Wild-type Cx43 Research Articles

Connexin 43-serine 282 modulates serine 279 phosphorylation in cardiomyocytes

Connexin 43 (Cx43) phosphorylation plays a pivotal role in cardiac electrical and contractile performance. In a previous study we have found that Cx43 phosphorylation at serine 282 (pS282) regulates cardiomyocyte survival. Considering that both sites are altered simultaneously in many studies, we designed this study to identify the status of S279 phosphorylation upon pS282 manipulation. In heterozygous mice with S282 gene substituted with alanine (S282A), we found ventricular arrhythmias with inhibition of Cx43 phosphorylation at both S282 and S279 in the hearts. In cultured neonatal rat ventricular myocytes (NRVMs), transfection of virus carrying S282A mutant also blocked Cx43 phosphorylation at both S279/282 and gap junction coupling, while expression of wild-type Cx43 or S279A did not. Further, NRVMs transfected with S282 phospho-mimicking mutant substituted with aspartate or treated with ATP exhibited promotions of Cx43 phosphorylation at S279/282 and intercellular communication. Therefore, this study demonstrated a regulatory role of Cx43-S282 on S279 phosphorylation in cardiomyocytes, and suggested an involvement of S279 in the Cx43-S282 mediated cardiomyocyte homeostasis.

Targeting MAPK phosphorylation of Connexin43 provides neuroprotection in stroke.

Connexin43 (Cx43) function is influenced by kinases that phosphorylate specific serine sites located near its C-terminus. Stroke is a powerful inducer of kinase activity, but its effect on Cx43 is unknown. We investigated the impact of wild-type (WT) and knock-in Cx43 with serine to alanine mutations at the protein kinase C (PKC) site Cx43S368A, the casein kinase 1 (CK1) sites Cx43S325A/328Y/330A, and the mitogen-activated protein kinase (MAPK) sites Cx43S255/262/279/282A (MK4) on a permanent middle cerebral artery occlusion (pMCAO) stroke model. We demonstrate that MK4 transgenic animals exhibit a significant decrease in infarct volume that was associated with improvement in behavioral performance. An increase in astrocyte reactivity with a concomitant decrease in microglial reactivity was observed in MK4 mice. In contrast to WT, MK4 astrocytes displayed reduced Cx43 hemichannel activity. Pharmacological blockade of Cx43 hemichannels with TAT-Gap19 also significantly decreased infarct volume in WT animals. This study provides novel molecular insights and charts new avenues for therapeutic intervention associated with Cx43 function.

PKA negatively modulates the migration enhancing effect of Connexin 43

Connexin 43 (Cx43) expression is associated with an increased cell migration and related changes of the actin cytoskeleton (enhanced filopodia formation). These effects are mediated by the C-terminal cytoplasmic part of Cx43 in a channel-independent manner. Since this part has been shown to interact with a variety of proteins and has multiple phosphorylation sites we analyzed here a potential role of the protein kinase A (PKA) for the Cx43 mediated increase in cell migration. Mutation of the PKA-phosphorylation site (substitution of three serines by alanine or glycine) resulted in a further increase in cell motility compared to wild-type Cx43, but with a loss of directionality. Likewise, cell motility was enhanced by PKA inhibition only in Cx43 expressing cells, while reduced in the presence of the PKA activator forskolin. In contrast, cell motility remained unaffected by stimulation with forskolin in cells expressing Cx43 with the mutated PKA phosphorylation site (Cx43-PKA) as well as in Cx-deficient cells. Moreover, PKA activation resulted in increased binding of PKA and VASP to Cx43 associated with an enhanced phosphorylation of VASP, an important regulatory protein of cell polarity and directed migration. Functionally, we could confirm these results in endothelial cells endogenously expressing Cx43. A Tat-Cx43 peptide containing the PKA phosphorylation site abolished the PKA dependent reduction in endothelial cell migration. Our results indicate that PKA dependent phosphorylation of Cx43 modulates cell motility and plays a pivotal role in regulating directed cell migration.

