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To identify signals that convey connexin oligomerization compatibility, we have aligned amino-acid sequences of alpha and beta group connexins (Cx) and compared the physico-chemical properties of each homologous amino-acid residue. Four positions were identified that consistently differed between alpha and beta-type connexins; two are located in the N-terminal domain (P1 and P2, corresponding to residues 12 and 13 of the Cx43 sequence), and two in the third trans-membrane-spanning domain TM3 (P3 and P4, corresponding to residues 152 and 153 of the Cx43 sequence). Replacement of each of these residues in Cx43 (an alpha-type connexin) with the corresponding residues of Cx32 (a beta-type connexin) resulted in the assembly of all variants into gap junctions; however, only the P4 variant was functional, as indicated by lucifer yellow dye transfer assays. The other three variants exerted a moderate to severe dose-dependent, dominant-negative effect on co-expressed wild-type (wt) Cx43 channel activity. Moreover, a significant dose-dependent, trans-dominant inhibition of channel activity was observed when either one of the N-terminal variants was co-expressed with wt Cx32. Assembly analyses indicated that dominant and trans-dominant inhibitory effects appeared to be based on the oligomerization of wt and variant connexins into mixed connexons. Interestingly, the identified N-terminal amino acids coincide with the position of naturally occurring, disease-causing missense mutations of several beta-connexin genes (Cx26, Cx30, Cx31, Cx32). Our results demonstrate that three of the identified discriminative amino-acid residues (positions 12, 13 and 152) are crucial for Cx43 channel function and suggest that the N-terminal amino-acid residues at position 12/13 are involved in the oligomerization compatibility of alpha and beta connexins.

Because in mammals the anterior pituitary lacks innervation, we investigated whether gap junctions established between selected cells within the gland are part of an intrapituitary mechanism to ensure physiological synchronization of cells involved in the control of hormone secretion. We report here the dynamics of anterior pituitary connexin 43 (Cx43)-gap junctions throughout the mink (Mustela vison) annual reproductive cycle and its relationship with the anterior pituitary prolactin (PRL) content that parallels variations in serum PRL levels documented in the literature. We found that PRL anterior pituitary levels were maximal in spring and during lactation and that they were minimal in autumn and winter. Anterior pituitary Cx43 levels were maximal during periods of high PRL secretion. During these periods, Cx43-positive gap junctions localized to stellate-shaped cells occupying the center of anterior pituitary follicles and to the rounded cells occupying the remaining follicles. Connexin 43-positive gap junctions were also observed between adjacent follicles. During periods of low PRL pituitary content, Cx43-positive gap junctions localized to the stellate cells but not to the cells of the remaining follicles. Moreover, Cx43 labeling was undetected between adjacent follicles. To assess between which cells within the mink anterior pituitary the Cx43 gap junctions were established, the different anterior pituitary cell populations were separated by a discontinuous Percoll gradient, and Western blot analyses of each cell population using Cx43 antibodies were performed. The immunoblots showed a Cx43 immunoreactive band associated with the cell layer enriched in S-100-positive, stellate-shaped cells. The result was confirmed by fluorescence microscopy studies that showed that Cx43-mediated gap junctions were established preferentially between the cultured S-100-positive, elongated cells. The results show that in mink stellate cells, the junctional machinery associated with the Cx43 protein varies in synchrony with the anterior pituitary PRL content throughout the mink annual reproductive cycle. It is suggested that the Cx43 gap junctions on the stellate cells play an important role in the synchronization of cellular activity within selected follicles of the anterior pituitary, thus contributing to the control of PRL secretion during the annual reproductive cycle.

