Projection structure of a N-terminal deletion mutant of connexin 26 channel with decreased central pore density

Gated gap junction channels are important cellular conduits for establishing and maintaining intercellular communication. The three-dimensional structure of a mutant human connexin 26 (Cx26M34A) by electron cryocrystallography revealed a plug-like density in the channel pore suggesting that physical blockage of the pore may be one mechanism of closure (Oshima et al. 2007, Proc Natl Acad Sci USA 104: 10034-10039). However, it remains to be determined what part of the sequence contributes to the plug. Here, we present the projection structure of an N-terminus deletion of Cx26M34A missing amino acids 2 to 7 (Cx26M34Adel2-7) crystallized in the same two-dimensional crystal form. A 10 A resolution projection map of Cx26M34Adel2-7 revealed that the plug density was dramatically reduced in comparison with that found in full-length Cx26 channel. The difference map between the deletion and full-length Cx26M34A channels strongly suggests that the N-terminus of connexin contributes to the plug for the physical closure of gap junction channels.     

Size selectivity between gap junction channels composed of different connexins

Gap junction channels are traditionally viewed as large, nonspecific pores connecting cells. Recently the diversity in the connexin family has drawn more attention to their permeability characteristics. Several studies have shown that both size and charge contribute to the permeability of gap junctional channels. We have used a graded series of neutral polyethylene glycol probes (PEGs), which eliminate charge contribution completely, to specifically assess the physical exclusion limits of gap junction channels formed by different connexins. Cx 26, 32 and 37 were expressed in paired Xenopus oocytes to form homotypic gap junctional channels. PEG probes were perfused intracellularly into one side of the oocyte pair. A reversible drop in conductance of the gap juctional channels indicated that the probe was small enough to enter the pore and hinder ion flux. Our data suggest that Cx32 channels have a size cut-off between PEG 400 (11.2 A) and PEG 300 (9.6 A) despite their relatively small single channel conductance (approximately 55 pS). Cx26 channels (approximately 130 pS single channel conductance) have a size exclusion limit around PEG 200 (8.0 A), while Cx37 channels show the most restricted size cut-off between PEG 200 (8.0 A) and TriEG (6.8 A), despite having the largest unitary conductance (approximately 300 pS).     

Growth-suppressing activity of the transfected Cx26 on BICR-M1Rk breast cancer cell line

There are accumulating evidences suggesting that connexin (Cx), a gap junction channel-forming protein, acts as a growth suppressor in various cancer cells, and this effect is attributed to the gap junction-mediated intercellular communication (GJIC). In order to characterize the relationship between the growth-arresting activity of Cx26 and its cytoplasmic localizations after expression, we linked a nuclear export signal (NES) sequence to Cx26 cDNA before transfecting into a rat breast cancer cell line. A confocal fluorescent microscopic observation revealed that the insertion of NES minimized the nuclear expression of Cx26, and increased its cytoplasmic expression, including plasma membrane junctions. Total cell counting and BrdUrd-labeling experiments showed that the growth of the breast cancer cells was inhibited by 74% upon transfection of Cx26-NES, whereas only 9% inhibition was observed with only Cx26 cDNA.     

Analysis of Trafficking,Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.     

Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions.

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.     

Growth inhibition by connexin26 expression in cultured rodent tumor cells

The Connexin (Cx) gene family acts as a tumor suppressor. However, it is unclear whether the tumor-suppressing activity acquired by Cx gene transfection is mainly due to the recovery of the gap junction-mediated intercellular communication (GJIC) or to other unknown mechanisms. In order to elucidate the mechanism of the Cx-induced tumor-suppressing activity, we transfected Cx26 cDNA into a rodent mammary tumor cell-line (BICR-M1Rk) in which Cx43 had been normally expressed and a typical pattern of GJIC had been observed. The exogenous Cx26 was mainly localized on the nuclear envelope, whereas most of the endogenous Cx43 resided at the plasma membrane of the transfected BICR-M1Rk. Consistent with the localization of Cx26, GJIC was not increased upon the transfection of Cx26 when it was assessed by a scrape-loading dye transfer technique. A conventional [3H]-thymidine incorporation study showed that the growth rate of the Cx26-transfected cells was significantly decreased (70%), compared to that of the control BICR-M1Rk. Therefore, our results clearly demonstrate that the exogenously expressed Cx26 in the BICR-M1Rk cancer cell-line exerts an anti-proliferate activity in a GJIC-independent manner.     

Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10(-7) M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx)32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes.     

Functional Role of the Carboxyl Terminal Domain of Human Connexin 50 in Gap Junctional Channels

Gap junction channels formed by connexin 50 (Cx50) are critical for maintenance of lens transparency. Because the C-terminus of Cx50 can be cleaved post-translationally, we hypothesized that channels formed by the truncated Cx50 exhibit altered properties or regulation. We used the dual whole-cell patch-clamp technique to investigate the macroscopic and single-channel properties of gap junctional channels formed by wild-type human Cx50 and a truncation mutant (Cx50A294stop) after transfection of N2A cells. Our results show that wild-type Cx50 formed functional gap junctional channels. The macroscopic Gjss-Vj relationship was well described by a Boltzmann equation with A of 0.10, V0 of 43.8 mV and Gjmin of 0.23. The single-channel conductance was 212 +/- 5 pS. Multiple long-lasting substates were observed with conductances ranging between 31 and 80 pS. Wild-type Cx50 gap junctional channels were reversibly blocked when pHi was reduced to 6.3. Truncating the C-terminus at amino acid 294 caused a loss of pHi sensitivity, but there were no significant changes in single-channel current amplitude or Gjss-Vj relationship. These results suggest that the C-terminus of human Cx50 is involved in pHi sensitivity, but has little influence over single-channel conductance, voltage dependence, or gating kinetics.     

Green fluorescent protein changes the conductance of connexin 43 (Cx43) hemichannels reconstituted in planar lipid bilayers

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.     

Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels

The functional diversity of gap junction intercellular channels arising from the large number of connexin isoforms is significantly increased by heterotypic interactions between members of this family. This is particularly evident in the rectifying behavior of Cx26/Cx32 heterotypic channels (. Proc. Natl. Acad. Sci. USA. 88:8410-8414). The channel properties responsible for producing the rectifying current observed for Cx26/Cx32 heterotypic gap junction channels were determined in transfected mouse neuroblastoma 2A (N2A) cells. Transfectants revealed maximum unitary conductances (gamma(j)) of 135 pS for Cx26 and 53 pS for Cx32 homotypic channels in 120 mM KCl. Anionic substitution of glutamate for Cl indicated that Cx26 channels favored cations by 2.6:1, whereas Cx32 channels were relatively nonselective with respect to charge. In Cx26/Cx32 heterotypic cell pairs, the macroscopic fast rectification of the current-voltage relationship was fully explained at the single-channel level by a rectifying gamma(j) that increased by a factor of 2.9 as the transjunctional voltage (V(j)) changed from -100 to +100 mV with the Cx26 cell as the positive pole. A model of electrodiffusion of ions through the gap junction pore based on Nernst-Planck equations for ion concentrations and the Poisson equation for the electrical potential within the junction is developed. Selectivity characteristics are ascribed to each hemichannel based on either pore features (treated as uniform along the length of the hemichannel) or entrance effects unique to each connexin. Both analytical GHK approximations and full numerical solutions predict rectifying characteristics for Cx32/Cx26 heterotypic channels, although not to the full extent seen empirically. The model predicts that asymmetries in the conductance/permeability properties of the hemichannels (also cast as Donnan potentials) will produce either an accumulation or a depletion of ions within the channel, depending on voltage polarity, that will result in rectification.     

Single channel behavior of recombinant beta 2 gap junction connexons reconstituted into planar lipid bilayers.

The beta 2 gap junction protein (Cx26) was expressed in an insect cell line by infection with a baculovirus vector containing the rat beta 2 cDNA. Isolated beta 2 gap junction connexons were reconstituted into planar lipid bilayers. Single channel activity was observed with a unitary conductance of 35-45 pS in 200 mM KCl. Channels with conductance values of 60 pS and 90-110 pS also coexisted with the lower conducting channel suggesting that there are channels with different conductance properties within a population of connexons. Channel activity was observed at voltages of up to 150 mV. Furthermore, the characterization of these channel properties from the beta 2 connexons that were generated by this heterologous expression system has provided the basis for identifying an endogenous beta 2 connexon channel in material reconstituted from native rat liver gap junctions.     

