Modeling and analysis of voltage gating of gap junction channels at a single-channel level.

The advancement of single-channel-level recording via the patch-clamp technique has provided a powerful means of assessing the detailed behaviors of various types of ion channels in native and exogenously expressed cellular environments. However, such recordings of gap junction (GJ) channels are hampered by unique challenges that are related to their unusual intercellular configuration and natural clustering into densely packed plaques. Thus, the methods for reliable cross-correlation of data recorded at macroscopic and single-channel levels are lacking in studies of GJs. To address this issue, we combined our previously published four-state model (4SM) of GJ channel gating by voltage with maximum likelihood estimation (MLE)-based analyses of electrophysiological recordings of GJ channel currents. First, we consider evaluation of single-channel characteristics and the methods for efficient stochastic simulation of single GJ channels from the kinetic scheme described by 4SM using data obtained from macroscopic recordings. We then present an MLE-based methodology for extraction of information about transition rates for GJ channels and, ultimately, gating parameters defined in 4SM from recordings with visible unitary events. The validity of the proposed methodology is illustrated using stochastic simulations of single GJ channels and is extended to electrophysiological data recorded in cells expressing connexin 43 tagged with enhanced green fluorescent protein.

Four-State Model for Simulating Kinetic and Steady-State Voltage-Dependent Gating of Gap Junctions.

Gap junction (GJ) channels, formed of connexin (Cx) proteins, provide a direct pathway for metabolic and electrical cell-to-cell communication. These specialized channels are not just passive conduits for the passage of ions and metabolites but have been shown to gate robustly in response to transjunctional voltage, Vj, the voltage difference between two coupled cells. Voltage gating of GJs could play a physiological role, particularly in excitable cells, which can generate large transients in membrane potential during the propagation of action potentials. We present a mathematical/computational model of GJ channel voltage gating to assess properties of GJ channels that takes into account contingent gating of two series hemichannels and the distribution of Vj across each hemichannel. From electrophysiological recordings in cell cultures expressing Cx43 or Cx45, the principal isoforms expressed in cardiac tissue, various data sets were fitted simultaneously using global optimization. The results showed that the model is capable of describing both steady-state and kinetic properties of homotypic and heterotypic GJ channels composed of these Cxs. Moreover, mathematical analyses showed that the model can be simplified to a reversible two-state system and solved analytically using a rapid equilibrium assumption. Given that excitable cells are arranged in interconnected networks, the equilibrium assumption allows for a substantial reduction in computation time, which is useful in simulations of large clusters of coupled cells. Overall, this model can serve as a tool for the studying of GJ channel gating and its effects on the spread of excitation in networks of electrically coupled cells.

Human diseases associated with connexin mutations.

Gap junctions and hemichannels comprised of connexins impact many cellular processes. Significant advances in our understanding of the functional role of these channels have been made by the identification of a host of genetic diseases caused by connexin mutations. Prominent features of connexin disorders are the inability of other connexins expressed in the same cell type to compensate for the mutated one, and the ability of connexin mutants to dominantly influence the activity of other wild-type connexins. Functional studies have begun to identify some of the underlying mechanisms whereby connexin channel mutation contributes to the disease state. Detailed mechanistic understanding of these functional differences will help to facilitate new pathophysiology driven therapies for the diverse array of connexin genetic disorders. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome.

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.

An Ala/Glu difference in E1 of Cx26 and Cx30 contributes to their differential anionic permeabilities.