Lens fiber cell gap junctions contain alpha(3) (Cx46) and alpha(8) (Cx50) connexins. To examine the roles of the two different connexins in lens physiology, we have genetically engineered mice lacking either alpha(3) or alpha(8) connexin. Intracellular impedance studies of these lenses were used to measure junctional conductance and its sensitivity to intracellular pH. In Gong et al. 1998, we described results from alpha(3) connexin knockout lenses. Here, we present original data from alpha(8) connexin knockout lenses and a comparison with the previous results. The lens has two functionally distinct domains of fiber cell coupling. In wild-type mouse lenses, the outer shell of differentiating fibers (see 1, DF) has an average coupling conductance per area of cell-cell contact of approximately 1 S/cm(2), which falls to near zero when the cytoplasm is acidified. In the inner core of mature fibers (see 1, MF), the average coupling conductance is approximately 0.4 S/cm(2), and is insensitive to acidification of the cytoplasm. Both connexin isoforms appear to contribute about equally in the DF since the coupling conductance for either heterozygous knockout (+/-) was approximately 70% of normal and 30-40% of the normal for both -/- lenses. However, their contribution to the MF was different. About 50% of the normal coupling conductance was found in the MF of alpha(3) +/- lenses. In contrast, the coupling of MF in the alpha(8) +/- lenses was the same as normal. Moreover, no coupling was detected in the MF of alpha(3) -/- lenses. Together, these results suggest that alpha(3) connexin alone is responsible for coupling MF. The pH- sensitive gating of DF junctions was about the same in wild-type and alpha(3) connexin -/- lenses. However, in alpha(8) -/- lenses, the pure alpha(3) connexin junctions did not gate closed in the response to acidification. Since alpha(3) connexin contributes about half the coupling conductance in DF of wild-type lenses, and that conductance goes to zero when the cytoplasmic pH drops, it appears alpha(8) connexin regulates the gating of alpha(3) connexin. Both connexins are clearly important to lens physiology as lenses null for either connexin lose transparency. Gap junctions in the MF survive for the lifetime of the organism without protein turnover. It appears that alpha(3) connexin provides the long-term communication in MF. Gap junctions in DF may be physiologically regulated since they are capable of gating when the cytoplasm is acidified. It appears alpha(8) connexin is required for gating in DF.

We used electron cryo-microscopy and image analysis to examine frozen-hydrated, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal truncation mutant of the cardiac gap junction channel formed by 43-kDa alpha(1) connexin. To our knowledge this is the first example of a structural analysis of a membrane protein that has been accomplished using microgram amounts of starting material. The recombinant alpha(1) connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously assembled gap junction plaques. Detergent treatment with Tween 20 and 1,2-diheptanoyl-sn-phosphocholine resulted in well-ordered 2D crystals. A three-dimensional density (3D) map with an in-plane resolution of approximately 7.5 A revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. In the extracellular gap the aqueous channel was bounded by a continuous wall of protein that formed a tight electrical and chemical seal to exclude exchange of substances with the extracellular milieu.

A series of arachidonic acid amides including anandamide and arachidonamide that act as potent inhibitors of the rat glial cell gap junction is described.

Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an α-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane α-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.

Connexin alpha 3 (Cx46 or Gja3) gene targeted null mice developed lens nuclear cataracts shortly after birth. A large variance in the cataracts was observed in alpha 3 null sibs on a mixed 129SvJae X C57BL/6J F3 background. This suggested that the genetic background might influence the cataract phenotype. Therefore, we placed the alpha 3 null mutation into a 129SvJae background, and also backcrossed the mutation for six generations into 129SvJ and C57BL/6J backgrounds. While alpha 3 nulls on the two 129 backgrounds contained severe cataracts associated with gamma crystallin cleavage, alpha 3 nulls on the C57B16 background had far milder cataracts with no detectable gamma crystallin cleavage. These findings suggest that a genetic modifier exists that influences gamma crystallin stability, and that gamma crystallin breakdown is associated with severe nuclear cataracts.

