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1.
2.
Probes against the retinal cGMP-gated cation channel were generated by PCR amplification of cDNA from rat and bovine retina. Southern and Northern analyses showed that the channel is encoded by a single gene that gives rise to a single mRNA species of 3.2 kb. Low levels of cGMP-gated channel RNA were detected in postnatal day 1 (PN 1) retinas and the amount increased to adult levels over the next two weeks of development. Screening of a number of tissues by Northern blot hybridization and by PCR amplification showed the channel to be expressed by heart and kidney as well as retina, but not by cerebellum, cerebral cortex, liver, muscle, olfactory bulb, spleen, testes or thymus.  相似文献   

3.
The cGMP-gated cation channel is a member of a new family of channel proteins that appear to be directly regulated by cyclic nucleotides. A protein with a subunit molecular mass of 78 kDa that exhibits cGMP-gated calcium flux when reconstituted into phospholipid-containing vesicles has been purified using 8-bromo-cGMP-agarose affinity chromatography. This channel activity is sensitive to the inhibitor l-cis-diltiazem. Treatment of the reconstituted channel with trypsin abolishes the l-cis-diltiazem sensitivity. Apparent endogenous proteolysis can also result in smaller molecular weight polypeptides that exhibit cGMP-gated channel activity but are insensitive to l-cis-diltiazem. These results show that the channel can bind cGMP and that it contains a l-cis-diltiazem inhibitory domain that is distinct from the cGMP-binding domain.  相似文献   

4.
The molecular properties and orientation of the cGMP-gated cation channel of bovine rod outer segment membranes were studied using biochemical and immunochemical methods. Western blots labeled with anti-channel monoclonal antibodies indicate that the channel has a subunit Mr of 63,000 in bovine rod outer segment membranes prepared in the presence and absence of protease inhibitors and in rod outer segments from other mammalian retinas. The channel has an apparent Mr of 78,000 in both COS-1 cells and Xenopus oocytes expressing the cloned cDNA. NH2-terminal sequence analysis indicates that the lower Mr of the channel in rod outer segments is caused by the absence of the first 92 amino acids predicted by cDNA sequence analysis. Immunofluorescent and immunogold labeling has confirmed that the 63,000 form of the channel is present in rod outer segments. These results indicate that photoreceptor cell-specific co-translational or post-translational cleavage of the NH2-terminal segment of the channel occurs prior or during the incorporation of the channel into the rod outer segment plasma membrane. Immunogold labeling studies using site-directed antibodies also indicate that the NH2-terminal segment of the rod outer segment channel is exposed on the cytoplasmic side of the plasma membrane.  相似文献   

5.
A 240-kDa protein exhibiting immunochemical cross-reactivity with red blood cell spectrin has been shown to be directly associated with the 63-kDa cGMP-gated channel of bovine rod outer segments. When detergent-solubilized, chromatographically purified channel preparations were treated with Sepharose beads coupled to either an anti-240-kDa monoclonal antibody (PMs 4B2) or an anti-63-kDa channel monoclonal antibody (PMc 1D1), both the 240-kDa protein and the 63-kDa channel protein were concomitantly immunoprecipitated as analyzed by Western blotting of sodium dodecyl sulfate gels. Both of these antibody-Sepharose matrices also removed cGMP-gated channel activity as measured by functional reconstitution. In control studies anti-rhodopsin monoclonal antibody (Rho 1D4)-Sepharose beads removed residual rhodopsin, but not the 63/240-kDa complex or channel activity. Western blotting of purified rod outer segment disk and plasma membrane fractions and immunogold-dextran labeling of lysed rod outer segments indicated that the 240-kDa polypeptide, like the 63-kDa channel, is preferentially localized to the plasma membrane as visualized by electron microscopy. The 240-kDa protein does not appear to be directly involved in the cGMP-gated channel activity, but it may be part of a cytoskeletal system that serves to maintain the organization of the 63-kDa channel complex within the rod outer segment plasma membrane.  相似文献   