Mouse Cx50, a functional member of the connexin family of gap junction proteins, is the lens fiber protein MP70.

The crystalline lens is an attractive system to study the biology of intercellular communication; however, the identity of the structural components of gap junctions in the lens has been controversial. We have cloned a novel member of the connexin family of gap junction proteins, Cx50, and have shown that it is likely to correspond to the previously described lens fiber protein MP70. The N-terminal amino acid sequence of MP70 closely matches the sequence predicted by the clone. Cx50 mRNA is detected only in the lens, among the 12 organs tested, and this distribution is indistinguishable from that of MP70 protein. A monoclonal antibody directed against MP70 and an anti-Cx50 antibody produced against a synthetic peptide identify the same proteins on western blots and produce identical patterns of immunofluorescence on frozen sections of rodent lens. We also show that expression of Cx50 in paired Xenopus oocytes induces high levels of voltage-dependent conductance. This indicates that Cx50 is a functional member of the connexin family with unique physiological properties. With the cloning of Cx50, all known participants in gap junction formation between various cell types in the lens are available for study and reconstitution in experimental systems.     

Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication

The mechanism by which gap junction proteins, connexins, act as potent tumor suppressors remains poorly understood. In this study human breast tumor cells were found to exhibit diverse gap junction phenotypes including (a) undetectable Cx43 and no intercellular communication (HBL100); (b) low levels of Cx43 and sparse intercellular communication (MDA-MB-231); and (c) significant levels of Cx43 and moderate intercellular communication (Hs578T). Although retroviral delivery of Cx43 and Cx26 cDNAs to MDA-MB-231 cells did not achieve an expected substantial rescue of intercellular communication, overexpression of connexin genes did result in a dramatic suppression of tumor growth when connexin-expressing MDA-MB-231 cells were implanted into the mammary fat pad of nude mice. Subsequent immunolocalization studies on xenograph sections revealed only cytoplasmic stores of Cx43 and no detectable gap junctions. Moreover, DNA array and Western blot analysis demonstrated that overexpression of Cx43 or Cx26 in MDA-MB-231 cells down-regulated fibroblast growth factor receptor-3. Surprisingly, these results suggest that Cx43 and Cx26 induce their tumor-suppressing properties by a mechanism that is independent of significant gap junctional intercellular communication and possibly through the down-regulation of key genes involved in tumor growth. Moreover, our studies show that retroviruses are effective vehicles for delivering connexins to human breast tumor cells, facilitating potential gene therapy applications.     

Cochlear gap junctions coassembled from Cx26 and 30 show faster intercellular Ca2+ signaling than homomeric counterparts

The importance of connexins (Cxs) in cochlear functions has been demonstrated by the finding that mutations in Cx genes cause a large proportion of sensorineural hearing loss cases. However, it is still unclear how Cxs contribute to the cochlear function. Recent data (33) obtained from Cx30 knockout mice showing that a reduction of Cx diversity in assembling gap junctions is sufficient to cause deafness suggest that functional interactions of different subtypes of Cxs may be essential in normal hearing. In this work we show that the two major forms of Cxs (Cx26 and Cx30) in the cochlea have overlapping expression patterns beginning at early embryonic stages. Cx26 and Cx30 were colocalized in most gap junction plaques in the cochlea, and their coassembly was tested by coimmunoprecipitation. To compare functional differences of gap junctions with different molecular configurations, homo- and heteromeric gap junctions composed of Cx26 and/or Cx30 were reconstituted by transfections in human embryonic kidney-293 cells. The ratio imaging technique and fluorescent tracer diffusion assays were used to assess the function of reconstituted gap junctions. Our results revealed that gap junctions with different molecular configurations show differences in biochemical coupling, and that intercellular Ca²⁺ signaling across heteromeric gap junctions consisting of Cx26 and Cx30 was at least twice as fast as their homomerically assembled counterparts. Our data suggest that biochemical permeability and the dynamics of intercellular signaling through gap junction channels, in addition to gap junction-mediated intercellular ionic coupling, may be important factors to consider for studying functional roles of gap junctions in the cochlea. cochlea; coassembly; deafness     

Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances

Several independent lines of investigation indicate that intercellular communication through gap junctions modulates bladder physiology and, moreover, that altered junctional communication may contribute to detrusor overactivity. However, as far as we are aware, there are still no direct recordings of gap junction-mediated intercellular currents between human or rat detrusor myocytes. Northern and Western blots were used to identify connexin expression in frozen human bladder tissue and short-term cultured human detrusor myocytes. Double whole cell patch (DWCP) recording revealed that human detrusor myocyte cell pairs were well coupled with an average junctional conductance of 6.5 ± 4.6 nS (ranging from 0.1 to 15 nS, n = 22 cell pairs). Macroscopic gap junction channel currents in human detrusor myocytes exhibited voltage dependence similar to homotypic connexin43. The normalized transjunctional conductance-voltage (G_j-V_j) relationship was symmetrical and well described by a two-state Boltzmann relation (G_min 0.33, V₀ = 63.6 mV, Z = 0.117 or equal to 2.95 gating charges), suggestive of a bilateral voltage-gated mechanism. In symmetric 165 mM CsCl, the measured single-channel slope conductance was 120 pS for the fully open channel and 26 pS for the major substate. Occasionally, other subconductance states were also observed. The single-channel mean open time declined with increasing V_j, accounting for the V_j-dependent decline of macroscopic junctional current. Qualitatively similar electrophysiological characteristics were observed in DWCP of freshly isolated rat detrusor myocytes. These data confirm and extend previous observations and are consistent with reports in other smooth muscle cells types in which Cx43-mediated intercellular communication has been identified. bladder function; intercellular communication; smooth muscle     

Gap junctions in cultured astrocytes: single-channel currents and characterization of channel-forming protein.

Currents from gap junction channels were recorded from pairs of astrocytes in primary culture using the double whole-cell recording technique. In weakly coupled pairs, single-channel events could be resolved without pharmacological uncoupling treatment. Under these conditions, unitary conductance was 56 +/- 7 pS, and except for multiples of this value, no other level of conductance was observed consistently. To characterize the type of junctional protein constituting astrocyte gap junction channels, immunological and biochemical experiments were carried out on the same material. Specific cDNA probes for three connexins identified in mammals (Cx26, Cx32, and Cx43) showed that only Cx43 mRNA was expressed in cultured astrocytes. The presence of Cx43 protein in cultured astrocytes was demonstrated by immunoblotting, immunofluorescence, and immunogold labeling using anti-peptide antibodies specific to Cx43. These results strongly suggest that gap junctions in astrocytes have a 50-60 pS unitary conductance associated with channels composed of Cx43 protein.     

Slow intercellular Ca(2+) signaling in wild-type and Cx43-null neonatal mouse cardiac myocytes

Focal mechanical stimulation of single neonatal mouse cardiac myocytes in culture induced intercellular Ca(2+) waves that propagated with mean velocities of approximately 14 micrometer/s, reaching approximately 80% of the cells in the field. Deletion of connexin43 (Cx43), the main cardiac gap junction channel protein, did not prevent communication of mechanically induced Ca(2+) waves, although the velocity and number of cells communicated by the Ca(2+) signal were significantly reduced. Similar effects were observed in wild-type cardiac myocytes treated with heptanol, a gap junction channel blocker. Fewer cells were involved in intercellular Ca(2+) signaling in both wild-type and Cx43-null cultures in the presence of suramin, a P(2)-receptor blocker; blockage was more effective in Cx43-null than in wild-type cells. Thus gap junction channels provide the main pathway for communication of slow intercellular Ca(2+) signals in wild-type neonatal mouse cardiac myocytes. Activation of P(2)-receptors induced by ATP release contributes a secondary, extracellular pathway for transmission of Ca(2+) signals. The importance of such ATP-mediated Ca(2+) signaling would be expected to be enhanced under ischemic conditions, when release of ATP is increased and gap junction channels conductance is significantly reduced.     

Analysis of Four Connexin26 Mutant Gap Junctions and Hemichannels Reveals Variations in Hexamer Stability

Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), Cx26T135A in M3 produces a closed channel; 2), Cx26M34A in M1 severely decreases channel activity; 3), Cx26P87L in M2 has been implicated in defective channel gating; and 4), Cx26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP₃ transfer. These mutations do not affect Cx26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, Cx26M34A and Cx26V84L are stable as hexamers or dodecamers, but Cx26T135A and Cx26P87L oligomers are not. This instability is also found in Cx26T135A and Cx26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type Cx26 and Cx26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of Cx26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating.