Altered CO₂ sensitivity of connexin26 mutant hemichannels in vitro

Abstract

Connexin26 (Cx26) mutations underlie human pathologies ranging from hearing loss to keratitis ichthyosis deafness (KID) syndrome. Cx26 hemichannels are directly gated by CO 2 and contribute to the chemosensory regulation of breathing. The KID syndrome mutation A88V is insensitive to CO 2, and has a dominant negative action on the CO 2 sensitivity of Cx26WT hemichannels, and reduces respiratory drive in humans. We have now examined the effect of further human mutations of Cx26 on its sensitivity to CO 2 . Mutated Cx26 subunits, carrying one of A88S, N14K, N14Y, M34T, or V84L, were transiently expressed in HeLa cells. The CO 2‐dependence of hemichannel activity, and their ability to exert dominant negative actions on cells stably expressing Cx26WT, was quantified by a dye‐loading assay. The KID syndrome mutation, N14K, abolished the sensitivity of Cx26 to CO 2. Both N14Y and N14K exerted a powerful dominant negative action on the CO 2 sensitivity of Cx26WT. None of the other mutations (all recessive) had a dominant negative action. A88S shifted the affinity of Cx26 to slightly higher levels without reducing its ability to fully open to CO 2. M34T did not change the affinity of Cx26 for CO 2 but reduced its ability to open in response to CO 2. V84L had no effect on the CO 2‐sensitivity of Cx26. Some pathological mutations of Cx26 can therefore alter the CO 2 sensitivity of Cx26 hemichannels. The loss of CO 2 sensitivity could contribute to pathology and consequent reduced respiratory drive could be an unrecognized comorbidity of these pathologies.

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.…”

“…Thus, the KIDS mutations not only prevent CO2dependent opening of the hemichannel, but also prevent CO2-dependent closing of the gap junction, at least when combined with M151L. Interestingly, the effect of N14K on hemichannel sensitivity to CO2 is less than the other KIDS mutations (de Wolf et al, 2016) and this parallels the trend in our data that permeation of NBDG through the Cx26 N14K,M151L gap junctions, while still occurring, may be slightly slowed at the higher level of PCO2 ( Figure 6C, D).…”

“…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).…”

DFNB1 Non-syndromic Hearing Impairment: Diversity of Mutations and Associated Phenotypes

“…Auditory transduction involves high energy consumption in the cochlea and concomitant CO 2 production, and thus CO 2 may evoke Cx26 hemichannel-dependent ATP signaling for coordination of supporting cell activity. At least two DFNB1 mutations interfere with CO 2 -mediated hemichannel opening, which would impair signaling: p.(Met34Thr), which reduces the ability of the hemichannel to open in response to CO 2 , and p.(Ala88Ser; Frei et al, 2002 ), with a reduced affinity for CO 2 (de Wolf et al, 2016 ).…”

Consensus interpretation of the Met34Thr and Val37Ile variants in GJB2 by the ClinGen Hearing Loss Expert Panel

“…Furthermore, c.101T>C transfected HeLa cells did not transfer Lucifer yellow (a fluorescent dye) to neighbors across gap junctions compared to wild-type connexin 26 transfected cells 20,22,24 and the ability to transfer neurobiotin was also reduced. 21 However, c.101T>C transfected HeLa cells was able to load the dye in response to non-physiological zero extracellular Ca 2+ stimulus 25 , suggesting that the Met34Thr variant connexin may retain some residual function under unusual circumstances. The atomic structure of the human connexin 26 gap junction channel revealed that the methyl group in Met34 in the first transmembrane domain interacts with the tryptophan at amino acid position 3 in the amino-terminal helix of an adjacent protomer to stabilize the hexameric channel 26 , and alteration of the residue is predicted to impact function 27 .…”