A novel M163L mutation in connexin 26 causing cell death and associated with autosomal dominant hearing loss

Matos, Tiago; Caria, Helena; Simões-Teixeira, Helena; Aasen, Trond; Dias, Óscar; Andrea, Mário; Kelsell, David P.; Fialho, Graça

doi:10.1016/j.heares.2008.03.004

Cited by 29 publications

(35 citation statements)

References 17 publications

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“…Mutations of single amino acids are also prone to impairment of trafficking and stability of proteins (28). To ensure that the functionally proper mutants Panx1-C136S and Panx1-C426S were trafficked correctly, we performed several control experiments.…”

Section: Discussionmentioning

confidence: 99%

Intracellular Cysteine 346 Is Essentially Involved in Regulating Panx1 Channel Activity

Bunse

Schmidt

Prochnow

et al. 2010

Journal of Biological Chemistry

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Pannexins constitute a family of proteins exhibiting predominantly hemichannel activity. Pannexin channels have been suggested to participate in a wide spectrum of biological functions such as propagation of calcium waves, release of IL-1␤, and responses to ischemic conditions. At present, the molecular mechanisms regulating pannexin hemichannel activity are essentially unknown. Because cysteines have been shown to constitute key elements in regulating hemichannel properties of the connexin-type we performed site-directed mutagenesis of intracellular cysteine residues of Panx1. Cysteine to serine exchange (Cys 3 Ser) at the C-terminal position amino acid 346 led to a constitutively leaky hemichannel and subsequently to cell death. Increased channel activity was demonstrated by dye uptake and electrophysiological profiling in injected Xenopus laevis oocytes and transfected N2A cells. Mutations of the remaining intracellular cysteines did not result in major changes of Panx1 channel properties. From these data we conclude that the Cys-346 residue is important for proper functioning of the Panx1 channel.In addition to connexins, a second family of proteins, termed pannexins (Panx) 3 (1), is suggested to form hemichannels. The pannexin channel family consists of three members: Panx1, which is abundantly expressed in different organs of the vertebrate organism, Panx2, which is found mainly in the central nervous system, and Panx3, which is primarily expressed in skin and cartilage (2-8). Panx1 is capable of forming gap junctions and hemichannels in the Xenopus laevis oocytes expression system (9, 10), but it is still a matter of debate whether pannexinformed gap junctions do exist under in vivo conditions. Apparently, Panx1 primarily forms hemichannels (9,11,12). Among others, a role of Panx1 hemichannels was suggested in the initiation and propagation of calcium waves (13), the release of interleukin-1␤ due to interaction with the P2X7 receptor (14 -16), neuronal cell death after ischemia (17, 18), and in activation of inflammasomes (19). Panx1 hemichannels open in response to a rise in intracellular Ca 2ϩ , to depolarization to membrane potentials above Ϫ20 mV, and to mechanical stretch (10,11,20).Even though there is no direct sequence homology between pannexins and connexins, both protein types possess the same topology with four transmembrane domains and intracellularly located N and C termini (for review, see Ref. 21). Significant differences between the two protein families are the number of extracellular cysteines and the fact that Panx1 is glycosylated at the second extracellular loop (9, 22). Deglycosylation with N-glycosidase F enhanced gap junctional coupling, indicating that glycosylation might be a regulatory key element to distinguish between gap junctional coupling and hemichannel activity (23).Besides the importance of extracellular cysteines in gap junction channel formation of connexins (24), intracellular cysteine residues have been suggested to be responsible for sensitivity of Cx43 and Cx46 hemi...

