Correlative studies of gating in Cx46 and Cx50 hemichannels and gap junction channels

Transjunctional voltage (V(j)) gating of gap junction (GJ) channels formed of connexins has been proposed to occur by gating of the component hemichannels. We took advantage of the ability of Cx46 and Cx50 to function as unapposed hemichannels to identify gating properties intrinsic to hemichannels and how they contribute to gating of GJ channels. We show that Cx46 and Cx50 hemichannels contain two distinct gating mechanisms that generate reductions in conductance for both membrane polarities. At positive voltages, gating is similar in Cx46 and Cx50 hemichannels, primarily showing increased transitioning to long-lived substates. At negative voltages, Cx46 currents deactivate completely and the underlying single hemichannels exhibit transitions to a fully closed state. In contrast, Cx50 currents do not deactivate completely at negative voltages and the underlying single hemichannels predominantly exhibit transitions to various substates. Transitions to a fully closed state occur, but are infrequent. In the respective GJ channels, both forms of gating contribute to the reduction in conductance by V(j). However, examination of gating of mutant hemichannels and GJ channels in which the Asp at position 3 was replaced with Asn (D3N) showed that the positive hemichannel gate predominantly closes Cx50 GJs, whereas the negative hemichannel gate predominantly closes Cx46 GJs in response to V(j). We also report, for the first time, single Cx50 hemichannels in oocytes to be inwardly rectifying, high conductance channels (gamma = 470 pS). The antimalarial drug mefloquine, which selectively blocks Cx50 and not Cx46 GJs, shows the same selectivity in Cx50 and Cx46 hemichannels indicating that the actions of such uncoupling agents, like voltage gating, are intrinsic hemichannel properties.     

Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.     

Opening hemichannels in nonjunctional membrane stimulates gap junction formation

We studied gap junction formation in pairs of Xenopus laevis oocytes expressing connexins that form functional hemichannels and found no correlation between junctional conductance (G(j)) and whole-cell hemichannel conductances (G(hemi)) within the first few hours of pairing. However, opening hemichannels to a threshold current stimulated a rapid G(j) increase. Moreover, cx46 hemichannel current stimulated cx40 G(j) even though cx40 and cx46 do not form heteromeric or heterotypic gap junctions. Initial growth rate and final steady-state level of stimulated G(j) were proportional to the product of hemichannel conductances. External calcium affected the growth rate of stimulated G(j) but not the final steady-state value. Time constants of formation were short in low [Ca(2+)](out) (3 min in 200 micro M Ca(2+)) and long in high [Ca(2+)](out) (15 min in 1 mM Ca(2+)), but in oocyte pairs pretreated with lectins to reduce steric hindrance imposed by large membrane glycoproteins the time constant was short and Ca(2+)-independent. We suggest that hemichannel activity stimulates G(j) by collapsing the extracellular volume between membranes to allow the end-to-end binding between hemichannels. These studies suggest the possibility that functional hemichannels could trigger or enhance junctional formation in vivo in response to appropriate stimuli.     

Connexins are mechanosensitive

Connexins form gap junction channels that provide a hydrophilic path between cell interiors. Some connexins, particularly the lens connexins, Cx46 and Cx50 and their orthologs, can form functional hemichannels in nonjunctional membranes. These hemichannels are a nonselective conduit to the extracellular medium and may jeopardize cell survival. The physiological function of hemichannels has remained elusive, but it has been postulated that hemichannels are involved in ATP-release caused by mechanical stimulation. Here we show with single-channel and whole cell electrophysiological studies that Cx46 hemichannels are mechanosensitive, like other families of ion channels and membrane-bound enzymes. The hemichannel response to mechanical stress is bipolar. At negative potentials stress opens the channel, and at positive potentials stress closes it. Physiologically, Cx46 hemichannels may assist accommodation of the ocular lens by providing a transient path for volume flow as the lens changes shape. mechanical stress; lens     

Regulation of connexin hemichannels by monovalent cations

Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+.     

Distribution of Connexin50 Channels and Hemichannels in Lens Fibers: A Structural Approach

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.     

Distribution of Connexin50 Channels and Hemichannels in Lens Fibers: A Structural Approach

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.     

Distribution of connexin50 channels and hemichannels in lens fibers: a structural approach

A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques.     