Two closely related connexins, Cx26 and Cx30, share widespread expression in the cochlear cellular networks. Gap junction channels formed by these connexins have been shown to have different permeability profiles, with Cx30 showing a strongly reduced preference for anionic tracers. The pore-forming segment of the first extracellular loop, E1, identified by computational studies of the Cx26 crystal structure to form a parahelix and a narrowed region of the pore, differs at a single residue at position 49. Cx26 contains an Ala and Cx30, a charged Glu at this position, and cysteine scanning in hemichannels identified this position to be pore-lining. To assess whether the Ala/Glu difference affects permeability, we modeled and quantified Lucifer Yellow transfer between HeLa cell pairs expressing WT Cx26 and Cx30 and variants that reciprocally substituted Glu and Ala at position 49. Cx26(A49E) and Cx30(E49A) substitutions essentially reversed the Lucifer Yellow permeability profile when accounting for junctional conductance. Moreover, by using a calcein efflux assay in single cells, we observed a similar reduced anionic preference in undocked Cx30 hemichannels and a reversal with reciprocal Ala/Glu substitutions. Thus, our data indicate that Cx26 and Cx30 gap junction channels and undocked hemichannels retain similar permeability characteristics and that a single residue difference in their E1 domains can largely account for their differential permeabilities to anionic tracers. The higher anionic permeability of Cx26 compared with Cx30 suggests that these connexins may serve distinct signaling functions in the cochlea, perhaps reflected in the vastly higher prevalence of Cx26 mutations in human deafness.

A pore locus in the E1 domain differentially regulates Cx26 and Cx30 hemichannel function.

Connexins (Cxs) function as gap junction (GJ) channels and hemichannels that mediate intercellular and transmembrane signaling, respectively. Here, we investigated the proximal segment of the first extracellular loop, E1, of two closely related Cxs, Cx26 and Cx30, that share widespread expression in the cochlea. Computational studies of Cx26 proposed that this segment of E1 contains a parahelix and functions in gating. The sequence of the parahelix is identical between Cx26 and Cx30 except for an Ala/Glu difference at position 49. We show through cysteine-scanning and mutational analyses that position 49 is pore-lining and interacts with the adjacent Asp50 residue to impact hemichannel functionality. When both positions 49 and 50 are charged, as occurs naturally in Cx30, the hemichannel function is dampened. Co-expression of Cx30 with Cx26(D50N), the most common mutation associated with keratitis-ichthyosis-deafness syndrome, results in robust hemichannel currents indicating that position 49-50 interactions are relevant in heteromerically assembled hemichannels. Cysteine substitution at position 49 in either Cx26 or Cx30 results in tonic inhibition of hemichannels, both through disulfide formation and high-affinity metal coordination, suggestive of a flexible region of the pore that can narrow substantially. These effects are absent in GJ channels, which exhibit wild-type functionality. Examination of postnatal cochlear explants suggests that Cx30 expression is associated with reduced propagation of Ca2+ waves. Overall, these data identify a pore locus in E1 of Cx26 and Cx30 that impacts hemichannel functionality and provide new considerations for understanding the roles of these connexins in cochlear function.

The N-terminal half of the connexin protein contains the core elements of the pore and voltage gates.

Connexins form channels with large aqueous pores that mediate fluxes of inorganic ions and biological signaling molecules. Studies aimed at identifying the connexin pore now include a crystal structure that provides details of putative pore-lining residues that need to be verified using independent biophysical approaches. Here we extended our initial cysteine-scanning studies of the TM1/E1 region of Cx46 hemichannels to include TM2 and TM3 transmembrane segments. No evidence of reactivity was observed in either TM2 or TM3 probed with small or large thiol-modifying reagents. Several identified pore residues in E1 of Cx46 have been verified in different Cx isoforms. Use of variety of thiol reagents indicates that the connexin hemichannel pore is large and flexible enough, at least in the extracellular part of the pore funnel, to accommodate uncommonly large side chains. We also find that that gating characteristics are largely determined by the same domains that constitute the pore. These data indicate that biophysical and structural studies are converging towards a view that the N-terminal half of the Cx protein contains the principal components of the pore and gating elements, with NT, TM1 and E1 forming the pore funnel.

The Amino Terminal Domain and Modulation of Connexin36 Gap Junction Channels by Intracellular Magnesium Ions.

