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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 19, 2004 as doi:10.1096/fj.03-0763fje. |
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* Department of Biological Sciences, University at Buffalo, State University of New York, Buffalo, New York, USA;
Centre for Cutaneous Research, Barts, and the London School of Medicine and Dentistry, Queen Mary, London, UK
2Correspondence: E-mail: nicholsonb{at}uthscsa.edu
SPECIFIC AIMS
The M34T mutation of the Cx26 gap junction gene causes sensorineural deafness, but there is controversy over the underlying mechanism, including reports that protein trafficking problems prevent localization to cell junctions, that channels localize properly but have a reduced permeability, and that dye permeability is unaffected by the mutation. We set out to reconcile some of these discrepancies by examining localization of this mutant directly in patient-derived tissue and by studying channel properties electrophysiologically after expression in Xenopus oocytes.
PRINCIPAL FINDINGS
1. Cx26M34T localizes to regions of cell–cell contact in both transfected HeLa cells and in cells derived from a homozygotic individual, suggesting that the mutation does not impair translation or trafficking of Cx26
2. Although M34T/M34T homotypic channels did not induce intercellular coupling, heterotypic pairings with Cx26 (Cx26/M34T) revealed functional channels with gating properties significantly different than those of wtCx26; transjunctional currents were induced, rather than reduced, by application of transjunctional voltage
3. PCR analysis of DNA and RNA from a heterozygotic individual confirmed that wild-type and mutant alleles are transcribed with equal efficiency
4. Coinjection of equimolar levels of wt and mutant cRNAs did not reduce wild-type coupling or change its gating properties. However, when overexpressed (e.g., RNA for M34T:WT=2:1), M34T did cause a decrease in Cx26 coupling. This suggests that previous inconsistencies in the literature regarding the dominant/recessive nature of the allele in expression systems may have occurred as a result of varied levels of protein expression
SUMMARY
Great interest in the gap junction field has been generated by the diverse array of diseases linked to mutations of different connexin genes. Of these, by far the most common has been inherited, asymptomatic sensorineural deafness, where over 50% of all cases worldwide appear to be associated with mutations in Cx26. One of the earliest reported mutations of Cx26 associated with deafness is M34T.
To ultimately understand aspects of connexin function that are important in the etiology of this widespread human disease, it is important to understand precise defects in channel function that can produce disease symptoms. In the case of M34T, it also seems particularly important to resolve previous discrepancies in the literature, as this mutation has generated a great deal of controversy in regard to its ability to form functional (dye-permeable) channels in exogenous expression systems, and its dominant/recessive nature. We have addressed both questions by presenting the first analysis of functional channels formed by this mutant in Xenopus oocytes, while also following expression of wild-type and mutant proteins in both exogenous, transfected systems, and also in patient-derived keratinocytes.
In HeLa cells transfected with Cx26M34T, connexin protein was found at both cell–cell interfaces as well as in punctate intracellular pools. This pattern was essentially identical to that of wtCx26 in the same cells. As shown in Fig. 1
B, M34T also localizes to cell–cell boundaries in sweat glands from a homozygotic M34T individual, where its expression patterns were again very similar to those of wild-type protein (Fig. 1A
). It appears that the M34T mutation does not disrupt translation or trafficking of Cx26.
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To study properties of assembled Cx26M34T junctions, we used the Xenopus oocyte system. In oocytes we were able to study channel function under conditions that can mimic both homozygotic and heterozygotic conditions. Cx26/Cx26 homotypic channels behaved as previously reported, inactivating slowly in response to transjunctional voltages > +/– 80 mV (actual transjunctional currents mediated by Cx26/Cx26 channel, Fig. 2
A, and corresponding conductance-voltage plot, Fig. 2
D). In contrast, when the mutant was expressed in both oocytes, no currents were detectable, consistent with previous findings (M34T/M34T; Fig. 3
). When the mutant was paired heterotypically with a wild-type-expressing oocyte, we readily detected currents that revealed significant changes in voltage gating of the mutant hemichannel that explain lack of currents from homotypic mutant channels. Mutant channels have their gating response inverted, such that they reside in a low conductance state at resting potentials (as would be experienced by most coupled hair cells in the ear) and are only opened by applied transjunctional voltage, as illustrated in Fig. 3
and in actual transjunctional currents recorded from Xenopus oocytes in Fig. 2B
(conductance-voltage plot in Fig. 2E
). Lack of homotypic currents from these channels is likely to be the result of at least one hemichannel being locked in the low conductance state at all voltages. This lower conductance state is also consistent with previous observations of reduced dye flux through these mutant channels.
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Almost all connexin-associated deafness is manifested as a recessive disease. The M34T mutation was an exception to this in that it was initially reported to be dominant. This seemed consistent with the initial expression of this mutation, where it was reported to have dominant negative effects over wild-type Cx26 in oocytes and to show trafficking defects that also affected coexpressed wtCx26 in transfected cells. However subsequent genetic screens revealed families more consistent with a recessive phenotype for this mutation. Expression studies in different cell lines also yielded variable results, from reduced function in HeLa cells to essentially wild-type expression patterns and coupling in insect cells.
To determine whether altered gating behavior of the M34T channel was related to its dominant/recessive behavior, M34T was coexpressed with wtCx26. To mimic the situation in vivo, where wild-type and mutant alleles are transcribed with equal efficiency in heterozygous individuals (results not shown), Cx26 and M34T RNA’s were coinjected at equimolar levels. Under these conditions, no effects on expression levels or channel properties of wt Cx26 were seen, consistent with the more recently reported recessive phenotype (Fig. 3
, and Fig. 2C, F
). When the ratio of mutant to wild-type RNA was increased (e.g., M34T:wtCx26 RNA
2:1), some decrement in current was observed, although this was not correlated with any consistent effect on gating behavior. This offers an explanation for occasional dominant effects observed in exogenous systems, where expression levels of mutant and wild-type RNAs were not controlled. It could also explain occasional "dominant" phenotypes in some families, if the two alleles are expressed at different levels. In several families, the M34T mutation has been linked to changes in the 5', noncoding end of the gene that could influence transcription.
CONCLUSIONS AND SIGNIFICANCE
This comprehensive analysis of the Cx26M34T deafness-associated mutant reveals novel findings that help to clarify several inconsistencies in the literature. The defect in the mutant protein’s function that leads to disease is not associated with biosynthesis, or even ablation of channel function, but rather a shift in the gating response of the channel that results in a low conductance (and permeability) version of the channel predominating in situ. This latter observation also serves to stress the relationship between voltage-gating behavior of gap junction channels and their role in disease, particularly as this property has proven highly sensitive to mutagenesis. In terms of controversy regarding the dominant or recessive nature of this allele, we clearly show that when equal transcripts are used, no dominant negative effects are seen, consistent with the most extensive genetic studies on this disease. Contrary reports may well be explained by the observation here that overexpression of the mutant can cause a reduction in wt junctional conductance, although not its properties.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0763fje; 
2 Current address: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, USA
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