Molecular mechanisms underlying enhanced hemichannel function of a cataract-associated Cx50 mutant

Connexin-50 (Cx50) is among the most frequently mutated genes associated with congenital cataracts. Although most of these disease-linked variants cause loss of function because of misfolding or aberrant trafficking, others directly alter channel properties. The mechanistic bases for such functional defects are mostly unknown. We investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system. T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Coexpression of mutant T39R with wild-type Cx50 (to mimic the heterozygous state) resulted in hemichannel currents whose properties were indistinguishable from those induced by T39R alone, suggesting that the mutant had a dominant effect. Furthermore, when T39R was coexpressed with Cx46, it produced hemichannels with increased activity, particularly at negative potentials, which could potentially contribute to its pathogenicity in the lens. In contrast, coexpression of wild-type Cx50 with Cx46 was associated with a marked reduction in hemichannel activity, indicating that it may have a protective effect. All-atom molecular dynamics simulations indicate that the R39 substitution can form multiple electrostatic salt-bridge interactions between neighboring subunits that could stabilize the open-state conformation of the N-terminal (NT) domain while also neutralizing the voltage-sensing residue D3 as well as residue E42, which participates in loop gating. Together, these results suggest T39R acts as a dominant gain-of-function mutation that produces leaky hemichannels that may cause cytotoxicity in the lens and lead to development of cataracts.     

Connexin Hemichannels in Astrocytes: An Assessment of Controversies Regarding Their Functional Characteristics

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a ‘docked’ counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.     

Lens connexins alpha3Cx46 and alpha8Cx50 interact with zonula occludens protein-1 (ZO-1)

Connexin alpha1Cx43 has previously been shown to bind to the PDZ domain-containing protein ZO-1. The similarity of the carboxyl termini of this connexin and the lens fiber connexins alpha3Cx46 and alpha8Cx50 suggested that these connexins may also interact with ZO-1. ZO-1 was shown to be highly expressed in mouse lenses. Colocalization of ZO-1 with alpha3Cx46 and alpha8Cx50 connexins in fiber cells was demonstrated by immunofluorescence and by fracture-labeling electron microscopy but showed regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate with alpha3Cx46 and alpha8Cx50, and pull-down experiments showed that the second PDZ domain of ZO-1 was involved in this interaction. Transiently expressed alpha3Cx46 and alpha8Cx50 connexins lacking the COOH-terminal residues did not bind to the second PDZ domain but still formed structures resembling gap junctions by immunofluorescence. These results indicate that ZO-1 interacts with lens fiber connexins alpha3Cx46 and alpha8Cx50 in a manner similar to that previously described for alpha1Cx43. The spatial variation in the interaction of ZO-1 with lens gap junctions is intriguing and is suggestive of multiple dynamic roles for this association.     

Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision

Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H₂S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H₂S.     

4-Hydroxynonenal induces Cx46 hemichannel inhibition through its carbonylation

Hemichannels formed by connexins mediate the exchange of ions and signaling molecules between the cytoplasm and the extracellular milieu. Under physiological conditions hemichannels have a low open probability, but in certain pathologies their open probability increases, which can result in cell damage. Pathological conditions are characterized by the production of a number of proinflammatory molecules, including 4-hydroxynonenal (4-HNE), one of the most common lipid peroxides produced in response to inflammation and oxidative stress. The aim of this work was to evaluate whether 4-HNE modulates the activity of Cx46 hemichannels. We found that 4-HNE (100 μM) reduced the rate of 4′,6-diamino-2-fenilindol (DAPI) uptake through hemichannels formed by recombinant human Cx46 fused to green fluorescent protein, an inhibition that was reversed partially by 10 mM dithiothreitol. Immunoblot analysis showed that the recombinant Cx46 expressed in HeLa cells becomes carbonylated after exposure to 4-HNE, and that 10 mM dithiothreitol reduced its carbonylation. We also found that Cx46 was carbonylated by 4-HNE in the lens of a selenite-induced cataract animal model. The exposure to 100 μM 4-HNE decreased hemichannel currents formed by recombinant rat Cx46 in Xenopus laevis oocytes. This inhibition also occurred in a mutant expressing only the extracellular loop cysteines, suggesting that other Cys are not responsible for the hemichannel inhibition by carbonylation. This work demonstrates for the first time that Cx46 is post-translationally modified by a lipid peroxide and that this modification reduces Cx46 hemichannel activity.     

