Focus on Molecules: Rod photoreceptor cGMP-gated cation channel (original) (raw)
Related papers
Journal of Biological Chemistry, 1996
The full-length cDNA for the -subunit of the human rod photoreceptor cyclic nucleotide-gated channel has been shown to encode a 1251-amino acid (ϳ140 kDa) polypeptide which, like its bovine counterpart, has an unusual bipartite structure. The C-terminal part corresponds to the previously reported "subunit 2" of the human rod channel and contains the structural features of other cyclic nucleotide-gated channel subunits including six putative membrane spanning segments, a cyclic nucleotide binding domain, a voltage-sensor motif, and a pore region. The N-terminal part contains the human homolog of the bovine glutamic acid-rich protein called GARP. Western blots indicate that both the native and heterologously expressed human -subunit migrate anomalously as a 220-kDa polypeptide by SDS-gel electrophoresis. Two other GARP variants, full-length GARP (f-GARP) and truncated GARP (t-GARP), are also present in human, bovine, and rat rod outer segments and migrate as 120-140-and 55-62-kDa polypeptides, respectively. The bovine f-GARP and t-GARP cDNAs code for proteins containing 590 amino acids and 299 amino acids, respectively. The first 571 amino acids of f-GARP and the first 291 amino acids of t-GARP are identical to the corresponding N-terminal amino acid sequence of the bovine -subunit. The two GARP variants, themselves, are not tightly associated with the rod channel. These results indicate that mammalian rod outer segments contain three alternatively spliced variants of GARP, one of which constitutes the N-terminal part of the rod channel -subunit.
FEBS Letters, 1996
Human and bovine rod photoreceptor cGMP-gated cation channel consists of two subunits: a (63 kDa) and p (240 kDa). The human p subunit was shown to consist partly of sequence encoded by the cDNA clone hRCNC2b. Here we present the complete sequence of the human b subunit and demonstrate that the previously reported human CAR1 gene encoding a glutamate-rich protein (hGARP) encodes its Ntenniual portion. Using PCR, RNA blot and genomic DNA analysis, we provide evidence that the l3 subunit is produced from a complex locus on chromosome 16 which is also capable of generating independent transcripts corresponding to GARl and the C-terminal two-thirds of the fl subunit. The results indicate that the p subunit of the cGMP-gated cation channel is produced from an unusual locus consisting of more than one transcription unit.
Neuron, 1995
The cyclic nucleotide-gated channel from rod photoreceptors is composed of two distinct subunits (~ and I~)-The properties of the ~ subunit, which can form functional channels by itself, are modified by coexpression with a homologous polypeptide, designated the I~ subunit. However, the a subunit from rod photoreceptor membranes copurifies with a 240 kDa protein that is significantly larger than this putative ~ subunit. We now demonstrate by peptide sequencing and by cloning and functional expression of cDNA that the 240 kDa protein represents the complete p subunit with an unusual bipartite structure. The N-terminal part is essentially identical to a glutamic acid-rich protein (GARP), whereas the C-terminal part is highly homologous to the previously cloned human "p subunit." Expression of the complete p subunit in HEK 293 cells results in a polypeptide with the same apparent molecular weight as the 240 kDa protein of the native rod channel. Coexpression of the ~ subunit with the fulllength p subunit yields hetero-oligomeric channels with properties characteristic of the native channel.
Molecular properties of the cGMP-gated channel of rod photoreceptors
Vision Research, 1998
The cGMP-gated channel of the rod photoreceptor cell plays a key role in phototransduction by controlling the flow of Na + and Ca 2 + into the outer segment in response to light-induced changes in cGMP concentrations. The rod channel is composed of two homologous subunits designated as h and i. Each subunit contains a core region of six putative membrane spanning segments, a cGMP binding domain, a voltage sensor-like motif and a pore region. In addition the i-subunit contains an extended N-terminal region that is identical in sequence to a previously cloned retinal glutamic acid rich protein called GARP. Three spliced variants of GARP (the GARP part of the i channel subunit; full length free GARP; and a truncated form of GARP) are expressed in rod cells and localized within the outer segments. Immunoaffinity chromatography has been used to purify the channel from detergent solubilized rod outer segments. A significant fraction of the rod Na + /Ca 2 +-K + exchanger copurifies with the channel as measured by western blotting suggesting that the channel can interact with the exchanger under certain conditions.