Electrical synapses between neurons in the mammalian CNS are predominantly formed of the connexin36 (Cx36) gap junction (GJ) channel protein. Unique among GJs formed of a number of other members of the Cx gene family, Cx36 GJs possess a high sensitivity to intracellular Mg2+ that can robustly act to modulate the strength of electrical synaptic transmission. Although a putative Mg2+ binding site was previously identified to reside in the aqueous pore in the first extracellular (E1) loop domain, the involvement of the N-terminal (NT) domain in the atypical response of Cx36 GJs to pH was shown to depend on intracellular levels of Mg2+. In this study, we examined the impact of amino acid substitutions in the NT domain on Mg2+ modulation of Cx36 GJs, focusing on positions predicted to line the pore funnel, which constitutes the cytoplasmic entrance of the channel pore. We find that charge substitutions at the 8th, 13th, and 18th positions had pronounced effects on Mg2+ sensitivity, particularly at position 13 at which an A13K substitution completely abolished sensitivity to Mg2+. To assess potential mechanisms of Mg2+ action, we constructed and tested a series of mathematical models that took into account gating of the component hemichannels in a Cx36 GJ channel as well as Mg2+ binding to each hemichannel in open and/or closed states. Simultaneous model fitting of measurements of junctional conductance, gj, and transjunctional Mg2+ fluxes using a fluorescent Mg2+ indicator suggested that the most viable mechanism for Cx36 regulation by Mg2+ entails the binding of Mg2+ to and subsequent stabilization of the closed state in each hemichannel. Reduced permeability to Mg2+ was also evident, particularly for the A13K substitution, but homology modeling of all charge-substituted NT variants showed only a moderate correlation between a reduction in the negative electrostatic potential and a reduction in the permeability to Mg2+ ions. Given the reported role of the E1 domain in Mg2+ binding together with the impact of NT substitutions on gating and the apparent state-dependence of Mg2+ binding, this study suggests that the NT domain can be an integral part of Mg2+ modulation of Cx36 GJs likely through the coupling of conformational changes between NT and E1 domains.

Metabolic inhibition increases activity of connexin-32 hemichannels permeable to Ca2+ in transfected HeLa cells.

Numerous cell types express functional connexin (Cx) hemichannels (HCs), and membrane depolarization and/or exposure to a divalent cation-free bathing solution (DCFS) have been shown to promote HC opening. However, little is known about conditions that can promote HC opening in the absence of strong depolarization and when extracellular divalent cation concentrations remain at physiological levels. Here the effects of metabolic inhibition (MI), an in vitro model of ischemia, on the activity of mouse Cx32 HCs were examined. In HeLa cells stably transfected with mouse Cx32 (HeLa-Cx32), MI induced an increase in cellular permeability to ethidium (Etd). The increase in Etd uptake was directly related to an increase in levels of Cx32 HCs present at the cell surface. Moreover, MI increased membrane currents in HeLa-Cx32 cells. Underlying these currents were channels exhibiting a unitary conductance of approximately 90 pS, consistent with Cx32 HCs. These currents and Etd uptake were blocked by HC inhibitors. The increase in Cx32 HC activity was preceded by a rapid reduction in mitochondrial membrane potential and a rise in free intracellular Ca(2+) concentration ([Ca(2+)](i)). The increase in free [Ca(2+)](i) was prevented by HC blockade or exposure to extracellular DCFS and was virtually absent in parental HeLa cells. Moreover, inhibition of Cx32 HCs expressed by HeLa cells in low-confluence cultures drastically reduced cell death induced by oxygen-glucose deprivation, which is a more physiological model of ischemia-reperfusion. Thus HC blockade could reduce the increase in free [Ca(2+)](i) and cell death induced by ischemia-like conditions in cells expressing Cx32 HCs.

Connexin hemichannels and cochlear function.