Structural determinants for the differences in voltage gating of chicken Cx56 and Cx45.6 gap-junctional hemichannels

The voltage- and calcium-dependent gating properties of two lens gap-junctional hemichannels were compared at the macroscopic and single channel level. In solutions containing zero added calcium and 1 mM Mg, chicken Cx56 hemichannels were mostly closed at negative potentials and application of depolarizing voltage clamp steps elicited a slowly activating outward current. In contrast, chicken Cx45.6 hemichannels were predominantly open at negative potentials and rapidly closed in response to application of large depolarizing potentials. Another difference was that macroscopic Cx45.6 currents were much smaller in size than the hemichannel currents induced by oocytes with similar amounts of cRNA for Cx56. The aim of this study was to identify which regions of the connexins were responsible for the differences in voltage-dependent gating and macroscopic current amplitude by constructing a series of chimeric Cx45.6-Cx56 channels. Our results show that two charged amino acids that are specific for the alpha3-group connexins (R9 in the N-terminus and E43 in the first extracellular loop) are important determinants for the difference in voltage-dependent gating between Cx45.6 and Cx56 hemichannels; the first transmembrane-spanning domain, M1, is an important determinant of macroscopic current magnitude; R9 and E43 are also determinants of single channel conductance and rectification.     

Gap junction channels formed by coexpressed connexin40 and connexin43

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.     

Connexin diversity and gap junction regulation by pHi

The molecular mechanisms controlling pH-sensitivity of gap junctions formed of two different connexins are yet to be determined. We used a proton-sensitive fluorophore and electrophysiological techniques to correlate changes in intracellular pH (pHi) with electrical coupling between connexin-expressing Xenopus oocytes. The pH sensitivities of alpha 3 (connexin46), alpha 2 (connexin38), and alpha 1 (connexin43) were studied when these proteins were expressed as: 1) nonjunctional hemichannels (for alpha 3 and alpha 2), 2) homotypic gap junctions, and 3) heterotypic gap junctions. We found that alpha 3 hemichannels are sensitive to changes in pHi within a physiological range (pKa = 7.13 +/- 0.03; Hill coefficient = 3.25 +/- 1.73; n = 8; mean +/- SEM); an even more alkaline pKa was obtained for alpha 2 hemichannels (pKa = 7.50 +/- 0.03; Hill coefficient = 3.22 +/- 0.66; n = 13). The pH sensitivity curves of alpha 2 and alpha 3 homotypic junctions were indistinguishable from those recorded from hemichannels of the same connexin. Based on a comparison of pKa values, both alpha 3 and alpha 2 gap junctions were more pHi-dependent than alpha 1. The pH sensitivity of alpha 2-containing heterotypic junctions could not be predicted from the behavior of the two connexons in the pair. When alpha 2 was paired with alpha 3, the pH sensitivity curve was similar to that obtained from alpha 2 homotypic pairs. Yet, pairing alpha 2 with alpha 1 shifted the curve similar to homotypic alpha 1 channels. Pairing alpha 2 with a less pH sensitive mutant of alpha 1 (M257) yielded the same curve as when alpha 1 was used. However, the pH sensitivity curve of alpha 3/alpha 1 channels was similar to alpha 3/alpha 3, while alpha 3/M257 was indistinguishable from alpha 3/alpha 1. Our results could not be consistently predicted by a probabilistic model of two independent gates in series. The data show that dissimilarities in the pH regulation of gap junctions are due to differences in the primary sequence of connexins. Moreover, we found that pH regulation is an intrinsic property of the hemichannels, but pH sensitivity is modified by the interactions between connexons. These interactions should provide a higher level of functional diversity to gap junctions that are formed by more than one connexin.     

Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.     

Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26

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.     

Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels

Gap junction channels may be comprised of either connexin or pannexin proteins (innexins and pannexins). Membrane topologies of both families are similar, but sequence similarity is lacking. Recently, connexin-like sequences have been identified in mammalian and zebrafish genomes that have only four conserved cysteines in the extracellular domains (Cx23), a feature of the pannexins. Phylogenetic analyses of the non-canonical "C4" connexins reveal that these sequences are indeed connexins. Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro.     

No model in mind: A model-free approach for studying ion channel gating

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 Ca²⁺, 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 Ca²⁺ 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.