Journal of Biological Chemistry
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 640-residue C-terminal portion that is well conserved in three mammalian species. Significant sequence similarity (69%) of the visual cGMPgated 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 6.2 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 6.
Genomic organization of the human rod photoreceptor cGMP-gated cation channel β-subunit gene
We previously reported that the CNGB1 locus encoding the rod photoreceptor cGMP-gated channel b-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 100 kb of genomic DNA on chromosome 16. The b-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 79 bp 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 2.5 kb 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.
Biochemistry, 2003
Proper control of intracellular free Ca 2+ is thought to involve subsets of proteins that colocalize to mediate coordinated Ca 2+ entry and Ca 2+ extrusion. The outer segments of vertebrate rod and cone photoreceptors present one example: Ca 2+ influx is exclusively mediated via cGMP-gated channels (CNG), whereas the Na + /Ca 2+-K + exchanger (NCKX) is the only Ca 2+ extrusion protein present. In situ, a rod NCKX homodimer and a CNG heterotetramer are thought to be part of a single protein complex. However, NCKX-NCKX and NCKX-CNG interactions have been described so far only in bovine rod outer segment membranes. We have used thiol-specific cross-linking and co-immunoprecipitation to examine NCKX self-assembly and CNG-NCKX co-assembly after heterologous expression of either the rod or cone NCKX/CNG isoforms. Co-immunoprecipitation clearly demonstrated both NCKX homooligomerization and interactions between NCKX and CNG. The NCKX-NCKX and NCKX-CNG interactions were observed for both the rod and the cone isoforms. Thiol-specific cross-linking led to rod NCKX1 dimers and to cone NCKX2 adducts of an apparent molecular weight higher than that predicted for a NCKX2 dimer. The mass of the cross-link product critically depended on the location of the particular cysteine residue used by the cross-linker, and we cannot exclude that NCKX forms a higher oligomer. The NCKX-NCKX and NCKX-CNG interactions were not isoform-specific (i.e., rod NCKX could interact with cone NCKX, rod NCKX could interact with cone CNGA, and vice versa). Deletion of the two large hydrophilic loops from the NCKX protein did not abolish the NCKX oligomerization, suggesting that it is mediated by the highly conserved transmembrane spanning segments. Na + /Ca 2+ exchange plays a crucial role in Ca 2+ homeostasis of most cells (recently reviewed in ref 1). Vertebrate rod photoreceptor cells express a distinct K +-dependent Na + / Ca 2+-K + exchanger that utilizes the K + outward gradient in addition to the Na + inward gradient to transport Ca 2+ out of the cell (NCKX) 1 (2, 3). There is very little homology between the amino acid sequences of the NCKX and the K +-independent Na + /Ca 2+ exchanger (NCX) (4). In the past few years, NCKX cDNAs have been cloned from retinal rod and cone photoreceptors from several vertebrate species (4-8), from nonretinal cells (9-11), and from lower organisms such as Drosophila (12), Caenorhabditis elegans (13), and sea urchin (14). In the outer segments of rod and cone photoreceptors, Ca 2+ enters the cell exclusively through the cGMP-gated channels (CNG) in the plasma membrane. The CNG channels of cone and rod photoreceptors differ, however, significantly in the cyclic nucleotide affinity, Ca 2+ permeability, and Ca 2+ blockage (15, 16). Moreover, Ca 2+ extrusion occurs much faster in cones than in rods (17, 18). The specific properties of both the CNG channel and NCKX and their spatial localization in the cell bring about the characteristic differences between the two types of photoreceptors. In the outer segments of bovine rod photoreceptors, NCKX1 has been reported to occur as a dimer (19), and this dimer has been shown to be part of a larger complex with CNG as well as other proteins (20-22). Here, we have used chemical crosslinking and co-immunoprecipitation to examine oligomerization of rod and cone NCKX expressed in insect cells, with and without coexpression with the respective CNG channel. Recently, it has been shown that the rod CNG channel is a heteromultimer containing three CNGA1 subunits and one CNGB1 subunit (23-25). Furthermore, the CNGA1 subunit interacts with NCKX1 in bovine rod photoreceptors (21). The CNGA, but not the CNGB subunit by itself, forms a functional cGMP-gated channel, whereas the CNGB subunit imparts several additional properties to the CNG channel † This work was supported by a visiting scientist grant (P.J.B.