Connexins play vital roles in hearing, including promoting cochlear development and sustaining auditory function in the mature cochlea. Mutations in connexins expressed in the cochlear epithelium, Cx26 and Cx30, cause sensorineural deafness and in the case of Cx26, is one of the most common causes of non-syndromic, hereditary deafness. Connexins function as gap junction channels and as hemichannels, which mediate intercellular and transmembrane signaling, respectively. Both channel configurations can play important, but very different roles in the cochlea. The potential roles connexin hemichannels can play are discussed both in normal cochlear function and in promoting pathogenesis that can lead to hearing loss.

Charges dispersed over the permeation pathway determine the charge selectivity and conductance of a Cx32 chimeric hemichannel.

Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexin, Cx32*43E1, which forms unapposed hemichannels in Xenopus oocytes, can result in a threefold difference in unitary conductance and alter the direction and amount of open channel current rectification. Here, we determine the charge selectivity of Cx32*43E1 unapposed hemichannels containing negative and/or positive charge substitutions at the 2nd, 5th and 8th positions in the N-terminus. Unlike Cx32 intercellular channels, which are weakly anion selective, the Cx32*43E1 unapposed hemichannel is moderately cation selective. Cation selectivity is maximal when the extracellular surface of the channel is exposed to low ionic strength solutions implicating a region of negative charge in the first extracellular loop of Cx43 (Cx43E1) in influencing charge selectivity analogous to that reported. Negative charge substitutions at the 2nd, 5th and 8th positions in the intracellular N-terminus substantially increase the unitary conductance and cation selectivity of the chimeric hemichannel. Positive charge substitutions at the 5th position decrease unitary conductance and produce a non-selective channel while the presence of a positive charge at the 5th position and negative charge at the 2nd results in a channel with conductance similar to the parental channel but with greater preference for cations. We demonstrate that a cysteine substitution of the 8th residue in the N-terminus can be modified by a methanthiosulphonate reagent (MTSEA-biotin-X) indicating that this residue lines the aqueous pore at the intracellular entrance of the channel. The results indicate that charge selectivity of the Cx32*43E1 hemichannel can be determined by the combined actions of charges dispersed over the permeation pathway rather than by a defined region that acts as a charge selectivity filter.

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+.

Functional properties of mouse connexin30.2 expressed in the conduction system of the heart.

Gap junction channels composed of connexin (Cx) 40, Cx43, and Cx45 proteins are known to be necessary for impulse propagation through the heart. Here, we report mouse connexin30.2 (mCx30.2) to be a new cardiac connexin that is expressed mainly in the conduction system of the heart. Antibodies raised to the cytoplasmic loop or the C-terminal regions of mCx30.2 recognized this protein in mouse heart as well as in HeLa cells transfected with wild-type mCx30.2 or mCx30.2 fused with enhanced green fluorescent protein (mCx30.2-EGFP). Immunofluorescence analyses of adult hearts yielded positive signals within the sinoatrial node, atrioventricular node, and A-V bundle of the cardiac conduction system. Dye transfer studies demonstrated that mCx30.2 and mCx30.2-EGFP channels discriminate poorly on the basis of charge, but do not allow permeation of tracers >400 Da. Both mCx30.2 and mCx30.2-EGFP gap junctional channels exhibited weak sensitivity to transjunctional voltage (Vj) and a single channel conductance of approximately 9 pS, which is the lowest among all members of the connexin family measured in HeLa cell transfectants. HeLa mCx30.2-EGFP transfectants when paired with cells expressing Cx40, Cx43, or Cx45 formed functional heterotypic gap junction channels that exhibited low unitary conductances (15 to 18 pS), rectifying open channel I-V relations and asymmetric Vj dependence. The electrical properties of homo- and hetero-typic junctions involving mCx30.2 may contribute to slow propagation velocity in nodal tissues and directional asymmetry of excitation spread in the AV nodal region.

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.

Pathogenesis of X-linked Charcot-Marie-Tooth disease: differential effects of two mutations in connexin 32.

