Syndromic deafness mutations at Asn 14 differentially alter the open stability of Cx26 hemichannels.
ABSTRACT: Connexin 26 (Cx26) is a transmembrane protein that forms hexameric hemichannels that can function when unopposed or dock to form intercellular gap junction channels. Aberrantly functioning unopposed hemichannels are a common feature of syndromic deafness associated with mutations in Cx26. In this study, we examine two different mutations at the same position in the N-terminal domain of Cx26, N14K and N14Y, which have been reported to produce different phenotypes in patients. We find that both N14K and N14Y, when expressed alone or together with wild-type (WT) Cx26, result in functional hemichannels with widely disparate functional properties. N14K currents are robust, whereas N14Y currents are small. The two mutants also exhibit opposite shifts in voltage-dependent loop gating, such that activation of N14K and N14Y is shifted in the hyperpolarizing and depolarizing directions, respectively. Deactivation kinetics suggests that N14K stabilizes and N14Y destabilizes the open state. Single N14K hemichannel recordings in low extracellular Ca(2+) show no evidence of stable closing transitions associated with loop gating, and N14K hemichannels are insensitive to pH. Together, these properties cause N14K hemichannels to be particularly refractory to closing. Although we find that the unitary conductance of N14K is indistinguishable from WT Cx26, mutagenesis and substituted cysteine accessibility studies suggest that the N14 residue is exposed to the pore and that the differential properties of N14K and N14Y hemichannels likely result from altered electrostatic interactions between the N terminus and the cytoplasmic extension of TM2 in the adjacent subunit. The combined effects that we observe on loop gating and pH regulation may explain the unusual buccal cutaneous manifestations in patients carrying the N14K mutation. Our work also provides new considerations regarding the underlying molecular mechanism of loop gating, which controls hemichannel opening in the plasma membrane.
Project description:A group of human mutations within the N-terminal (NT) domain of connexin 26 (Cx26) hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. In this study, we explore the molecular basis by which N14K, an NT KID mutant, promotes gain of function. In macroscopic and single-channel recordings, we find that the N14K mutant favors the open conformation of hemichannels, shifts calcium and voltage sensitivity, and slows deactivation kinetics. Multiple copies of MD simulations of WT and N14K hemichannels, followed by the Kolmogorov-Smirnov significance test (KS test) of the distributions of interaction energies, reveal that the N14K mutation significantly disrupts pairwise interactions that occur in WT hemichannels between residue K15 of one subunit and residue E101 of the adjacent subunit (E101 being located at the transition between transmembrane segment 2 [TM2] and the cytoplasmic loop [CL]). Double mutant cycle analysis supports coupling between the NT and the TM2/CL transition in WT hemichannels, which is disrupted in N14K mutant hemichannels. KS tests of the ? carbon correlation coefficients calculated over MD trajectories suggest that the effects of the N14K mutation are not confined to the K15-E101 pairs but extend to essentially all pairwise residue correlations between the NT and TM2/CL interface. Together, our data indicate that the N14K mutation increases hemichannel open probability by disrupting interactions between the NT and the TM2/CL region of the adjacent connexin subunit. This suggests that NT-TM2/CL interactions facilitate Cx26 hemichannel closure.
Project description:Mutations in connexin 26 (Cx26) hemichannels can lead to syndromic deafness that affects the cochlea and skin. These mutations lead to gain-of-function hemichannel phenotypes by unknown molecular mechanisms. In this study, we investigate the biophysical properties of the syndromic mutant Cx26G12R (G12R). Unlike wild-type Cx26, G12R macroscopic hemichannel currents do not saturate upon depolarization, and deactivation is faster during hyperpolarization, suggesting that these channels have impaired fast and slow gating. Single G12R hemichannels show a large increase in open probability, and transitions to the subconductance state are rare and short-lived, demonstrating an inoperative fast gating mechanism. Molecular dynamics simulations indicate that G12R causes a displacement of the N terminus toward the cytoplasm, favoring an interaction between R12 in the N terminus and R99 in the intracellular loop. Disruption of this interaction recovers the fast and slow voltage-dependent gating mechanisms. These results suggest that the mechanisms of fast and slow gating in connexin hemichannels are coupled and provide a molecular mechanism for the gain-of-function phenotype displayed by the syndromic G12R mutation.
