974, P29973; β-subunit Q9UMG2, Q80XL8) arranged around a central pore. The αand β-subunits are the products of separate genes whose loci are designated CNGA1 and CNGB1. The CNGB1 locus is complex encoding multiple alternatively spliced transcripts that are expressed in several diverse tissues (Ardell et al., 2000). For example, an olfactory tissue specific transcript encodes a βsubunit variant that is required for normal channel function of the olfactory cyclic nucleotide gated channel.

Genomic organization of the human rod photoreceptor cGMP-gated cation channel beta-subunit gene.

We previously reported that the CNGB1 locus encoding the rod photoreceptor cGMP-gated channel beta-subunit is complex, comprising non-overlapping transcription units that give rise to at least six transcripts (Ardell, M.D., Aragon, I., Oliveira, L., Porche, G.E., Burke, E., Pittler, S.J., 1996. The beta subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit. FEBS Lett. 389, 213-218). To further understand the transcriptional regulation of this extraordinarily complex locus, and to develop a screen for defects in the gene in patients with hereditary disease, we determined its genomic organization and DNA sequence. The CNGB1 locus consists of 33 exons, which span approximately 100kb of genomic DNA on chromosome 16. The beta-subunit comprises two domains, an N-terminal glutamic acid-rich segment (GARP), and a C-terminal channel-like portion. Two additional exons encoding a short GARP transcript and a truncated channel-like transcript have been identified. A major transcription start point was identified 79bp upstream of the initiator ATG. To begin analysis of the basis for the generation of multiple transcripts, and to identify promoters driving expression in retina, approximately 2.5kb of the upstream region were sequenced. Putative cis-elements, which can bind the retina-specific transcription factors Crx and Erx, were found immediately upstream of the transcription start point, and may be important for gene expression in this tissue. From our analysis, a model is reported to account for at least four of the retinal transcripts.

Developmental expression of the rat rod photoreceptor cGMP-gated cation channel.

The appearance of cGMP-gated cation channel protein in the postnatal rat retina has been studied by fluorescence immunocytochemistry of radial retinal sections and immunoblots of retinal membrane proteins. Channel immunoreactivity was first detectable with RCNGC1-7H2 monoclonal antibody at postnatal day 7 (PN7) by both methods. Immunocytochemical label in retinal sections was localized to the outer segments, and immunoreactivity increased with increasing age. We also compared the developmental appearance of the cGMP-gated cation channel to that of other phototransduction proteins and developmental markers. RET-P2, a monoclonal antibody recognizing the 39-kDa rds/peripherin disc protein, first labeled outer segments at PN7, coincident with cGMP-gated cation channel expression. Double labeling of the same section of PN7 rat retina with RET-P2 and R309 (a polyclonal antiserum against the rod cGMP-gated cation channel) revealed identical patterns of labelling. Similarly, double labeling with RCNGC1-7H2 and an antibody against the rod cGMP-phosphodiesterase gave coincident labeling, suggesting coordinate expression mechanisms of phototransduction proteins with each other and with outer segment structural proteins.

Substituted cGMP analogs can act as selective agonists of the rod photoreceptor cGMP-gated cation channel.

Cyclic nucleotide-gated (CNG) channels are expressed in many cell types in both the nervous system and nonexcitable tissues. In order to understand the roles of cGMP-gated channels, and to distinguish actions of cGMP mediated through CNG channels from those through cGMP-dependent protein kinase (G-kinase), several new cGMP analogs were tested for potency as CNG channel agonists. Using Xenopus oocytes expressing the rat rod cGMP-gated ion channel alpha-subunit, we showed that an analog containing a pCPT group at the 8-position, 8-pCPT-cGMP, was 80 times more potent than cGMP and 14 times more potent than 8-Br-cGMP. 8-pCPT-cGMP is the most potent CNG channel agonist so far described and also has the advantages of much better membrane permeability as well as much higher resistance to PDE-hydrolysis, as compared with 8-Br-cGMP. Modification of both 8-Br-cGMP and 8-pCPT-cGMP by introduction of a sulphur atom into the cyclic phosphate group gave smaller changes in agonist efficiency. Both Sp-8-Br-cGMPS and Sp-8-pCPT-cGMPS acted as agonists of CNG channels and are also G-kinase activators. In contrast, Rp-8-Br-cGMPS was a channel agonist, with an EC50 of 173.5 microM, but a G-kinase antagonist with a Ki of 4 microM. Finally, Rp-8-pCPT-cGMPS was a channel agonist and showed additional noncompetitive antagonist activity at higher concentrations. The results suggest that 8-pCPT-cGMPS is a highly potent photoreceptor CNG channel agonist with high membrane permeability and PDE-resistance and furthermore Rp-8-Br-cGMPS can be used to test whether the actions of cGMP are selectively mediated by CNG channels.

Characterization of canine rod photoreceptor cGMP-gated cation channel α-subunit gene and exclusion of its involvement in the hereditary retinal dystrophy of Swedish Briards

The nucleotide sequence of the canine rod photoreceptor cGMP-gated cation channel α-subunit ( cCNCG1) cDNA has been determined. The open reading frame consists of 2073 nucleotides, which encode a putative protein of 691 amino acids. In addition, we have established the exon/intron boundaries of the cCNCG1 gene and determined the complete sequence of six introns of a total of eight. The exon/intron organization (location and length of exons and introns) of the cCNCG1 gene is very similar to that of the human rod photoreceptor cGMP-gated cation channel α-subunit gene. We used single-strand conformation polymorphism analysis to search for potential pathogenic sequence changes in the cCNCG1 gene in a Swedish Briard and Briard–Beagle dog kindred, in which an autosomal recessive retinal dystrophy is segregating, a disease which shows phenotypic similarities to retinitis pigmentosa, a heterogeneous group of hereditary and progressive retinal degeneration in human. In intron 3, we found several DNA polymorphisms, which do not cosegregate with the affected status of the dogs, thus excluding cCNCG1 as a candidate gene for the retinal dystrophy in this strain of Swedish Briards.