X-linked Charcot-Marie-Tooth disease is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding connexin 32 (Cx32). Cx32 is expressed at the paranodes and Schmidt-Lantermann incisures of myelinating Schwann cells in which it is believed to form a reflexive pathway between the abaxonal and adaxonal cytoplasmic domains. Patients with the Val181Ala (V181A) mutation have a severe peripheral neuropathy. Experiments using a nude mouse xenograft system show that Schwann cells expressing only this mutant form of Cx32 are profoundly impaired in their ability to support the earliest stages of regeneration of myelinated fibers. Coupling between paired Xenopus oocytes expressing V181A is reduced compared with the coupling between oocytes expressing wild-type human Cx32 (32WT), and protein levels assayed by Western blot are substantially lower. Immunocytochemisty shows that Neuro2a cells expressing the V181A mutant have very few gap junction plaques compared with cells expressing 32WT; Cx32 protein levels are lower in these cells than in those expressing 32WT. Because failure of normal regeneration is evident before formation of myelin, loss of function of Cx32 may impact on the function of precursors of the myelinating Schwann cell before the formation of the hypothesized reflexive pathway. The Glu102Gly (E102G) mutation leads to a milder phenotype. Early regeneration is normal in grafts with Schwann cells expressing the E102G mutant. The only abnormality detected in the behavior of its channel is increased sensitivity to acidification-induced closure, a property that may lead to reduced gap junction coupling during periods of metabolic stress. This restricted functional abnormality may explain the relatively mild phenotype seen in the xenograft model and in E102G patients.

Gap junction channel gating.

Over the last two decades, the view of gap junction (GJ) channel gating has changed from one with GJs having a single transjunctional voltage-sensitive (V(j)-sensitive) gating mechanism to one with each hemichannel of a formed GJ channel, as well as unapposed hemichannels, containing two, molecularly distinct gating mechanisms. These mechanisms are termed fast gating and slow or 'loop' gating. It appears that the fast gating mechanism is solely sensitive to V(j) and induces fast gating transitions between the open state and a particular substate, termed the residual conductance state. The slow gating mechanism is also sensitive to V(j), but there is evidence that this gate may mediate gating by transmembrane voltage (V(m)), intracellular Ca(2+) and pH, chemical uncouplers and GJ channel opening during de novo channel formation. A distinguishing feature of the slow gate is that the gating transitions appear to be slow, consisting of a series of transient substates en route to opening and closing. Published reports suggest that both sensorial and gating elements of the fast gating mechanism are formed by transmembrane and cytoplamic components of connexins among which the N terminus is most essential and which determines gating polarity. We propose that the gating element of the slow gating mechanism is located closer to the central region of the channel pore and serves as a 'common' gate linked to several sensing elements that are responsive to different factors and located in different regions of the channel.

Conductance and permeability of the residual state of connexin43 gap junction channels.

We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell-cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (V(j)) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher V(j)s that are negative on the side of gated hemichannel. The degree of rectification increases when Cl(-) is replaced by Asp(-) and decreases when K(+) is replaced by TEA(+). These data are consistent with an increased anionic selectivity of the residual state. The V(j)-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast V(j)-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.

Connexin-GFPs shed light on regulation of cell-cell communication by gap junctions.

Important roles for connexins have emerged from studies linking connexin mutations to human disease. Use of connexins tagged with GFP have provided a clearer picture of the mechanisms that govern connexin channel function and it is now evident that functional forms of connexin channel include cell-cell channels and unapposed hemichannels. Clustering appears to be a requirement for opening of cell-cell channels and suggests that dynamic changes occur in plaques (clusters) as they form and grow that are critical for channel function. In particular, recruitment or generation of 'silent' channels has gained support as a mechanism by which coupling can be dynamically regulated within formed plaques. Two distinct voltage sensitive gating mechanisms appear to be built-into each hemichannel, one putatively located at the cytoplasmic entrance and the other at the extracellular end, each differing in sensitivity, kinetics and degree of channel/hemichannel closure. The extracellular gate may also be that which opens unapposed hemichannels in the plasma membrane and be the final target of many known chemical agents that act as uncouplers of cell-cell communication. An understanding of the structural requirements for regulation via gating and clustering represents an important preclinical step in the design of therapeutic agents to treat disorders arising from connexin channel and hemichannel dysfunction.