Fiber cells of the lens are interconnected by an extensive network of gap junctions containing α 3 (Cx46) and α 8 (Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous α 3 knockout (−/−), heterozygous (+/−), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that α 8 remained at similar levels in the three classes of lenses, whereas α 3 was approximately 50% of the normal level in the +/− lenses, and it was absent from the −/− lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In −/− lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (≈−65 mV). The DF remained coupled, but the conductance was reduced to 30–35% of normal. However, the gap junctions in the DF of α 3 −/− lenses remained sensitive to pH. We conclude that α 3 connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and has been shown to effect serotonergic systems and block gap junction communication in a structurally specific manner. Herein, the structural features of oleamide required for inhibition of the gap junction-mediated chemical and electrical transmission in rat glial cells are defined. The effective inhibitors fall into two classes of fatty acid primary amides of which oleamide and arachidonamide are the prototypical members. Of these two, oleamide constitutes the most effective, and its structural requirements for inhibition of the gap junction are well defined. It requires a chain length of 16–24 carbons of which 16–18 carbons appears optimal, a polarized terminal carbonyl group capable of accepting but not necessarily donating a hydrogen bond, a Δ 9 cis double bond, and a hydrophobic methyl terminus. Within these constraints, a range of modifications are possible, many of which may be expected to improve in vivo properties. A select set of agents has been identified that serves both as oleamide agonists and as inhibitors of fatty acid amide hydrolase, which is responsible for the rapid inactivation of oleamide.

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction–mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18β-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme activity that degrades neuromodulatory fatty acid amides, including oleamide and anandamide. A single 2.5-kb FAAH mRNA is distributed throughout the rat CNS and accumulates progressively between embryonic day 14 and postnatal day 10, remains high until postnatal day 30, then decreases into adulthood. FAAH enzymatic activity, as measured in dissected brain regions, was well correlated with the distribution of its messenger RNA. In situ hybridization revealed profound distribution of FAAH mRNA in neuronal cells throughout the CNS. The most prominent signals were detected in the neocortex, hippocampal formation, amygdala, and cerebellum. The FAAH distribution in the CNS suggests that degradation of neuromodulatory fatty acid amides at their sites of action influences their effects on sleep, euphoria, and analgesia.

To examine the possible role of gap junctions in mouse skin tumor progression, we generated a panel of mouse skin tissue samples exhibiting normal, hyperplastic, or neoplastic changes and characterized the expression of the gap-junction genes connexin 43 (Cx43) and connexin 26 (Cx26) by in situ hybridization and immunohistochemical analyses. In normal skin, these two gap junction genes were differentially expressed; Cx43 was found predominantly in the less differentiated lower spinous layers, whereas Cx26 was found in terminally differentiating upper spinous and granular layers. In hyperplastic epidermis exhibiting an expansion of the differentiated upper layer, i.e., epidermis with a thickened granular layer or in which the granular layer was replaced with keratinocytes exhibiting tricholemmal differentiation, expression of Cx43 and Cx26 remained segregated in the lower and upper spinous layers, respectively. However, in papillomas, Cx26 was localized in the lower but not upper spinous layer, an expression pattern identical to that of Cx43. In addition, the overall expression levels of both Cx43 and Cx26 appeared to be greatly elevated in the papillomas. It is interesting that such marked alteration in the pattern of Cx26 expression occurred within the context of hyperplastic changes histologically identical to those seen in the nonpapillomous hyperplasias. Interestingly, in neoplastic skin lesions containing a squamous cell carcinoma, Cx43 and Cx26 expression was extinguished. Moreover, expression of Cx43 was also significantly reduced in adjacent apparently nonneoplastic tissues. Overall, these observations show that perturbations in gap-junction gene expression are associated with skin hyperplasia and neoplasia. Such findings suggest a possible role for gap junctions in the malignant conversion of mouse epidermal cells.