6.
Tetraalkylammonium compounds and other organic cations were used to probe the structure of the internal and external mouths of the pore of cGMP-gated cation channels from rod and cone photoreceptors. Both rod and cone channels were blocked by tetramethyl- through tetrapentylammonium from the intracellular side in a voltage-dependent fashion at millimolar to micromolar concentrations. The dissociation constant at 0 mV (KD(O)) decreased monotonically with increasing carbon chain length from approximately 80 mM (TMA) to approximately 80 microM (TPeA), where the dissociation constant in rod channels is approximately 50% that of cone channels. N-Methyl-D-glucamine and the buffer Tris also blocked the cone channel in a voltage-dependent fashion at millimolar concentrations, but with lower affinity than similarly sized tetraalkylammonium blockers. Block by tetrahexylammonium (THxA) was voltage-independent, suggesting that the diameter of the intracellular mouth of these channels is less than the size of THxA but larger than TPeA. The location of the binding site for intracellular blockers was approximately 40% across the voltage-drop from the intracellular side. The addition of one carbon to each of the alkyl side chains increased the binding energy by approximately 4 kJ mol-1, consistent with hydrophobic interactions between the blocker and the pore. Cone, but not rod, channels were blocked by millimolar concentrations of extracellular TMA. The location of the extracellular binding site was approximately 13% of the voltage drop from the extracellular side. In cone channels, the two blocker binding sites flank the location of the cation binding site proposed previously.  相似文献   

7.
Bauer PJ  Krause E 《Biochemistry》2005,44(5):1624-1634
Cyclic nucleotide-gated channels of photoreceptors and olfactory sensory neurons are tetramers consisting of A and B subunits. Here, the accessibility of the cysteines of the bovine rod cyclic nucleotide-gated channel is examined as a function of ligand binding. N-Ethylmaleimide-modified cysteines of both subunits were identified by mass spectrometry after trypsin digestion. In the absence of ligand, the intracellular carboxy-terminal cysteines of both subunits were accessible to N-ethylmaleimide. Activation of the channel abolished the accessibility of Cys505 of the A subunit and Cys1104 of the B subunit, with both being conserved cysteines of the cyclic nucleotide-binding sites. The cysteine of the pore loop of the B subunit was also found to be modified by this reagent in the absence of ligand. The total number of accessible cysteines of each subunit was determined by mass shifting upon modification with polyethylene glycol maleimide. In the absence of cyclic nucleotides, this hydrophilic reagent only weakly labeled cysteines of the A subunit but readily labeled at least three cysteines of the B subunit. Ligand binding exposed two cysteines of the A subunit and one cysteine of the B subunit to chemical modification. Double-modification experiments suggest that some of these cysteines are in or close to membrane-spanning domains. However, these cysteines could not yet be identified. Together, the cysteine accessibility of the native rod cyclic nucleotide-gated channel varies markedly upon ligand binding, thus indicating major structural rearrangements, which are of functional importance for channel activation.  相似文献   

8.
9.
Calmodulin (CaM) controls the activity of the rod cGMP-gated ion channel by decreasing the apparent cGMP affinity. We have examined the mechanism of this modulation using electrophysiological and biochemical techniques. Heteromeric channels, consisting of alpha- and beta-subunits, display a high CaM sensitivity (EC50 </=5 nM) similar to the native channel. Using surface plasmon resonance spectroscopy, we identified two unconventional CaM-binding sites (CaM1 and CaM2), one in each of the N- and the C-terminal regions of the beta-subunit. Ca2+ co-operatively stimulates binding of CaM to these sites exactly within the range of [Ca2+] occurring during a light response. Deletion of the N-terminal CaM1 site results in channels that are no longer CaM-sensitive, whereas deletion of CaM2 has only minor effects. We discuss different models to explain the high-affinity binding of CaM.  相似文献   

10.
The rod cGMP-gated channel is localized in the plasma membrane of rod photoreceptor outer segments, where it plays a central role in phototransduction. It consists of alpha- and beta-subunits that assemble into a heterotetrameric protein. Each subunit contains structural features characteristic of nucleotide-gated channels, including a cGMP-binding domain, multiple membrane-spanning segments, and a pore region. In addition, the beta-subunit has a large glutamic acid- and proline-rich region called GARP that is also expressed as two soluble protein variants. Using monoclonal antibodies in conjunction with immunoprecipitation, cross-linking, and electrophoretic techniques, we show that the cGMP-gated channel associates with the Na/Ca-K exchanger in the rod outer segment plasma membrane. This complex and soluble GARP proteins also interact with peripherin-2 oligomers in the rim region of outer segment disc membranes. These results suggest that channel/peripherin protein interactions mediated by the GARP part of the channel beta-subunit play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel.exchanger complex in the rod outer segment plasma membrane.  相似文献   