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Section: Discussionmentioning

confidence: 99%

Intracellular Cysteine 346 Is Essentially Involved in Regulating Panx1 Channel Activity

Bunse

Schmidt

Prochnow

et al. 2010

Journal of Biological Chemistry

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“…Therefore, the dominant-negative effect of the p.D46E mutation may be due to defective docking of the two opposing connexin hemichannels. In many cases, mutations in extracellular domains are associated with dominant inheritance and syndromic phenotype, such as skin disorders and other ectodermal abnormalities (Deng et al, 2006;Martinez et al, 2009;Marziano et al, 2003;Matos et al, 2008;Melchionda et al, 2005;Sun et al, 2005). Two different mutations in the EC1 (p.W44C and p.W44S) have been reported to be associated with dominantly inherited nonsyndromic hearing loss and to exert a dominant-negative effect on gap junction permeability (Martin et al, 1999;Marziano et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Different functional consequences of two missense mutations in theGJB2gene associated with non-syndromic hearing loss

Choi

Park²,

Lee

et al. 2009

Hum. Mutat.

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Mutations in the GJB2 gene, which encodes the gap junction (GJ) protein connexin26 (Cx26), are the most common cause of inherited non-syndromic hearing loss (NSHL). We identified two missense mutations, p.D46E (c.138T>G) and p.T86R (c.257C>G), of GJB2 in Korean HL families. The novel p.D46E mutation exhibited autosomal dominant inheritance, while the p.T86R mutation, which is exclusively found in Asians, segregated with an autosomal recessive pattern. Thus, we sought to elucidate the pathogenic nature of such different inherited patterns of HL. We studied protein localization and gap junction functions in cells transfected with wild-type or mutant Cx26 tagged with fluorescent proteins, which allowed visual confirmation of homozygous or heterozygous mutant GJs. The Cx26-D46E mutant was targeted to the plasma membrane, but this mutant protein failed to transfer Ca 2+ or propidium iodide intercellularly, suggesting disruption of both ionic and biochemical coupling. Heterozygous GJs also showed dysfunctional intercellular couplings and hemichannel opening, confirming the dominant-negative nature of the p.D46E mutation. The Cx26-T86R mutant protein did not form GJs, since the mutated protein was confined in the cytoplasm and not transported to the cell membrane. When Cx26-T86R was co-expressed with Cx26-WT, ionic and biochemical coupling was normal, consistent with the recessive nature of the mutation. These studies revealed distinct pathogenic mechanisms of two GJB2 mutations identified in Korean families.

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“…On the other hand, it is reported that the altered Cx hemichannel function may lead to cell death and thus causes cataracts [Minogue et al, 2009]. The Cx46G2D significantly inhibited the hemichannel permeability, may therefore lead to ionic and biochemical changes, disrupted the cells' ability to maintain homeostasis [Diekmann et al, 2010;Matos et al, 2008], and ultimately resulted in apoptosis.…”

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confidence: 99%

A novelGJA3mutation associated with congenital nuclear pulverulent and posterior polar cataract in a chinese family

et al. 2011

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Congenital cataract (CC) is the leading cause of visual disability in children. To date, mutations in many genes have been linked to CC. In a four-generation Chinese family with congenital nuclear pulverulent and posterior polar cataracts, we detected a heterozygous c.5G>A transition in the second exon of GJA3, resulting in the substitution of a highly conserved glycine with aspartic acid (p.G2D) at the N-terminus of the connexin46 (Cx46) protein. Wild type (wt) and mutant Cx46 plasmids were transfected into HeLa cells to examine the molecular basis of cataract formation. Unlike wt Cx46, Cx46G2D mutant formed gap junction plaques inefficiently, changed hemichannel permeability, and caused apoptosis. These results suggest that the glycine residue at the second position of the N-terminus is important for gap junction plaque formation and hemichannel function.

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A novel M163L mutation in connexin 26 causing cell death and associated with autosomal dominant hearing loss

Cited by 29 publications

References 17 publications

Intracellular Cysteine 346 Is Essentially Involved in Regulating Panx1 Channel Activity

Intracellular Cysteine 346 Is Essentially Involved in Regulating Panx1 Channel Activity

Different functional consequences of two missense mutations in theGJB2gene associated with non-syndromic hearing loss

A novelGJA3mutation associated with congenital nuclear pulverulent and posterior polar cataract in a chinese family

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