Connexin46 mutations linked to congenital cataract show loss of gap junction channel function (original) (raw)
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An Aberrant Sequence in a Connexin46 Mutant Underlies Congenital Cataracts
Journal of Biological Chemistry, 2005
An increasing number of diseases have been mapped to genes coding for ion channel proteins, including the gap junction proteins, connexins. Here, we report on the identification of an amino acid sequence underlying the behavior of a non-functional mutant connexin46 (CX46) associated with congenital cataracts. The mutant protein, CX46fs380, is 31 amino acids longer than CX46 and contains 87 aberrant amino acids in its C terminus. When expressed in mammalian cells, the mutant CX46 was not found at gap junctional plaques, but it showed extensive co-localization with markers for ERGIC and Golgi. The severe reductions in function and formation of gap junctional plaques were transferred to other connexins by creating chimeras containing the last third (or more) of the aberrant C terminus of the CX46 mutant. This sequence also impaired trafficking of a CD8 chimera. Site-directed mutagenesis of a diphenylalanine restored appositional membrane localization and function. These results suggest a novel mechanism in which a mutation causes disease by generating a motif that leads to retention within the synthetic/secretory pathway.
Properties of two cataract-associated mutations located in the NH2 terminus of connexin 46
AJP: Cell Physiology, 2013
Mutations in connexin 46 are associated with congenital cataracts. The purpose of this project was to characterize cellular and functional properties of two congenital cataract-associated mutations located in the NH2 terminus of connexin 46: Cx46D3Y and Cx46L11S, which we found localized to gap junctional plaques like wild-type Cx46 in transfected HeLa cells. Dual two-microelectrode-voltage-clamp studies of Xenopus oocyte pairs injected with wild-type or mutant rat Cx46 showed that oocyte pairs injected with D3Y or L11S cRNA failed to induce gap junctional coupling, whereas oocyte pairs injected with Cx46 showed high levels of coupling. D3Y, but not L11S, functionally paired with wild-type Cx46. To determine whether coexpression of D3Y or L11S affected the junctional conductance produced by wild-type lens connexins, we studied pairs of oocytes coinjected with equal amounts of mutant and wild-type connexin cRNA. Expression of D3Y or L11S almost completely abolished gap junctional cou...
PLoS ONE, 2013
Connexin channels play a critical role in maintaining metabolic homeostasis and transparency of the lens. Mutations in connexin genes are linked to congenital cataracts in humans. The G143R missense mutation on connexin (Cx) 46 was recently reported to be associated with congenital Coppock cataracts. Here, we showed that the G143R mutation decreased Cx46 gap junctional coupling in a dominant negative manner; however, it significantly increased gap junctional plaques. The G143R mutant also increased hemichannel activity, inversely correlated with the level of Cx46 protein on the cell surface. The interaction between cytoplasmic loop domain and C-terminus has been shown to be involved in gating of connexin channels. Interestingly, the G143R mutation enhanced the interaction between intracellular loop and Cx46. Furthermore, this mutation decreased cell viability and the resistance of the cells to oxidative stress, primarily due to the increased hemichannel function. Together, these results suggest that mutation of this highly conserved residue on the cytoplasmic loop domain of Cx46 enhances its interaction with the C-terminus, resulting in a reduction of gap junction channel function, but increased hemichannel function. This combination leads to the development of human congenital cataracts.
Molecular mechanism underlying a Cx50-linked congenital cataract
The American journal of physiology, 1999
Mutations in gap junctional channels have been linked to certain forms of inherited congenital cataract (D. Mackay, A. Ionides, V. Berry, A. Moore, S. Bhattacharya, and A. Shiels. Am. J. Hum. Genet. 60: 1474-1478, 1997; A. Shiels, D. Mackay, A. Ionides, V. Berry, A. Moore, and S. Bhattacharya. Am. J. Hum. Genet. 62: 526-532, 1998). We used the Xenopus oocyte pair system to investigate the functional properties of a missense mutation in the human connexin 50 gene (P88S) associated with zonular pulverulent cataract. The associated phenotype for the mutation is transmitted in an autosomal dominant fashion. Xenopus oocytes injected with wild-type connexin 50 cRNA developed gap junctional conductances of approximately 5 microS 4-7 h after pairing. In contrast, the P88S mutant connexin failed to form functional gap junctional channels when paired homotypically. Moreover, the P88S mutant functioned in a dominant negative manner as an inhibitor of human connexin 50 gap junctional channels w...
American journal of physiology. Cell physiology, 2018
The connexins are members of a family of integral membrane proteins which form gap junction channels between apposed cells and/or hemichannels across the plasma membranes. The importance of the Arginine at position 76 (Arg) in the structure and/or function of Cx46 is highlighted by its conservation across the entire connexin family and the occurrence of pathogenic mutations at this (or the corresponding homologous) residue in a number of human diseases. Two mutations at Arg in Cx46 are associated with cataracts in humans, highlighting the importance of this residue. We examined the expression levels and macroscopic and single channel properties of human Cx46 and compared them to those for two pathogenic mutants, namely R76H and R76G. To gain further insight into the role of charge at this position, we generated two additional non-naturally occurring mutants, R76K (charge-conserving) and R76E (charge-inverting). We found that when expressed exogenously in Neuro2a cells, all four muta...
Properties of Two Cataract Associated Mutations Located in the N-Terminus of Connexin46
Biophysical Journal, 2012
architecture of Cx26. Results obtained from our simulations show that the studied mutations modify the position of the constriction zone in the hemmi-chanel, change the diameter of the pore and produce rearragements on the electrostatic potential inside the channel. These changes are related to an increase on the freedom of movement of the N-terminal helix and trans-membrane helix 1 (TM1) of each sub-unit. These results provide relevant clues and insights about the effect of these mutations over the hemi-channel perm-selectivity and conductance.
Connexin Mutants and Cataracts
Frontiers in Pharmacology, 2013
The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8) have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating) or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death) and formation of cytoplasmic accumulations (that may act as light scattering particles). These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues.