The Effects of a Mutant Connexin 26 on Epidermal Differentiation

To elucidate the mode of action of dominant mutant connexins in causing inherited skin diseases, transgenic mice were produced that express the true Vohwinkel syndrome-associated mutant Cx26 (D66H), from a keratin 10 promoter, specifically in the suprabasal epidermal keratinocytes. Following birth, the transgenic mice developed keratoderma similar to that of human carriers of Cx26 (D66H). Expression of the transgene resulted in a loss of Cx26 and Cx30 at intercellular junctions of epidermal keratinocytes and accumulation of these connexins in the cytoplasm. Injection of primary mouse keratinocytes with Lucifer Yellow showed no difference in terms of dye spreading between transgenic and non transgenic keratinocytes in vitro. Expression of the mutant Cx26 (D66H) did not interfere with the formation of the epidermal water barrier during late embryonic development. Attempts to produce transgenic mice expressing the wild type form of Cx26 from the K10 promoter failed to produce viable animals although transgenic embryos were recovered at days 9 and 12 of gestation, suggesting that the transgene might be embryonic lethal.     

Connexin 43在细胞死亡中的作用

缝隙连接蛋白在细胞膜表面聚合形成半通道,部分两两结合构成缝隙连接通道,两者与胚胎发育、肿瘤发生及某些心脑血管疾病有关。Connexin 43(Cx43)在心肌细胞和神经细胞高表达,在多种缺血性心脑疾病及缺血再灌注损伤的病理过程中具有重要作用。近来有研究发现,Cx43也存在于线粒体和细胞核,分别参与心肌保护和细胞分化。该文以心肌细胞和神经细胞为主讨论近年来Cx43在细胞死亡中的作用的研究进展。     

Cx26基因重表达抑制人鼻咽癌细胞系HNE1的恶性增殖

间隙连接蛋白 (Cx)基因在胚胎发育、细胞生长、分化以及细胞内环境的稳定过程中起重要调节作用 .肿瘤发生与Cx基因的表达及功能异常密切相关 ,肿瘤细胞常存在Cx基因表达下调或缺失 .将人Cx2 6基因编码区cDNA序列 ,亚克隆于真核表达载体pcDNA3 1(+) ,采用脂质体转染 ,将重组表达载体pcDNA3 1(+) Cx2 6转入鼻咽癌细胞系HNE1,使Cx2 6基因在HNE1中重表达 ,探讨Cx2 6基因对鼻咽癌细胞系HNE1的生物学功能的影响 .研究结果表明 :Cx2 6基因的重表达 ,抑制HNE1细胞生长 ,细胞周期阻滞于G0 G1期 ,HNE1细胞的克隆形成能力下降 ,裸鼠致瘤能力减弱 .     

Connexin 32 Fused to the Green Fluorescent Protein Retains its Ability to Control the Proliferation of Thyroid Cells

Cell-to-cell exchanges of signaling molecules are thought to be involved in the control of cell proliferation. Connexins, which are encoded by a family of genes expressed in a cell type-specific manner, are considered as tumor suppressors. Thyroid epithelial cells co-express connexin 32 (Cx32) and connexin 43 (Cx43) that form distinct and delocalized gap junctions in vivo. The communication-deficient rat thyroid-derived cell lines, FRTL-5 and FRT, stably transfected with the Cx32 cDNA, have a reduced proliferation rate related to a prolonged G1 cell cycle phase. To determine whether Cx32-gap junctions exert the same regulatory role in vivo, we have undertaken a program of production of transgenic mice over-expressing Cx32 specifically in thyrocytes. To this purpose, we designed a vector in which the Cx32 cDNA was fused to the gene encoding the enhanced green fluorescent protein (EGFP) and placed under the control of a strong and thyroid-specific promoter, the thyroglobulin gene promoter (pTg). In stably transfected FRTL-5 cells, the Cx32/EGFP chimeric protein forms functional gap junction channels and induces the same proliferation slowdown as native Cx32. The pTg-Cx32/EGFP construct should thus allow us to obtain the thyroid-targeted over-expression of Cx32 in the mouse to investigate the involvement of Cx32-gap junctions in thyroid growth, functional activity and propensity to form tumors.     

Selective Effect of PDGF on Connexin43 versus Connexin40 Comprised Gap Junction Channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g_j and γ_j, respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g_j in Cx40/Cx40 pairs, but decreased g_j in the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g_j suggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_j involved a decrease in both γ_j and Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

Selective Effect of PDGF on Connexin43 versus Connexin40 Comprised Gap Junction Channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g_jand γ_j, respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g_jin Cx40/Cx40 pairs, but decreased g_jin the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g_jsuggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_jinvolved a decrease in both γ_jand Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

Conductive and Kinetic Properties of Connexin45 Hemichannels Expressed in Transfected HeLa Cells

Human HeLa cells transfected with mouse connexin Cx45 were used to examine the conductive and kinetic properties of Cx45 hemichannels. The experiments were carried out on single cells using a voltage-clamp method. Lowering the [Ca²⁺]_o revealed an extra current. Its sensitivity to extracellular Ca²⁺ and gap junction channel blockers (18α-glycyrrhetinic acid, palmitoleic acid, heptanol), and its absence in non-transfected HeLa cells suggested that it is carried by Cx45 hemichannels. The conductive and kinetic properties of this current, I _hc, were determined adopting a biphasic pulse protocol. I _hc activated at positive V _m and deactivated partially at negative V _m. The analysis of the instantaneous I _hc yielded a linear function g _hc,inst = f(V _m) with a hint of a negative slope (g _hc,inst: instantaneous conductance). The analysis of the steady-state I _hc revealed a sigmoidal function g _hc,ss = f(V _m) best described with the Boltzmann equation: V _m,0 = −1.08 mV, g _hc,min = 0.08 (g _hc,ss: steady-state conductance; V _{m, 0}:V _m at which g _hc,ss is half-maximally activated; g _hc,min: minimal conductance; major charge carriers: K⁺ and Cl⁻). The g _hc was minimal at negative V _m and maximal at positive V _m. This suggests that Cx45 connexons integrated in gap junction channels are gating with negative voltage. I _hc deactivated exponentially with time, giving rise to single time constants, τ_d. The function τ_d = f(V _m) was exponential and increased with positive V _m (τ_d = 7.6 s at V _m = 0 mV). The activation of I _hc followed the sum of two exponentials giving rise to the time constants, τ_a1 and τ_a2. The function τ_a1 = f(V _m) and τ_a2 = f(V _m) were bell-shaped and yielded a maximum of ≅ 0.6 s at V _m ≅ −20 mV and ≅ 4.9 s at V _m ≅ 15 mV, respectively. Neither τ_a1 = f(V _m) nor τ_a2 = f(V _m) coincided with τ_d = f(V _m). These findings conflict with the notion that activation and deactivation follow a simple reversible reaction scheme governed by first-order voltage-dependent processes.     

An Update on Connexin Genes and their Nomenclature in Mouse and Man

Gap junctions, composed of connexin protein subunits, allow direct communication through conduits between neighboring cells. Twenty and twenty-one members of the connexin gene family are likely to be expressed in the mouse and human genome, respectively, 19 of which can be grouped into sequence-orthologous pairs. Their gene structure appears to be relatively simple. In most cases, an untranslated exon1 is separated by an intron of different lengh from exon2 that includes the uninterrupted coding region and the 3'-untranslated region. However, there are several exceptions to this scheme, since some mouse connexin genes contain different 5'-untranslated regions spliced either in an alternative and/or consecutive manner. Additionally, in at least 3 mouse and human connexin genes (mCx36, mCx39, mCx57 and hCx31.3, hCx36, as well as hCx40.1) the reading frame is spliced together from 2 different exons. So far, there are two nomenclatures to classify the known connexin genes: The “Gja/Gjb” nomenclature, as it is currently adopted by the NCBI data base, contains some inconsistencies compared to the “Cx” nomenclature. Here we suggest some minor corrections to co-ordinate the “Gja/Gjb” nomenclature with the “Cx” nomenclature. Furthermore, this short review contains an update on phenotypic correlations between connexin deficient mice and patients bearing mutations in their orthologous connexin genes.     

An Update on Connexin Genes and their Nomenclature in Mouse and Man

Gap junctions, composed of connexin protein subunits, allow direct communication through conduits between neighboring cells. Twenty and twenty-one members of the connexin gene family are likely to be expressed in the mouse and human genome, respectively, 19 of which can be grouped into sequence-orthologous pairs. Their gene structure appears to be relatively simple. In most cases, an untranslated exon1 is separated by an intron of different lengh from exon2 that includes the uninterrupted coding region and the 3′-untranslated region. However, there are several exceptions to this scheme, since some mouse connexin genes contain different 5′-untranslated regions spliced either in an alternative and/or consecutive manner. Additionally, in at least 3 mouse and human connexin genes (mCx36, mCx39, mCx57 and hCx31.3, hCx36, as well as hCx40.1) the reading frame is spliced together from 2 different exons. So far, there are two nomenclatures to classify the known connexin genes: The “Gja/Gjb” nomenclature, as it is currently adopted by the NCBI data base, contains some inconsistencies compared to the “Cx” nomenclature. Here we suggest some minor corrections to co-ordinate the “Gja/Gjb” nomenclature with the “Cx” nomenclature. Furthermore, this short review contains an update on phenotypic correlations between connexin deficient mice and patients bearing mutations in their orthologous connexin genes.     

Mouse horizontal cells do not express connexin26 or connexin36

Gap junctions between neurons function as electrical synapses, and are present in all layers of mammalian and teleost retina. These synapses are largest and most prominent between horizontal cells where they function to increase the receptive field of a single neuron beyond the width of its dendrites. Receptive field size and the extent of gap junctional coupling between horizontal cells is regulated by ambient light levels and may mediate light/dark adaptation. Furthermore, teleost horizontal cell gap junction hemichannels may facilitate a mechanism of feedback inhibition between horizontal cells and cone photoreceptors. As a prelude to using mouse genetic models to study horizontal cell gap junctions and hemichannels, we sought to determine the connexin complement of mouse horizontal cells. Cx36, Cx37, Cx43, Cx45 and Cx57 mRNA could be detected in mouse retina by RT-PCR. Microscopy was used to further examine the distribution of Cx26 and Cx36. Cx26 immunofluorescence and a beta-gal reporter under regulatory control of the Cx36 promoter did not colocalize with a horizontal cell marker, indicating that these genes are not expressed by horizontal cells. The identity of the connexin(s) forming electrical synapses between mouse horizontal cells and the connexin that may form hemichannels in the horizontal cell telodendria remains unknown.     

Properties of Connexin26 Hemichannels Expressed in Xenopus Oocytes

1. Hemichannels formed by connexin26 (Cx26) on the horizontal cell dendrites that invaginate cone terminals in the vertebrate retina have been implicated in the feedback mechanism by which horizontal cells regulate transmitter release from cone photoreceptors. However, their membrane properties had not been studied previously, and it was unclear whether they could subserve their purported function at the membrane potentials over which horizontal cells operate. 2. We used the two-electrode voltage clamp technique to record the membrane currents and pharmacological properties of Cx26 hemichannels formed in the Xenopus oocyte expression system. 3. Oocytes expressing Cx26 exhibited large membrane conductances over a broad range of hyperpolarizing and depolarizing membrane potentials, and displayed little evidence of voltage-dependent gating, indicating that the hemichannels are constitutively open. The Cx26-mediated nonjunctional currents were relatively insensitive to quinine, a cinchona alkaloid that opens hemichannels formed by several other connexins. However, the hemichannel currents were blocked by carbenoxolone, a rise in extracellular calcium, or lowering intracellular pH. The currents could also be suppressed by reducing extracellular pH, and by the chloride channel blocker NPPB through its direct interaction with Cx26 hemichannels. 4. These findings provide a basis with which to evaluate the in situ pharmacological studies that attempt to assess the putative role of Cx26 hemichannels in the feedback pathway in the distal retina.     

Shear Stress and Cyclic Circumferential Stretch, But Not Pressure, Alter Connexin43 Expression in Endothelial Cells

Hemodynamic forces play a critical role in atherogenesis, as evidenced by the focal pattern of development of atherosclerotic lesions. Whereas disturbed flow in the branches and curved regions of large arteries is proatherogenic, laminar flow in the straight parts of vessels is atheroprotective. In addition, hypertension and age-related changes in arterial stiffness are important risk factors of the disease. Hemodynamic forces induce various changes in the structure and function of vascular endothelium, many of which reflect alterations in gene expression. Endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vascular wall. Using an in vitro perfusion system, we investigated the effects of pulsed unidirectional and oscillatory flows in combination with different levels of hydrostatic pressure and circumferential stretch on the expression of Cx43 in endothelial cells. Our results show that shear stress and circumferential stretch, but not pressure, modulate the expression of Cx43. In view of the distribution of this protein along the vascular tree, our findings provide new insights into the role of mechanical forces on gap junctional communication in regions prone to the development of atherosclerosis.     

Connexin45 Interacts with Zonula Occludens-1 in Osteoblastic Cells

Connexin43(Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.     

Connexin45 Interacts with Zonula Occludens-1 in Osteoblastic Cells

Connexin43(Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.     

Short-Range Functional Interaction Between Connexin35 and Neighboring Chemical Synapses

Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication—chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.     

Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32

Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Fusion of GFP to the Carboxyl Terminus of Connexin43 Increases Gap Junction Size in HeLa Cells

The pattern of gap junctional coupling between cells is thought to be important for the proper function of many types of tissues. At present, little is known about the molecular mechanisms that control the size and distribution of gap junctions. We addressed this issue by expressing connexin43 (Cx43) constructs in HeLa cells, a connexin-deficient cell line. HeLa cells expressing exogenously introduced wild-type Cx43 formed small, punctate gap junctions. By contrast, cells expressing Cx43-GFP formed large, sheet-like gap junctions. These results suggest that the GFP tag, which is fused to the carboxyl terminus of Cx43, alters gap junction size by masking the carboxyl terminal amino acids of Cx43 that comprise a zonula occludins-1 (ZO-1) binding site. We are currently testing this hypothesis using deletion and dominant-negative constructs that directly target the interaction between Cx43 and ZO-1.     

Deafness induced by Connexin 26 (GJB2) deficiency is not determined by endocochlear potential (EP) reduction but is associated with cochlear developmental disorders

Connexin 26 (Cx26, GJB2) mutations are the major cause of hereditary deafness and are responsible for >50% of nonsyndromic hearing loss. Mouse models show that Cx26 deficiency can cause congenital deafness with cochlear developmental disorders, hair cell degeneration, and the reduction of endocochlear potential (EP) and active cochlear amplification. However, the underlying deafness mechanism still remains undetermined. Our previous studies revealed that hair cell degeneration is not a primary cause of hearing loss. In this study we investigated the role of EP reduction in Cx26 deficiency-induced deafness. We found that the EP reduction is not associated with congenital deafness in Cx26 knockout (KO) mice. The threshold of auditory brainstem response (ABR) in Cx26 KO mice was even greater than 110 dB SPL, demonstrating complete hearing loss. However, the EP in Cx26 KO mice varied and not completely abolished. In some cases, the EP could still remain at higher levels (>70 mV). We further found that the deafness in Cx26 KO mice is associated with cochlear developmental disorders. Deletion of Cx26 in the cochlea before postnatal day 5 (P5) could cause congenital deafness. The cochlea had developmental disorders and the cochlear tunnel was not open. However, no congenital deafness was found when Cx26 was deleted after P5. The cochlea also displayed normal development and the cochlear tunnel was open normally. These data suggest that congenital deafness induced by Cx26 deficiency is not determined by EP reduction and may result from cochlear developmental disorders.