Interaction of 4.1G and cGMP-gated channels in rod photoreceptor outer segments
2013
In photoreceptors, the assembly of signaling molecules into macromolecular complexes is important for phototransduction and maintaining the structural integrity of rod outer segments (ROSs). However, the molecular composition and formation of these complexes are poorly understood. Using immunoprecipitation and mass spectrometry, 4.1G was identified as a new interacting partner for the cyclic-nucleotide gated (CNG) channels in ROSs. 4.1G is a widely expressed multifunctional protein that plays a role in the assembly and stability of membrane protein complexes. Multiple splice variants of 4.1G were cloned from bovine retina. A smaller splice variant of 4.1G selectively interacted with CNG channels not associated with peripherin-2-CNG channel complex. A combination of truncation studies and domain-binding assays demonstrated that CNG channels selectively interacted with 4.1G through their FERM and CTD domains. Using immunofluorescence, labeling of 4.1G was seen to be punctate and partially colocalized with CNG channels in the ROS. Our studies indicate that 4.1G interacts with a subset of CNG channels in the ROS and implicate this protein-protein interaction in organizing the spatial arrangement of CNG channels in the plasma membrane of outer segments.
Biochemistry, 2003
Proper control of intracellular free Ca 2+ is thought to involve subsets of proteins that colocalize to mediate coordinated Ca 2+ entry and Ca 2+ extrusion. The outer segments of vertebrate rod and cone photoreceptors present one example: Ca 2+ influx is exclusively mediated via cGMP-gated channels (CNG), whereas the Na + /Ca 2+ -K + exchanger (NCKX) is the only Ca 2+ extrusion protein present. In situ, a rod NCKX homodimer and a CNG heterotetramer are thought to be part of a single protein complex. However, NCKX-NCKX and NCKX-CNG interactions have been described so far only in bovine rod outer segment membranes. We have used thiol-specific cross-linking and co-immunoprecipitation to examine NCKX self-assembly and CNG-NCKX co-assembly after heterologous expression of either the rod or cone NCKX/CNG isoforms. Co-immunoprecipitation clearly demonstrated both NCKX homooligomerization and interactions between NCKX and CNG. The NCKX-NCKX and NCKX-CNG interactions were observed for both the rod and the cone isoforms. Thiol-specific cross-linking led to rod NCKX1 dimers and to cone NCKX2 adducts of an apparent molecular weight higher than that predicted for a NCKX2 dimer. The mass of the cross-link product critically depended on the location of the particular cysteine residue used by the cross-linker, and we cannot exclude that NCKX forms a higher oligomer. The NCKX-NCKX and NCKX-CNG interactions were not isoform-specific (i.e., rod NCKX could interact with cone NCKX, rod NCKX could interact with cone CNGA, and vice versa). Deletion of the two large hydrophilic loops from the NCKX protein did not abolish the NCKX oligomerization, suggesting that it is mediated by the highly conserved transmembrane spanning segments. Na + /Ca 2+ exchange plays a crucial role in Ca 2+ homeostasis of most cells (recently reviewed in ref 1). Vertebrate rod photoreceptor cells express a distinct K + -dependent Na + / Ca 2+ -K + exchanger that utilizes the K + outward gradient in addition to the Na + inward gradient to transport Ca 2+ out of the cell (NCKX) 1 (2, 3). There is very little homology between the amino acid sequences of the NCKX and the K + -independent Na + /Ca 2+ exchanger (NCX) (4). In the past few years, NCKX cDNAs have been cloned from retinal rod and cone photoreceptors from several vertebrate species (4-8), from nonretinal cells (9-11), and from lower organisms such as Drosophila , Caenorhabditis elegans (13), and sea urchin (14).