Connexin43 mutations linked to skin disease have augmented hemichannel activity

Mutations in the gene (GJA1) encoding connexin43 (Cx43) are responsible for several rare genetic disorders, including non-syndromic skin-limited diseases. Here we used two different functional expression systems to characterize three Cx43 mutations linked to palmoplantar keratoderma and congenital alopecia-1, erythrokeratodermia variabilis et progressiva, or inflammatory linear verrucous epidermal nevus. In HeLa cells and Xenopus oocytes, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D all formed functional gap junction channels with the same efficiency as wild-type Cx43, with normal voltage gating and a unitary conductance of ~110 pS. In HeLa cells, all three mutations also localized to regions of cell-cell contact and displayed a punctate staining pattern. In addition, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D significantly increase membrane current flow through formation of active hemichannels, a novel activity that was not displayed by wild-type Cx43. The increased membrane current was inhibited by either 2 mM calcium, or 5 µM gadolinium, mediated by hemichannels with a unitary conductance of ~250 pS, and was not due to elevated mutant protein expression. The three Cx43 mutations all showed the same gain of function activity, suggesting that augmented hemichannel activity could play a role in skin-limited diseases caused by human Cx43 mutations.

Roles of aberrant hemichannel activities due to mutant connexin26 in the pathogenesis of KID syndrome

Germline missense mutations in GJB2 encoding connexin (Cx) 26 have been found in keratitis, ichthyosis and deafness (KID) syndrome. We explored the effects of three mouse Cx26 mutants (Cx26-G12R, -G45E and -D50N) corresponding to KID syndrome-causative human mutants on hemichannel activities leading to cell death and the expression of immune response-associated genes. We analyzed the 3D images of cells expressing wild-type (WT) or mutant Cx26 molecules to demonstrate clearly the intracellular localization of Cx26 mutants and hemichannel formation. High extracellular Ca2+ conditions lead to the closure of gap junction hemichannels in Cx26-G12R or Cx26-G45E expressing cells, resulting in prohibition of the Cx26 mutant-induced cell death. Fluorescent dye uptake assays revealed that cells with Cx26-D50N had aberrantly high hemichannel activities, which were abolished by a hemichannel blocker, carbenoxolone and 18α-Glycyrrhetinic acid. These results further support the idea that abnormal hemichannel activities play important roles in the pathogenesis of KID syndrome. Furthermore, we revealed that the expressions of IL15, CCL5, IL1A, IL23R and TLR5 are down-regulated in keratinocytes expressing Cx26-D50N, suggesting that immune deficiency in KID syndrome expressing Cx26-D50N might be associated not only with skin barrier defects, but also with the down-regulated expression of immune response-related genes.

Alterations at Arg76 of human connexin 46, a residue associated with cataract formation, cause loss of gap junction formation but preserve hemichannel function.

The connexins are members of a family of integral membrane proteins that form gap junction channels between apposed cells and/or hemichannels across the plasma membranes. The importance of the arginine at position 76 (Arg76) in the structure and/or function of connexin 46 (Cx46) is highlighted by its conservation across the entire connexin family and the occurrence of pathogenic mutations at this (or the corresponding homologous) residue in a number of human diseases. Two mutations at Arg76 in Cx46 are associated with cataracts in humans, highlighting the importance of this residue. We examined the expression levels and macroscopic and single-channel properties of human Cx46 and compared them with those for two pathogenic mutants, namely R76H and R76G. To gain further insight into the role of charge at this position, we generated two additional nonnaturally occurring mutants, R76K (charge conserving) and R76E (charge inverting). We found that, when expressed exogenously in Neuro2a cells, all four mutants formed membrane hemichannels, inducing membrane permeability at levels comparable to those recorded in cells expressing the wild-type Cx46. In contrast, the number of gap-junction plaques and the magnitude of junctional coupling were reduced by all four mutations. To gain further insight into the role of Arg76 in the function of Cx46, we performed homology modeling of Cx46 and in silico mutagenesis of Arg76 to Gly, His, or Glu. Our studies suggest that the loss of interprotomeric interactions has a significant effect on the extracellular domain conformation and dynamics, thus affecting the hemichannel docking required for formation of cell-cell channels.

The syndromic deafness mutation G12R impairs fast and slow gating in Cx26 hemichannels.

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.