Project description:Connexin26 (Cx26) mutations underlie human pathologies ranging from hearing loss to keratitis ichthyosis deafness (KID) syndrome. Cx26 hemichannels are directly gated by CO2 and contribute to the chemosensory regulation of breathing. The KID syndrome mutation A88V is insensitive to CO2, and has a dominant negative action on the CO2 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 CO2 : Mutated Cx26 subunits, carrying one of A88S, N14K, N14Y, M34T, or V84L, were transiently expressed in HeLa cells. The CO2-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 CO2 Both N14Y and N14K exerted a powerful dominant negative action on the CO2 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 CO2 M34T did not change the affinity of Cx26 for CO2 but reduced its ability to open in response to CO2 V84L had no effect on the CO2-sensitivity of Cx26. Some pathological mutations of Cx26 can therefore alter the CO2 sensitivity of Cx26 hemichannels. The loss of CO2 sensitivity could contribute to pathology and consequent reduced respiratory drive could be an unrecognized comorbidity of these pathologies.
Project description:Mutations in the GJB2 gene-encoding connexin 26 (Cx26) have been linked to skin disorders and genetic deafness. However, the severity and type of the skin disorders caused by Cx26 mutations are heterogeneous. Here we explored the effect of Cx26 KID syndrome-associated mutations, G12R, S17F, and D50N on channel function. The Cx26 N14K mutation was also examined that is associated with deafness but has a skin disorder distinct from the KID syndrome mutations. The proteins were all expressed in Xenopus oocytes with levels equal to wild-type Cx26. The G12R, N14K, and D50N mutations resulted in larger hemichannel currents than the wild-type-expressing cells, but the S17F mutation resulted in a complete loss of hemichannel activity. Elevated hemichannel activity correlated with an increased cell death. This result could be reversed through the elevation of calcium (Ca2+) in the extracellular media. Functional gap junctions were only produced by paired N14K cells, which had a similar conductance level to wild type, even though they exhibited a complete loss of voltage sensitivity. This set of data confirms that aberrant hemichannel activity is a common feature of Cx26 mutations associated with KID syndrome, and this may contribute to a loss of cell viability and tissue integrity.
Project description:Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca(2+). This study presents two new possible contributing factors, pH and Zn(2+). Plasma pH levels and micromolar concentrations of Zn(2+) inhibit WT Cx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H(+) flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation by pH or Zn(2+). N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn(2+) responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn(2+). From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to close Cx26 hemichannels via this gating mechanism.
Project description:Connexin hemichannels are regulated by several gating mechanisms, some of which depend critically on the extracellular Ca(2+) concentration ([Ca(2+)]e). It is well established that hemichannel activity is inhibited at normal (?1 mM) [Ca(2+)]e, whereas lowering [Ca(2+)]e to micromolar levels fosters hemichannel opening. Atomic force microscopy imaging shows significant and reversible changes of pore diameter at the extracellular mouth of Cx26 hemichannels exposed to different [Ca(2+)]e, however, the underlying molecular mechanisms are not fully elucidated. Analysis of the crystal structure of connexin 26 (Cx26) gap junction channels, corroborated by molecular dynamics (MD) simulations, suggests that several negatively charged amino acids create a favorable environment for low-affinity Ca(2+) binding within the extracellular vestibule of the Cx26 hemichannel. In particular a highly conserved glutammic acid, found in position 47 in most connexins, is thought to undergo post translational gamma carboxylation (?Glu47), and is thus likely to play an important role in Ca(2+) coordination. ?Glu47 may also form salt bridges with two conserved arginines (Arg75 and Arg184 in Cx26), which are considered important in stabilizing the structure of the extracellular region. Using a combination of quantum chemistry methods, we analyzed the interaction between ?Glu47, Arg75 and Arg184 in a Cx26 hemichannel model both in the absence and in the presence of Ca(2+). We show that Ca(2+) imparts significant local structural changes and speculate that these modifications may alter the structure of the extracellular loops in Cx26, and may thus account for the mechanism of hemichannel closure in the presence of mM [Ca(2+)]e.
Project description:The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.