The β subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit

Human and bovine rod photoreceptor cGMP-gated cation channel consists of two subunits: α (63 kDa) and β (240 kDa). The human β subunit was shown to consist partly of sequence encoded by the cDNA clone hRCNC2b. Here we present the complete sequence of the human β subunit and demonstrate that the previously reported human GAR1 gene encoding a glutamate-rich protein (hGARP) encodes its N-terminal portion. Using PCR, RNA blot and genomic DNA analysis, we provide evidence that the β subunit is produced from a complex locus on chromosome 16 which is also capable of generating independent transcripts corresponding to GAR1 and the C-terminal two-thirds of the β subunit. The results indicate that the β subunit of the cGMP-gated cation channel is produced from an unusual locus consisting of more than one transcription unit.

Identification of competitive antagonists of the rod photoreceptor cGMP-gated cation channel: beta-phenyl-1,N2-etheno-substituted cGMP analogues as probes of the cGMP-binding site.

cGMP is the natural activator of the cyclic nucleotide-gated channel originally isolated from rod photoreceptors but now known to be expressed in a wide variety of neural and non-neural cells. To identify antagonists of cGMP action and to better understand the interaction between cGMP and the channel protein, experimental studies were undertaken using four synthetic cGMP analogues, PET-cGMP, 8-Br-PET-cGMP, Rp-8-Br-PET-cGMPS, and Sp-8-Br-PET-cGMPS. With excised patches from either Xenopus oocytes expressing a cloned rat rod channel alpha-subunit or from native Xenopus rod photoreceptors, Rp-8-Br-PET-cGMPS competitively suppressed the cGMP-induced current with an IC50 of 25 microM and Sp-8-Br-PET-cGMPS inhibited this current with an IC50 of 105 microM. On the expressed rat rod channel, 8-Br-PET-cGMP behaved as a very weak partial agonist at high concentrations and an antagonist (IC50 = 64 microM) at lower concentrations when coapplied with cGMP. PET-cGMP did not activate channel currents alone but showed a synergism when coapplied with subsaturating concentrations of cGMP. Because Sp-8-Br-PET-cGMPS is a potent activator of type I cGMP-dependent protein kinase, but a competitive antagonist of channel activation, it will be a useful reagent for discriminating between those effects of cGMP that are mediated by a protein kinase and those mediated by channel activation. Because the PET derivatives all contain a phenyl-substituted 5-membered ring system fused to the amino group in position 2 and the nitrogen in position 1 of the guanine ring, the results support the idea that N1 and N2 are important for channel activation. They also suggest a minor role for the cyclic phosphate group in binding or activation.

Isolation and characterization of the alpha-subunit of the rat rod photoreceptor cGMP-gated cation channel.

A combination of genomic and PCR clones has been used to derive the full-length coding sequence of the alpha-subunit of the rat rod photoreceptor cGMP-gated cation channel. The sequence encodes a protein of 683 amino acids with a predicted molecular weight of 79,221. The sequence shows extensive homology with other rod cGMP-gated channels and also with the rat olfactory cyclic nucleotide-gated cation channel. When the full-length sequence of the rat rod channel was expressed in Xenopus oocytes it gave a conductance that responded to cGMP with an EC(50) of 42 mu M. No response to 2 mM cAMP was detected. The conductance was decreased in the presence of increasing concentrations of calcium. Both monoclonal and polyclonal antibodies were generated against a C-terminal peptide of the rat rod channel. On immunoblots of adult rat retinal membranes the antibodies recognized a band of 71 kDa, suggesting that the rat channel may undergo proteolytic cleavage in the retina, as has previously been found for the bovine rod channel. Immunocytochemical labeling of adult rat retinal sections detected prominent labeling over the rod photoreceptor outer segments with both monoclonal and polyclonal antibodies.

Primary structure and chromosomal localization of human and mouse rod photoreceptor cGMP-gated cation channel.

We have determined the primary structures of the human and mouse retinal rod cGMP-gated cation channel by analysis of cDNA clones and amplified DNA. The open reading frames predicted polypeptides of 690 and 683 residues exhibiting 88% sequence similarity. Sequence comparison indicated that the rod channels consist of a variable 90-residue N-terminal region, a short highly charged segment rich in lysine and glutamate, and a 540-residue C-terminal portion that is well conserved in three mammalian species. Significant sequence similarity (59%) of the visual cGMP-gated channel to the olfactory cAMP-gated channel established the existence of a family of cyclic nucleotide-gated ion channel genes. RNA blot analysis revealed transcripts of 3.2 kilobases (kb) in human, mouse, and dog, 3.2, 4.6, and 5.2 kb in bovine, and 3.6 kb in fish. The human channel gene was mapped by polymerase chain reaction of somatic cell hybrid DNAs to chromosome 4 (p14-q13) near the centromere. The mouse channel gene locus (Cncg) was mapped by interspecific backcross haplotype analysis 0.9 centimorgan proximal of the Kit locus on chromosome 5.