Functional Characterization of Novel Atrial Fibrillation-Linked GJA5 (Cx40) Mutants.

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia. Recently, four novel heterozygous Cx40 mutations—K107R, L223M, Q236H, and I257L—were identified in 4 of 310 unrelated AF patients and a followup genetic analysis of the mutant carriers’ families showed that the mutants were present in all the affected members. To study possible alterations associated with these Cx40 mutants, including their cellular localization and gap junction (GJ) function, we expressed GFP-tagged and untagged mutants in connexin-deficient model cells. All four Cx40 mutants showed clustered localization at cell–cell junctions similar to that observed of wildtype Cx40. However, cell pairs expressing Cx40 Q236H, but not the other individual mutants, displayed a significantly lower GJ coupling conductance (Gj) than wildtype Cx40. Similarly, co-expression of Cx40 Q236H with Cx43 resulted in a significantly lower Gj. Transjunctional voltage-dependent gating (Vj gating) properties were also altered in the GJs formed by Q236H. Reduced GJ function and altered Vj gating may play a role in promoting the Q236H carriers to AF.

Intrathecal gene therapy in mouse models expressing CMT1X mutations.

Gap junction beta-1 (GJB1) gene mutations affecting the gap junction protein connexin32 (Cx32) cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a common inherited neuropathy. Targeted expression of virally delivered Cx32 in Schwann cells following intrathecal injection of lentiviral vectors in the Cx32 knockout (KO) mouse model of the disease has led to morphological and functional improvement. To examine whether this approach could be effective in CMT1X patients expressing different Cx32 mutants, we treated transgenic Cx32 KO mice expressing the T55I, R75W or N175D CMT1X mutations. All three mutants were localized in the perinuclear compartment of myelinating Schwann cells consistent with retention in the ER (T55I) or Golgi (R75W, N175D) and loss of physiological expression in the non-compact myelin. Following intrathecal delivery of the GJB1 gene we detected the virally delivered wild-type (WT) Cx32 in non-compact myelin of T55I KO mice, but only rarely in N175D KO or R75W KO mice, suggesting dominant-negative effects of the R75W and N175D mutants but not of the T55I mutant on co-expressed WT Cx32. GJB1 treated T55I KO mice showed improved motor performance, lower ratios of abnormally myelinated fibers and reduction of inflammatory cells in spinal roots and peripheral nerves compared with mock-treated littermates. Either partial (N175D KO) or no (R75W KO) improvement was observed in the other two mutant lines. Thus, certain CMT1X mutants may interfere with gene addition therapy for CMT1X. Whereas gene addition can be used for non-interfering CMT1X mutations, further studies will be needed to develop treatments for patients harboring interfering mutations.

Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover.

To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series of phosphomimetic and phosphorylation-dead mutants by mutating known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 located in the C-terminal domain of connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All connexin mutants were translated into stable, full-length proteins and assembled into GJs when expressed in HeLa or Madin-Darby canine kidney epithelial cells. However, mutants with S residues exchanged at positions 365, 368, and 373 exhibited a significantly altered ZO-1 interaction profile, while mutants with S residues exchanged at 255 and 279/282 did not. Unlike wild-type Cx43, in which ZO-1 binding is restricted to the periphery of GJ plaques, S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. Inability to disengage from ZO-1 correlated with increased GJ plaque size and increased connexin protein half-life, while maintaining GJ channels in an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations in clathrin-binding efficiency, suggesting that the inability to disengage from ZO-1 prevented maturation of functional into nonfunctional/endocytic channels, rather than ZO-1 interfering with GJ endocytosis directly. Collectively, our results indicate that ZO-1 binding regulates channel accrual, while disengagement from ZO-1 is critical for GJ channel closure and transitioning GJ channels for endocytosis. Intriguingly, these transitional ZO-1 binding/release and channel-aging steps are mediated by a series of hierarchical phosphorylation/dephosphorylation events at S373, S365, and S368, well-known Cx43 Akt, protein kinase A, and protein kinase C phosphorylation sites located in the vicinity of the ZO-1 binding site.