11.
We report the genomic structure of the human gamma adducin gene (ADD3). Adducin is a protein involved in cytoskeletal assembly and composed of alpha-beta or alpha-gamma subunits which share a high degree of homology between human and rat. Mutations in alpha subunit have been shown associated to both human and rat hypertension. The human ADD3 gene spans over 20 kb and is composed of at least 13 introns and 14 exons covering the entire coding region. The exon size ranges from 81 bp to greater than 293 bp and the intron size from 111 bp to longer than 3.2 kb. We also demonstrate the presence of an alternative splicing event around exon 13, whose sequence, position, and expression is analogous in rat Add3 gene. Moreover, human ADD3 amino acid sequence presents 91.9% of identity compared to rat sequence. Characterization of human ADD3 gene provides an important tool for mutation analysis.  相似文献   

12.
CD5 is a member of the family of receptors which contain extracellular domains homologous to the type I macrophage scavenger receptor cysteine-rich (SRCR) domain. Here, we compare the exon/intron organization of the human CD5 gene with its mouse homologue, as well as with the human CD6 gene, the closest related member of the SRCR superfamily. The human CD5 gene spans about 24.5 kb and consists of at least 11 exons. These exons are conserved in size, number, and structure in the mouse CD5 homologue. No evidence for the biallelic polymorphism reported in the mouse could be found among a population of 100 individuals of different ethnic origins. The human CD5 gene maps to the Chromosome (Chr) 11q12.2 region, 82 kb downstream from the human CD6 gene, in a head-to-tail orientation, a situation which recalls that reported at mouse Chr 19. The exon/intron organization of the human CD5 and CD6 genes was very similar, differing in the size of intron 1 and the number of exons coding for their cytoplasmic regions. While several isoforms, resulting from alternative splicing of the cytoplasmic exons, have been reported for CD6, we only found evidence of a cytoplasmic tailless CD5 isoform. The conserved structure of the CD5 and CD6 loci, both in mouse and human genomes, supports the notion that the two genes may have evolved from duplication of a primordial gene. The existence of a gene complex for the SRCR superfamily on human Chr 11q (and mouse Chr 19) still remains to be disclosed.  相似文献   

13.
The electrical response to light in retinal rods is mediated by cyclic nucleotide-gated, nonselective cation channels in the outer segment plasma membrane. Although cGMP appears to be the primary light-regulated second messenger, cellular levels of other substances, including Ca2+ and phosphatidylinositol-4,5-bisphosphate, are also sensitive to the level of illumination. We now show that diacylglycerol (DAG) analogs reversibly suppress the cGMP-activated conductance in excised patches from frog rod outer segments. This suppression did not require nucleoside triphosphates, indicating that a phosphorylation reaction was not involved. DAG was more effective at low than at high [cGMP]: with 50 microM 8-Br-cGMP, the DAG analog 1,2-dioctanoyl-sn-glycerol (1,2-DiC8) reduced the current with an IC50 of approximately 22 microM (Hill coefficient, 0.8), whereas with 1.2 microM 8-Br-cGMP, only approximately 1 microM 1,2-DiC8 was required to halve the current. DAG reduced the apparent affinity of the channels for cGMP: 4 microM 1,2-DiC8 produced a threefold increase in the K1/2 for channel activation by 8-Br-cGMP, as well as a threefold reduction in the maximum current, without changing the apparent stoichiometry or cooperativity of cGMP binding. Inhibition by 1,2-DiC8 was not relieved by supersaturating concentrations of 8-Br-cGMP, suggesting that DAG did not act by competitive inhibition of cGMP binding. Furthermore, DAG did not seem to significantly reduce single-channel conductance. A DAG analog similar to 1,2-DiC8--1,3-dioctanoyl-sn-glycerol (1,3-DiC8)--suppressed the current with the same potency as 1,2-DiC8, whereas an ethylene glycol of identical chain length (DiC8-EG) was much less effective.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
The gene encoding the beta-subunit of rod photoreceptor cGMP phosphodiesterase (gene symbol PDEB, homolog of the mouse rd gene) is mapped to human chromosome 4 using somatic cell hybrids and further localized to the chromosome band 4p16 using in situ hybridization. A mutation in the mouse gene underlies the recessive trait of retinal degeneration in the rd mouse. Thus, the human homolog is a candidate for lesions causing retinal degeneration.  相似文献   

