Molecular mechanisms underlying enhanced hemichannel function of a cataract-associated Cx50 mutant (original) (raw)
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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.
BioMed Research International, 2014
Connexin50 (Cx50) mutations are reported to cause congenital cataract probably through the disruption of intercellular transport in the lens. Cx50 mutants that undergo mistrafficking have generally been associated with failure to form functional gap junction channels; however, sometimes even properly trafficked mutants were found to undergo similar consequences. We hereby wanted to elucidate any structural bases of the varied functional consequences of Cx50 missense mutations through in silico approach. Computational studies have been done based on a Cx50 homology model to assess conservation, solvent accessibility, and 3dimensional localization of mutated residues as well as mutation-induced changes in surface electrostatic potential, H-bonding, and steric clash. This was supplemented with meta-analysis of published literature on the functional properties of connexin missense mutations. Analyses revealed that the mutation-induced critical alterations of surface electrostatic potential in Cx50 mutants could determine their fate in intracellular trafficking. A similar pattern was observed in case of mutations involving corresponding conserved residues in other connexins also. Based on these results the trafficking fates of 10 uncharacterized Cx50 mutations have been predicted. Further experimental analyses are needed to validate the observed correlation.
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...
A Mutant Connexin50 with Enhanced Hemichannel Function Leads to Cell Death
Investigative Ophthalmology & Visual Science, 2009
To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts. METHODS. The GJA8 gene was directly sequenced. Formation of functional channels was assessed by the two-microelectrode voltage-clamp method. Connexin protein levels and distribution were assessed by immunoblot analysis and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry. RESULTS. Direct sequencing of the GJA8 gene identified a 137 GϾT transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in the number of cells and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions. CONCLUSIONS. Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function. (Invest Ophthalmol Vis Sci. 2009;50:5837-5845)
Biophysical Journal, 1995
The gap-junctional protein rat connexin46 (Cx46) has the unusual ability to form voltage-gated channels in the nonjunctional plasma membrane of Xenopusoocytes (Paul et al., 1991; Ebihara and Steiner, 1993). These have been suggested to be gap-junctional hemichannels or connexons. The Xenopusoocyte system was used to characterize the functional properties of a closely related lens gap-junctional protein, chicken connexin56 (Cx56) (Rup et al., 1993) and to contrast them to those of rat Cx46. Single oocytes injected with either Cx56 or Cx46 cRNA developed time-dependent, outward currents that activated on depolarization. The currents induced by Cx56 and Cx46 showed differences in steady-state voltage dependence and in their degree of rectification. Furthermore, the voltage-dependent properties of the nonjunctional channels induced by the connexin cRNAs in external solutions containing low concentrations of calcium ions could account remarkably well for the behavior of the intercellular channels formed by Cx56 and Cx46 in paired oocytes. These results suggest that many of the voltage-dependent properties of the hemi-gap-junctional channels are retained by the intercellular channels.
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...
Cataracts Are Caused by Alterations of a Critical N-Terminal Positive Charge in Connexin50
Investigative Ophthalmology & Visual Science, 2008
PURPOSE. To elucidate the basis of the autosomal dominant congenital nuclear cataracts caused by the connexin50 mutant, CX50R23T, by determining its cellular distribution and functional behavior and the consequences of substituting other amino acids for arginine-23. METHODS. Connexin50 (CX50) mutants were generated by PCR and transfected into HeLa or N2a cells. Expressed CX50 protein was detected by immunoblot analysis and localized by immunofluorescence. Intercellular communication was assessed by microinjection of neurobiotin or by double wholecell patch-clamp recording. RESULTS. HeLa cells stably transfected with CX50R23T or wild-type CX50 produced immunoreactive CX50 bands of identical electrophoretic mobility. Whereas HeLa cells stably expressing CX50 contained abundant gap junction plaques, CX50R23T localized predominantly in the cytoplasm. HeLa cells expressing wild-type CX50 showed large gap junctional conductances and extensive transfer of neurobiotin, but those expressing CX50R23T did not show significant intercellular communication by either assay. Moreover, CX50R23T inhibited the function of coexpressed wild-type CX50. Three CX50R23 substitution mutants (CX50R23K, CX50R23L, and CX50R23W) formed gap junction plaques, whereas two mutant substitutions with negatively charged residues (CX50R23D, CX50R23E) did not form detectable plaques. Only the mutant with a positive charge substitution (CX50R23K) allowed neurobiotin transfer at levels similar to those of wild-type CX50; none of the other mutants induced transfer. CONCLUSIONS. These results suggest that replacement of amino acid 23 in CX50 by any residue that is not positively charged would lead to cataract formation.