Induction of cell death and gain-of-function properties of connexin26 mutants predict severity of skin disorders and hearing loss

Connexin26 (Cx26) is a gap junction protein that oligomerizes in the cell to form hexameric transmembrane channels called connexons. Cell surface connexons dock between adjacent cells to allow for gap junctional intercellular communication. Numerous autosomal dominant mutations in the Cx26-encoding GJB2 gene lead to many skin disorders and sensorineural hearing loss. Although some insights have been gained into the pathogenesis of these diseases, it is not fully understood how distinct GJB2 mutations result in hearing loss alone or in skin pathologies with comorbid hearing loss. Here we investigated five autosomal dominant Cx26 mutants (N14K, D50N, N54K, M163V, and S183F) linked to various syndromic or nonsyndromic diseases to uncover the molecular mechanisms underpinning these disease links. We demonstrated that when gap junction-deficient HeLa cells expressed the N14K and D50N mutants, they undergo cell death. The N54K mutant was retained primarily within intracellular compartments and displayed dominant or transdominant properties on wild-type Cx26 and coexpressed Cx30 and Cx43. The S183F mutant formed some gap junction plaques but was largely retained within the cell and exhibited only a mild transdominant reduction in gap junction communication when co-expressed with Cx30. The M163V mutant, which causes only hearing loss, exhibited impaired gap junction function and showed no transdominant interactions. These findings suggest that Cx26 mutants that promote cell death or exert transdominant effects on other connexins in keratinocytes will lead to skin diseases and hearing loss, whereas mutants having reduced channel function but exhibiting no aberrant effects on coexpressed connexins cause only hearing loss. Moreover, cell death-inducing GJB2 mutations lead to more severe syndromic disease.

Golgi-retained Cx32 mutants interfere with gene addition therapy for CMT1X

Numerous GJB1 gene mutations cause the X-linked form of Charcot-Marie-Tooth disease (CMT1X). GJB1 encodes connexin32 (Cx32), which forms trans-myelin gap junctions in Schwann cells. Most GJB1 mutations result in loss-of-function mechanisms, supporting the concept of gene replacement therapy. However, interactions between delivered wild type and endogenously expressed mutant Cx32 may potentially occur in the setting of gene replacement therapy. In order to screen for possible interactions of several representative CMT1X mutants with wild type Cx32 that may interfere with the functional gap junction formation, we established an in vitro screening method co-expressing in HeLa cells wild type Cx32 and one of eight different Cx32 mutants including A39P, A39V, T55I, R75W, M93V, L143P, N175D and R183S. Some of the Golgi-retained mutants hindered gap junction plaque assembly by Cx32 on the cell membrane, while co-immunoprecipitation analysis revealed a partial interaction of wild type protein with Golgi-retained mutants. Dye transfer studies confirmed that Golgi-retained R75W, M93V and N175D but not endoplasmic reticulum-retained T55I had a negative effect on wild type Cx32 function. Finally, in vivo intraneural delivery of the gene encoding the wild type Cx32 in mice bearing either the T55I or R75W mutation on Cx32 knockout background showed that virally delivered protein was correctly localized in mice expressing the endoplasmic reticulum-retained T55I whereas it did not traffic normally in mice expressing the Golgi-retained R75W. Thus, certain Golgi-retained Cx32 mutants may interfere with exogenously delivered Cx32. Screening for mutant-wild type Cx32 interactions should be considered prior to planning gene addition therapy for CMT1X.

Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26.

Revertant mosaicism (RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c.148G > A, p.Asp50Asn. At age 20, the patient developed spots of healthy-looking skin that grew in size and number within widespread erythrokeratodermic lesions. Ultra-deep sequencing of two healthy-looking skin biopsies identified five somatic nonsynonymous mutations, independently present in cis with the p.Asp50Asn mutation. Functional studies of Cx26 in HeLa cells revealed co-expression of Cx26-Asp50Asn and wild-type Cx26 in gap junction channel plaques. However, Cx26-Asp50Asn with the second-site mutations identified in the patient displayed no formation of gap junction channel plaques. We argue that the second-site mutations independently inhibit Cx26-Asp50Asn expression in gap junction channels, reverting the dominant negative effect of the p.Asp50Asn mutation. To our knowledge, this is the first time RM has been reported to result in the development of healthy-looking skin in a patient with KID syndrome.