15.
The permeation of monovalent cations through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions. For monovalent cations presented on the cytoplasmic side of the channel, the permeability ratios with respect to extracellular Na followed the sequence NH4 > K > Li > Rb = Na > Cs while the conductance ratios at +50 mV followed the sequence Na approximately NH4 > K > Rb > Li = Cs. These patterns are broadly similar to the amphibian rod channel. The symmetry of the channel was tested by presenting the test ion on the extracellular side and using Na as the common reference ion on the cytoplasmic side. Under these biionic conditions, the permeability ratios with respect to Na at the intracellular side followed the sequence NH4 > Li > K > Na > Rb > Cs while the conductance ratios at +50 mV followed the sequence NH4 > K approximately Na > Rb > Li > Cs. Thus, the channel is asymmetric with respect to external and internal cations. Under symmetrical 120 mM ionic conditions, the single-channel conductance at +50 mV ranged from 58 pS in NH4 to 15 pS for Cs and was in the order NH4 > Na > K > Rb > Cs. Unexpectedly, the single-channel current-voltage relation showed sufficient outward rectification to account for the rectification observed in multichannel patches without invoking voltage dependence in gating. The concentration dependence of the reversal potential for K showed that chloride was impermeant. Anomalous mole fraction behavior was not observed, nor, over a limited concentration range, were multiple dissociation constants. An Eyring rate theory model with a single binding site was sufficient to explain these observations.  相似文献   

16.
In order to investigate the lectin-binding properties of the photoreceptor cGMP-gated channel, solubilized and purified channel protein was incubated with immobilized lectins followed by reconstitution of unbound proteins. Of the lectins tested, only concanavalin A (ConA) was able to specifically sediment channel activity. A 240-kDa protein, which copurifies with the 63-kDa channel protein but does not bind ConA, was also found to be sedimented by the ConA-affinity matrix, thereby implicating that it is associated with the channel complex. Treatment of the purified channel protein with the enzyme glycopeptidase F in the presence of the denaturing detergent sodium dodecyl sulfate resulted in a rapid reduction of the apparent molecular mass by 1.90 kDa, and the abolition of ConA-binding. No intermediate molecular weight species were observed, suggesting that the channel protein is N-glycosylated at one site only. Under nondenaturing conditions, the kinetics of deglycosylation were distinctly two-phased: 50-60% deglycosylation was achieved rapidly; however, prolonged incubation was required to arrive at complete deglycosylation. Reconstitution experiments showed that deglycosylation had no significant effect on the kinetics of channel protein activation by cGMP.  相似文献   

17.
18.
Genomic organization of the human oestrogen receptor gene.   总被引:37,自引:4,他引:37       下载免费PDF全文
  相似文献   

19.
Genomic organization of the human amyloid beta-protein precursor gene   总被引:15,自引:0,他引:15  
S Yoshikai  H Sasaki  K Doh-ura  H Furuya  Y Sakaki 《Gene》1990,87(2):257-263
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20.
The intracellular Ca(2+) concentration in rod outer segments of vertebrate photoreceptors is controlled by Ca(2+) influx through cGMP-gated channels and by Ca(2+) efflux driven by Na/Ca-K exchangers. Previously, we suggested that channel and exchanger are associated (Bauer, P. J., and Drechsler, M. (1992) J. Physiol. (Lond. ) 451, 109-131). This suggestion has been thoroughly examined using a variety of biochemical approaches. First, we took advantage of the fact that cGMP-gated channels bind calmodulin (CaM). Using CaM affinity chromatographic purification of the channel in 10 mm CHAPS, a significant fraction of exchanger was co-eluted with the channel indicating a binding affinity between channel and exchanger. Binding of channel and exchanger was examined more directly by cross-linking of proteins in the rod outer segment membranes. Activation of the channel with cyclic 8-bromo-GMP lead to exposure of a cysteine, which allowed cross-linking of the channel to the exchanger with the thiol-specific reagent dl-1,4-bismaleimido-2,3-butanediol. Cleavage of the cross-links and electrophoretic analysis indicated that a cross-link between the alpha-subunit of the channel and the exchanger formed. Furthermore, a cross-link between two adjacent alpha-subunits of the channel was found, suggesting that the alpha-subunits of the native channel are dimerized. Further support for an interaction between alpha-subunit and exchanger was obtained by in vitro experiments. Specific binding of the exchanger to the alpha-subunit but not to the beta-subunit of the channel was observed in Western blots of purified channel incubated with purified exchanger. This study suggests that two exchanger molecules bind to one cGMP-gated channel and, more specifically, that binding of exchanger molecules occurs at the alpha-subunits, which in the native channel are dimerized. The implications of these findings regarding the possibility of local Ca(2+) signaling in vertebrate photoreceptors will be discussed.  相似文献   

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