Connexins and apoptotic transformation.

We examined the influence of connexin (Cx) expression on the development of apoptosis in HeLa parental cells (coupling deficient cell line) and HeLa cells expressing wild-type Cx43 and Cxs fused with enhanced green fluorescent protein (EGFP). EGFP was attached to the C-terminus of Cx32 and Cx43, Cx32-EGFP and Cx43-EGFP, respectively, and to the N-terminus of Cx32, EGFP-Cx32. All fusion proteins assembled into junctional plaques (JPs) at areas of cell-cell contact, but only the C-terminal fusion proteins formed functional gap junction (GJ) channels as well as hemichannels. In each cell line, apoptosis was induced by treatment with various agents including anisomycin, camptothecin, cis-platinum, colchicine, cycloheximide, etoposide, staurosporin and taxol. Using fluorescence microscopy, time-lapse imaging and dual whole-cell voltage clamp techniques, we correlated the changes in functional properties of GJ channels and Cx distribution with the progression of apoptosis based on cells' labeling with acridine orange and ethidium bromide (EB). The early phase of apoptosis (a viable apoptotic (VA) state) was characterized by shrinkage of the cells and by increased internalization of JPs accompanied by decreased cell-cell coupling. The apoptotic reagents had no direct effect on electrical cell-cell coupling. Transformation from a VA to a nonviable apoptotic (NVA) state was faster in HeLa cells expressing Cx43 or Cx43-EGFP than in HeLa parental cells. The potent GJ uncoupler, octanol, slowed the transition of HelaCx43-EGFP cells into a NVA state. In the absence of apoptotic reagents, the rate of EB uptake was higher in HeLaCx43-EGFP than in HeLa parental cells consistent with the presence of open Cx43-EGFP hemichannels. However, in both cell lines the rate of EB uptake decreased proportionally during the development of apoptosis suggesting that membrane permeability ascribed to Cx hemichannels is reduced. Cells expressing Cx32-EGFP and EGFP-Cx32 demonstrate the same apoptotic patterns as HeLaCx43-EGFP and HeLa parental cells, respectively. Intracellular levels of ATP in HeLaCx43-EGFP cells were substantially lower than in HeLa parental cells, and ATP added to the medium abolished the accelerated transition from a VA to a NVA state in HeLaCx43-EGFP cells. In summary, Cx32 or Cx43 accelerates transformation of cells into a NVA state or secondary necrosis and this depends on the ability of Cxs to form functional GJ channels and hemichannels.

Aberrant Cx26 hemichannels and keratitis-ichthyosis-deafness syndrome: insights into syndromic hearing loss.

Mutation of the GJB2 gene, which encodes the connexin 26 (Cx26) gap junction (GJ) protein, is the most common cause of hereditary, sensorineural hearing loss. Cx26 is not expressed in hair cells, but is widely expressed throughout the non-sensory epithelial cells of the cochlea. Most GJB2 mutations produce non-syndromic deafness, but a subset produces syndromic deafness in which profound hearing loss is accompanied by a diverse array of infectious and neoplastic cutaneous disorders that can be fatal. Although GJ channels, which are assembled by the docking of two, so-called hemichannels (HCs), have been the main focus of deafness-associated disease models, it is now evident that the HCs themselves can function in the absence of docking and contribute to signaling across the cell membrane as a novel class of ion channel. A notable feature of syndromic deafness mutants is that the HCs exhibit aberrant behaviors providing a plausible basis for disease that is associated with excessive or altered contributions of Cx26 HCs that, in turn, lead to compromised cell integrity. Here we discuss some of the aberrant Cx26 HC properties that have been described for mutants associated with keratitis-ichthyosis-deafness (KID) syndrome, a particularly severe Cx26-associated syndrome, which shed light on genotype-phenotype relationships and causes underlying cochlear dysfunction.