Transmembrane S1 Mutations in<i>CNGA3</i>from Achromatopsia 2 Patients Cause Loss of Function and Impaired Cellular Trafficking of the Cone CNG Channel

Patel, Kirti A.; Bartoli, Kristen M.; Fandino, Richard A.; Ngatchou, Anita N.; Woch, Gustaw; Carey, Jannette; Tanaka, Jacqueline C.

doi:10.1167/iovs.05-0179

Cited by 37 publications

(50 citation statements)

References 47 publications

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“…Besides affecting the apparent ligand sensitivity, impaired protein folding and/or trafficking resulting in a decreased channel density at the cell membrane is a common consequence of variants in the CNGA3 subunit. 32,33,42,43,45 Immunocytochemical analyses performed in this study demonstrated that the p.Cys319Arg variant also leads to a disturbed surface expression. For some of the CNGA3 variants, it was shown that this folding/trafficking defect could be rescued by the presence of the wild-type CNGB3 subunit in heteromeric channels or by lowering the cultivation temperature of the corresponding heterologous expression system.…”

Section: Discussionmentioning

confidence: 56%

“…We therefore reason that a few mutant channels might have escaped the quality control process in early cones and be sufficient to carry out residual cone responses in the young affected individuals, after which these cones rapidly degenerate with age. Numerous CNGA3 variants, causing achromatopsia, have been assessed functionally upon heterologous expression in Xenopus oocytes or HEK293 cells with the majority of mutant channels being non-functional in calcium imaging or patch clamp experiments, 29,32,33,[42][43][44][45] -similar to what has been observed for p.Cys319Arg mutant channels. Besides affecting the apparent ligand sensitivity, impaired protein folding and/or trafficking resulting in a decreased channel density at the cell membrane is a common consequence of variants in the CNGA3 subunit.…”

Section: Discussionmentioning

confidence: 95%

“…For some of the CNGA3 variants, it was shown that this folding/trafficking defect could be rescued by the presence of the wild-type CNGB3 subunit in heteromeric channels or by lowering the cultivation temperature of the corresponding heterologous expression system. 29,32,33,[42][43][44][45] Both approaches were also applied in the case of the p.Cys319Arg mutant channels, but here no improvement of channel functionality could be observed in calcium imaging experiments. As shown in this study, the alteration in p.Cys319Arg also affects protein stability, an observation which has so far only been described for two pore variants and one variant located in transmembrane domain 4 of CNGA3, 42,43 representing another mechanism how CNGA3 variants impair channel functionality.…”

Section: Discussionmentioning

confidence: 99%

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Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy

et al. 2014

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We assessed a large consanguineous Pakistani family (PKAB157) segregating early onset low vision problems. Funduscopic and electroretinographic evaluation of affected individuals revealed juvenile cone-rod dystrophy (CRD) with maculopathy. Other clinical symptoms included loss of color discrimination, photophobia and nystagmus. Whole-exome sequencing, segregation and haplotype analyses demonstrated that a transition variant (c.955T4C; p.(Cys319Arg)) in CNGA3 co-segregated with the CRD phenotype in family PKAB157. The ability of CNGA3 channel to influx calcium in response to agonist, when expressed either alone or together with the wild-type CNGB3 subunit in HEK293 cells, was completely abolished due to p.Cys319Arg variant. Western blotting and immunolocalization studies suggest that a decreased channel density in the HEK293 cell membrane due to impaired folding and/or trafficking of the CNGA3 protein is the main pathogenic effect of the p.Cys319Arg variant. Mutant alleles of the human cone photoreceptor cyclic nucleotide-gated channel (CNGA3) are frequently associated with achromatopsia. In rare cases, variants in CNGA3 are also associated with cone dystrophy, Leber's congenital amaurosis and oligo cone trichromacy. The identification of predicted p.(Cys319Arg) missense variant in CNGA3 expands the repertoire of the known genetic causes of CRD and phenotypic spectrum of CNGA3 alleles.

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Section: Discussionmentioning

confidence: 56%

Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

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Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy

et al. 2014

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“…Immunoblot Analysis of Protein Expression-The cells were grown in 35-mm dishes and harvested 48 h after transfection as previously described (33). Protein concentrations were determined using a Bradford assay.…”

Section: Methodsmentioning

confidence: 99%

Voltage Gating at the Selectivity Filter of the Ca2+ Release-activated Ca2+ Channel Induced by Mutation of the Orai1 Protein