Loss of Coupling Distinguishes GJB1 Mutations Associated with CNS Manifestations of CMT1X from Those Without CNS Manifestations

CMT1X, an X-linked inherited neuropathy, is caused by mutations in GJB1, which codes for Cx32, a gap junction protein expressed by Schwann cells and oligodendrocytes. Many GJB1 mutations cause central nervous system (CNS) abnormality in males, including stable subclinical signs and, less often, short-duration episodes characterized by motor difficulties and altered consciousness. However, some mutations have no apparent CNS effects. What distinguishes mutations with and without CNS manifestations has been unclear. Here we studied a total of 14 Cx32 mutations, 10 of which are associated with florid episodic CNS clinical syndromes in addition to peripheral neuropathy. The other 4 mutations exhibit neuropathy without clinical or subclinical CNS abnormalities. These “PNS-only” mutations (Y151C, V181M, R183C and L239I) form gap junction plaques and produce levels of junctional coupling similar to those for wild-type Cx32. In contrast, mutants with CNS manifestations (F51L, E102del, V139M, R142Q, R142W, R164W T55I, R164Q and C168Y) either form no morphological gap junction plaques or, if they do, produce little or no detectable junctional coupling. Thus, PNS and CNS abnormalities may involve different aspects of connexin function.

Comparative functional characterization of novel non-syndromic GJB2 gene variant p.Gly45Arg and lethal syndromic variant p.Gly45Glu.

We characterized a novel GJB2 missense variant, c.133G>A, p.Gly45Arg, and compared it with the only other variant at the same amino acid position of the connexin 26 protein (Cx26) reported to date: c.134G>A, p.Gly45Glu. Whereas both variants are associated with hearing loss and are dominantly inherited, p.Gly45Glu has been implicated in the rare fatal keratitis-ichthyosis-deafness (KID) syndrome, which results in cutaneous infections and septicemia with premature demise in the first year of life. In contrast, p.Gly45Arg appears to be non-syndromic. Subcellular localization experiments in transiently co-transfected HeLa cells demonstrated that Cx26-WT (wild-type) and p.Gly45Arg form gap junctions, whereas Cx26-WT with p.Gly45Glu protein does not. The substitution of a nonpolar amino acid glycine in wildtype Cx26 at position 45 with a negatively charged glutamic acid (acidic) has previously been shown to interfere with Ca2+ regulation of hemichannel gating and to inhibit the formation of gap junctions, resulting in cell death. The novel variant p.Gly45Arg, however, changes this glycine to a positively charged arginine (basic), resulting in the formation of dysfunctional gap junctions that selectively affect the permeation of negatively charged inositol 1,4,5-trisphosphate (IP3) and contribute to hearing loss. Cx26 p.Gly45Arg transfected cells, unlike cells transfected with p.Gly45Glu, thrived at physiologic Ca2+ concentrations, suggesting that Ca2+ regulation of hemichannel gating is unaffected in Cx26 p.Gly45Arg transfected cells. Thus, the two oppositely charged amino acids that replace the highly conserved uncharged glycine in p.Gly45Glu and p.Gly45Arg, respectively, produce strikingly different effects on the structure and function of the Cx26 protein.

Syndromic deafness mutations at Asn 14 differentially alter the open stability of Cx26 hemichannels.

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.

Specificity of the connexin W3/4 locus for functional gap junction formation

ABSTRACTThe N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (Vj) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the α- and δ-group connexins possess a G2 residue not found in the β- and γ-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Δ) affected the Vj gating, increased the single channel conductance (γj), and decreased the relative K+/Cl− permeability (PK/PCl) ratio of the Cx40 gap junction channel. The conserved α/β-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in Vj gating and noisy low γj gap junction channels that reduced the γj of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all α/β/δ/γ-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.

