Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicityin vitro

Abstract

The Cx26 mRNA has not been reported to undergo alternative splicing. In expressing a series of human keratitis ichthyosis deafness (KID) syndrome mutations of Cx26 (A88V, N14K and A40V), we found the production of a truncated mRNA product. These mutations, although not creating a cryptic splice site, appeared to activate a pre-existing cryptic splice site. The alternative splicing of the mutant Cx26 mRNA could be prevented by mutating the predicted 3′, 5′ splice sites and the branch point. The presence of a C-terminal fluorescent protein tag (mCherry or Clover) was necessary for this alternative splicing to occur. Strangely, Cx26 A88V could cause the alternative splicing of co-expressed WT Cx26—suggesting a trans effect. The alternative splicing of Cx26 A88V caused cell death, and this could be prevented by the 3′, 5′ and branch point mutations. Expression of the KID syndrome mutants could be rescued by combining them with removal of the 5′ splice site. We used this strategy to enable expression of Cx26 A40V-5′ and demonstrate that this KID syndrome mutation removed CO 2 sensitivity from the Cx26 hemichannel. This is the fourth KID syndrome mutation found to abolish the CO 2 -sensitivity of the Cx26 hemichannel, and suggests that the altered CO ­2 -sensitivity could contribute to the pathology of this mutation. Future research on KID syndrome mutations should take care to avoid using a C-terminal tag to track cellular localization and expression or if this is unavoidable, combine this mutation with removal of the 5′ splice site.

Opposing modulation of Cx26 gap junctions and hemichannels by CO2

“…The key result from our study is that Cx26 gap junctions are closed by a direct action of CO2 on the protein. Our prior publications have demonstrated the opening action of CO2 on Cx26 hemichannels (Huckstepp et al, 2010a;Meigh et al, 2013;Meigh et al, 2014;de Wolf et al, 2016;de Wolf et al, 2017;Cook et al, 2019;Dospinescu et al, 2019). This paper further shows that these diametrically opposite actions of CO2 on gap junctions and hemichannels depend on the same residues and presumably the same carbamate bridging mechanism.…”

“…We have previously shown that KID syndrome mutations A88V, N14K, and A40V ( Figure 5) abolish the ability of CO2 to open the mutant hemichannels (Meigh et al, 2014;de Wolf et al, 2016;Cook et al, 2019). Recently, we discovered that certain KID syndrome mutations induce alternative splicing of the Cx26 mutation when this is tagged with a fluorescent protein (Cook et al, 2019). This splicing results in poor expression and cell death but can be prevented by also mutating the 5' splice site (Cook et al, 2019).…”

“…Recently, we discovered that certain KID syndrome mutations induce alternative splicing of the Cx26 mutation when this is tagged with a fluorescent protein (Cook et al, 2019). This splicing results in poor expression and cell death but can be prevented by also mutating the 5' splice site (Cook et al, 2019). However, the 5' splice site cannot be silently mutated so we chose a conservative mutation, M151L ( Figure 5), which appeared to have little effect on Cx26 expression by itself.…”

“…However, the 5' splice site cannot be silently mutated so we chose a conservative mutation, M151L ( Figure 5), which appeared to have little effect on Cx26 expression by itself. Cx26 M151L hemichannels are blocked normally by extracellular Ca 2+ and they retain CO2sensitivity (Cook et al, 2019). Furthermore, Cx26 A40V,M151L hemichannels are insensitive to CO2 (Cook et al, 2019).…”

“…Cx26 M151L hemichannels are blocked normally by extracellular Ca 2+ and they retain CO2sensitivity (Cook et al, 2019). Furthermore, Cx26 A40V,M151L hemichannels are insensitive to CO2 (Cook et al, 2019).…”