Spassova

Hewavitharana

Fandino

et al. 2008

Journal of Biological Chemistry

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The Ca 2؉ release-activated Ca 2؉ (CRAC) channel is a plasma membrane (PM) channel that is uniquely activated when free Ca 2؉ level in the endoplasmic reticulum (ER) is substantially reduced. Several small interfering RNA screens identified two membrane proteins, Orai1 and STIM1, to be essential for the CRAC channel function. STIM1 appears to function in the PM and as the Ca 2؉ sensor in the ER. Orai1 is forming the pore of the CRAC channel. Despite the recent breakthroughs, a mechanistic understanding of the CRAC channel gating is still lacking. Here we reveal new insights on the structure-function relationship of STIM1 and Orai1. Our data suggest that the cytoplasmic coiled-coil region of STIM1 provides structural means for coupling of the ER membrane to the PM to activate the CRAC channel. We mutated two hydrophobic residues in this region to proline (L286P/L292P) to introduce a kink in the first ␣-helix of the coiled-coil domain. This STIM1 mutant caused a dramatic inhibition of the CRAC channel gating compared with the wild type. Structure-function analysis of the Orai1 protein revealed the presence of intrinsic voltage gating of the CRAC channel. A mutation of Orai1 (V102I) close to the selectivity filter modified CRAC channel voltage sensitivity. Expression of the Orai1 V102I mutant resulted in slow voltage gating of the CRAC channel by negative potentials. The results revealed that the alteration of Val 102 develops voltage gating in the CRAC channel. Our data strongly suggest the presence of a novel voltage gating mechanism at the selectivity filter of the CRAC channel.The CRAC 2 channel is activated by depletion of the intracellular Ca 2ϩ stores and mediates sustained Ca 2ϩ entry in hematopoietic cells (1-3) and some other cell types (1, 3). Until recently, the molecular identity of the machinery linking store depletion with Ca 2ϩ entry remained elusive. However, recent studies identified two proteins, STIM1 and Orai1, as being essential for CRAC function (4 -11). Although STIM1 resides predominantly in the ER and the plasma membrane (PM) (11), Orai1 is almost exclusively expressed in the PM (12). STIM1 functions as the Ca 2ϩ sensor in the ER that triggers the CRAC channel activation (5, 6, 11). However, its translocation to the PM upon activation (11, 13) and functional role in the PM have also been demonstrated (6,14). Even though a report by Mignen et al. (14) revealed that PM-STIM1 regulates another Ca 2ϩ channel (arachidonate-regulated Ca 2ϩ channels), the same group later revealed that arachidonate-regulated Ca 2ϩ channels have the same molecular identity as CRAC channels (Ref. 41; see also Ref. 15). STIM1 aggregates into puncta and translocates toward the PM, when the stores are depleted (5, 16). Physical incorporation of STIM1 molecules into the PM after Ca 2ϩ store depletion has been clearly demonstrated (11,13). The time course of the ER Ca 2ϩ depletion followed by CRAC activation correlates with the time course of STIM1 translocation that is ϳ52 s (5, 17).How STIM1 relays signals about ER C...

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“…It is worth mentioning that though lack of cone function in the absence of CNG channel (such as in CNGA3 Ϫ/Ϫ mice) causes opsin mislocalization, subunit mislocalization arising from diseaselinked missense mutations in CNGA3 has been reported extensively in heterologous expression system. Enhanced cytosolic aggregation and activation of ER stress were observed in many CNGA3 mutants, including those with mutations located in the subunit transmembrane S1 and S4 domains (45,46) and at the carboxyl terminals (47)(48)(49). Thus, the UPR and ER stress in CNGA3 missense (and loss of function) mutations could be caused not only by opsin mislocalization (due to lack of normal channel function/phototransduction) but also by mislocalization of mutant CNGA3 subunits, and the ER stress might be more severe than the situation of lack of CNGA3.…”

Section: Cnga3mentioning

confidence: 99%

Endoplasmic Reticulum Stress-associated Cone Photoreceptor Degeneration in Cyclic Nucleotide-gated Channel Deficiency

Thapa

Morris

et al. 2012

Journal of Biological Chemistry

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Background:Photoreceptors undergo degeneration when phototransduction is impaired. Results: The endoplasmic reticulum stress markers and processing of the associated caspases are elevated in retinas with cone photoreceptor CNG channel deficiency. Conclusion:The endoplasmic reticulum stress-associated apoptotic pathways play a crucial role in cone degeneration. Significance: Understanding of the mechanism(s) of photoreceptor degeneration is essential for development of therapeutic strategies.

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Transmembrane S1 Mutations inCNGA3from Achromatopsia 2 Patients Cause Loss of Function and Impaired Cellular Trafficking of the Cone CNG Channel

Cited by 37 publications

References 47 publications

Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy

Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy

Voltage Gating at the Selectivity Filter of the Ca2+ Release-activated Ca2+ Channel Induced by Mutation of the Orai1 Protein

Endoplasmic Reticulum Stress-associated Cone Photoreceptor Degeneration in Cyclic Nucleotide-gated Channel Deficiency

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