Polymorphism of the 86th amino acid in CX26 protein and hereditary deafness

ObjectiveTo investigate the membrane localization function of the CX26 protein when its 86th amino acid is Thr, Ser or Arg, and its relations to deafness. MethodsCX26-GFP protein with either Thr, Ser or Arg as the 86th amino acid was expressed in mouse SGN cells via the GFP fusion type lenti-virus expression system. The membrane localization of the fusion protein was observed under a fluorescence microscope. ResultsThe mutated protein of CX26 T86S was localized to cell membrane and form gap conjunction structures, showing no difference to the wild type CX26 protein (with Thr as the 86th amino acid). However, the gap conjunction structure disappeared when the mutation was CX26 T86A. ConclusionThese results indicate that the CX26 T86R mutation may be a cause of hearing loss, but CX26 T86S as a non-pathogenic polymorphism mutation does not affect functions of the CX26 protein. The results are in accordance with the results of clinical screening.

Molecular Determinants Underlying the Pathogenic Mechanism of Kid Syndrome Elicited by Cx26G12R Mutation

Several mutations in Cx26 promote keratitis ichthyosis deafness (KID), a rare disease in which patients exhibit deafness, skin and corneal disorders. Some KID mutations are located in the amino terminal segment of Cx26, which has been proposed as the putative voltage sensor. Most syndromic mutations elicit a gain of hemichannel function, however the mechanism underlying this phenomenon remains unclear. Here we examined the biophysical properties of the mutant G12R at macroscopic and single channel levels. We perform two-electrode voltage clamp to study the deactivation kinetics and apparent affinity for calcium of hemichannel currents. In contrast to wild-type (wt) Cx26, we found that the activation kinetics of the mutant lack saturation of the current upon depolarization pulses above +40 mV. Estimates of the deactivation time constant yield values of 5s and 10s for G12R and wtCx26 hemichannels, respectively; however the apparent affinity for calcium remains similar to wtCx26. Single channel recordings analysis showed that the mutant virtually eliminates the transitions to the subconductance state. Moreover, although the single channel conductance was similar to wtCx26 hemichannels (320 pS), the open probability and mean open times of G12R hemichannels become larger. Molecular dynamics indicates that the G12R induce an electrostatic effect and ddG calculations support protein destabilization.Supported by Fondecyt-3150634 to IEG; Fondecyt-3140590 to GC; Fondecyt-1130855 to ADM; Anillo-ACT1104 to ADM and CG; Fondecyt-1120802 to CG; Grant RO1-GM099490 to J.E.C; AP has a Conicyt Fellowship. The Centro Interdisciplinario de Neurociencias de Valparaiso is a Chilean Millennium Institute (P09-022-F).

Human Connexin 26 (Cx26) N14K Mutant Alters Hemichannel Calcium and Voltage Sensitivity

Unpaired hemichannels, those not forming gap junction channels, play an autocrine/paracrine role by releasing transmitters, such as glutamate and ATP, to the extracellular medium. Hemichannels are gated by changes in extracellular Ca+2 concentration and membrane voltage. Several human connexin mutations that produce diseases increase hemichannel opening (e.g., gain of function due to decreased sensitivity to extracellular Ca+2). Using Xenopusoocytes and two-electrode voltage clamp techniques, we studied the voltage and calcium sensitivity of a connexin 26 (Cx26) mutation, N14K, located in the N-terminal domain (NT). The NT is thought to fold into the pore and form part of the pore lining; position N14 is approximately at the “hinge” region at which the NT bends toward the pore. We observe a significant leftward shift in the voltage-dependence N14K (midpoint at - 6.7 mV ± 0.7) mutants with respect to wild type channels (midpoint at + 9.6 mV ± 0.9). Rearrangements of the NT are associated with voltage dependent sub-conductances in wild type Cx26 hemichannels, however, N14K mutant channels did not display voltage dependent sub-conductances. The apparent calcium affinity was also significantly reduced in N14K (KD = 0.74 mM) compared to wild type channels (KD = 0.34 mM). Deactivation kinetics in wild type channels show single mono-exponential relaxations that are accelerated as a function of calcium concentration, but not voltage. The Cx26 mutant N14K displays two slower components. One of these components is both Ca+2 and voltage dependent but the other is not. Overall, our data indicate that under physiological conditions the N14K mutation stabilizes the open state relative to wild type channels by reducing both calcium and voltage sensitivity. R01GM099490/R01GM101950.