Project description:Here, we show that human Connexin 26 (hCx26 or Cx26WT) hemichannel opening rapidly enables the transport of small molecules when triggered by temperature and by compensation of the Ca<sup>2+</sup> blockade with EDTA. Point mutations within Cx26 were analysed by a novel optical microarray-based Lucifer Yellow uptake assay or by two electrode voltage clamp (TEVC) on frog oocytes to monitor simultaneous activities of channel proteins. Point mutations L90P, F161S, R184P or K188N influenced the temperature-dependent activity drastically. Since several mutations blocked trafficking, the temperature-dependent activity of the recombinant synthesized and purified wild-type Cx26WT and Cx26K188N hemichannel was tested by liposome flux assay (LFA) and on a microarray-based Lucifer Yellow uptake assay under warm conditions (>30?°C). The data from TEVC measurements and dye flux experiments showed that the mutations gave no or only a weak activity at increased temperature (>30?°C). We conclude that the position K188 in the Cx26WT forms a temperature-sensitive salt bridge with E47 whereas the exchange to K188N destabilizes the network loop- gating filter, which was recently identified as a part of the flexible Ca<sup>2+</sup> binding site. We assume that the temperature sensitivity of Cx26 is required to protect cells from uncontrolled release or uptake activities through Cx26 hemichannels.
Project description:Mutations in GJB2 (connexin [Cx]26) cause either deafness or deafness associated with skin diseases. That different disorders can be caused by distinct mutations within the same gene suggests that unique channel activities are influenced by each class of mutation. We have examined the functional characteristics of two human mutations, Cx26-H73R and Cx26-S183F, causing palmoplantar keratoderma (PPK) and deafness. Both failed to form gap junction channels or hemichannels when expressed alone. Coexpression of the mutants with wild-type Cx43 showed a transdominant inhibition of Cx43 gap junction channels, without reductions in Cx43 protein synthesis. In addition, the presence of mutant Cx26 shifted Cx43 channel gating and kinetics toward a more Cx26-like behavior. Coimmunoprecipitation showed Cx43 being pulled down more efficiently with mutant Cx26 than wild-type, confirming the enhanced formation of heteromeric connexons. Finally, the formation of heteromeric connexons resulted in significantly increased Cx43 hemichannel activity in the presence of Cx26 mutants. These findings suggest a common mechanism whereby Cx26 mutations causing PPK and deafness transdominantly influence multiple functions of wild-type Cx43. They also implicate a role for aberrant hemichannel activity in the pathogenesis of PPK and further highlight an emerging role for Cx43 in genetic skin diseases.
Project description:Mutations in the GJB2 gene, which encodes Cx26, are the most common cause of sensorineural deafness. In syndromic cases, such as keratitis-ichthyosis-deafness (KID) syndrome, in which deafness is accompanied by corneal inflammation and hyperkeratotic skin, aberrant hemichannel function has emerged as the leading contributing factor. We found that D50N, the most frequent mutation associated with KID syndrome, produces multiple aberrant hemichannel properties, including loss of inhibition by extracellular Ca(2+), decreased unitary conductance, increased open hemichannel current rectification and voltage-shifted activation. We demonstrate that D50 is a pore-lining residue and that negative charge at this position strongly influences open hemichannel properties. Examination of two putative intersubunit interactions involving D50 suggested by the Cx26 crystal structure, K61-D50 and Q48-D50, showed no evidence of a K61-D50 interaction in hemichannels. However, our data suggest that Q48 and D50 interact and disruption of this interaction shifts hemichannel activation positive along the voltage axis. Additional shifts in activation by extracellular Ca(2+) remained in the absence of a D50-Q48 interaction but required an Asp or Glu at position 50, suggesting a separate electrostatic mechanism that critically involves this position. In gap junction (GJ) channels, D50 substitutions produced loss of function, whereas K61 substitutions functioned as GJ channels but not as hemichannels. These data demonstrate that D50 exerts effects on Cx26 hemichannel and GJ channel function as a result of its dual role as a pore residue and a component of an intersubunit complex in the extracellular region of the hemichannel. Differences in the effects of substitutions in GJ channels and hemichannels suggest that perturbations in structure occur upon hemichannel docking that significantly impact function. Collectively, these data provide insight into Cx26 structure-function and the underlying bases for the phenotypes associated with KID syndrome patients carrying the D50N mutation.