The objective of the present study was to examine the effects of the antiprogesterone RU486 on the expression levels of multiple gap junction (GJ) gene products and, in detail, on alpha 1 (connexin43 [Cx43]), in various regions of the rat implantation chamber during experimentally induced preterm labor at mid/late stages of gestation. Vaginal bleeding, but not expulsion of concepti, was observed in a majority of animals 24 h after a single injection of RU486 at Day 15 of gestation, and it persisted until animals were killed 48 h later. The bleeding was completely suppressed by R 5020, a synthetic progesterone with a high affinity for the progesterone receptor (PR). Various components of the implantation chamber (uterus, mesometrial stroma, placenta) and ovaries were isolated 24 and 48 h postinjection and analyzed for alpha 1 (Cx43), beta 1 (Cx32), and beta 2 (Cx26) connexin expression by immunohistochemistry and by Northern blots (alpha 1 only). Alpha 1 Connexin was present at high levels in the myometrium following the inhibition of progesterone action by RU486; accordingly, this effect was completely suppressed by R 5020. The blocking of the PR also had a dramatic effect on expression levels, size, and distribution of junctional plaques composed of beta 1 and beta 2 connexins in polarized luminal and glandular epithelium. The average size of junctional plaques was significantly reduced in the luminal epithelium after RU486 administration. This effect was inhibited in the presence of R 5020. At the RNA level, the alpha 1 transcript was markedly elevated in the uterus and in the ovaries 24 and 48 h after administration of RU486. An elevation was also observed in the mesometrial stroma, while no increase was detected in the placenta. The RU486-induced alpha 1 mRNA steady-state levels in various tissues were completely suppressed by R 5020. These results demonstrate the modulation of multiple connexins in various cell types of the implantation chamber upon blocking of PR action. The expression profile of the myometrial and epithelial GJs was similar to that previously observed in the estrogen-treated rat uterus.

Gap junctional communication is important in many physiological processes, including growth control, patterning, and the synchronization of cell-to-cell activities. It has been difficult to study the synthesis and assembly of gap junctions due to their low abundance. To overcome this limitation, baby hamster kidney cells (BHK) have been transfected with a human beta 1 (Cx32) connexin cDNA construct. Expression was placed under the control of the mouse metallothionein promoter that can be induced by heavy metals. The transfected cells were characterized by DNA, RNA and protein analysis, as well as by scrape loading to detect functional channels. Functional beta 1 connexin was detected only in cells transfected with beta 1 connexin cDNA in the correct orientation (beta 1-BHK). Analysis of the cells by light microscopic immunocytochemistry indicated that beta 1 connexin antigen was localized to the plasma membrane and to several intracellular compartments. Characterization with thin section electron microscopy revealed extensive areas of assembled double membrane gap junctions between cells (on the cell surface), in the endoplasmic reticulum (ER), and the nuclear envelope. This unusual intracellular distribution for assembled gap junction protein was confirmed by freeze fracture analysis, which revealed large particle aggregates, characteristic of gap junction plaques, on the fracture faces of all these membranes. The presence of gap junction particle aggregates in the ER suggests that the oligomerization of connexin can occur at its site of synthesis. Further, the process of assembly into double membrane junction structures in intracellular membranes may be driven by connexin protein concentration.

The expression and localization of three different connexins (alpha 1, Cx43; beta 1, Cx32; and beta 2, Cx26) were analyzed in human, mouse, and rat mammary glands by PCR analysis of reverse-transcribed RNA (RT-PCR) and indirect immunohistochemistry. For the rodent mammary gland, the study included different physiological stages of development during nonpregnancy, pregnancy, lactation, and postweaning. RT-PCR amplification revealed a constitutive expression of RNA for alpha 1 connexin in all three species. In contrast, both beta 1 and beta 2 transcripts were expressed only during lactation in the rodent mammary gland. Specifically, immunohistochemistry showed that the expression of all three connexins was restricted to specific cell types, and it varied according to the physiological activity of the organ. In particular, alpha 1 antigen was detected only between myoepithelial cells, the contractile cells surrounding alveoli and ductal systems in the mammary gland, while beta 1 and beta 2 antigens were localized solely at the basolateral border of alveolar secretory cells. The level of beta 1 and beta 2 connexins was increased in the rodent mammary gland during lactation. No staining for either beta connexin was detected in human mammary gland or in that of nonpregnant, pregnant, or postweaning rodents. The inducible coexpression of beta 1 and beta 2 antigens in luminal cells of the lactating rodent suggests a possible role for these connexins in the coordination of the secretory epithelium.

Enriched subcellular fractions of double membrane gap junctions (plaques) from rat livers were treated under reducing conditions with high salt and non-ionic detergent concentrations at high pH to obtain a preparation of structural 80-90 A complexes of oligomers (connexons). The isolated oligomers were chromatographically purified, and subsequently characterized immunologically, morphologically by electron microscopy, hydrodynamically by gel filtration and ultracentrifugation, spectroscopically by circular dichroism, and chemically via cross-linking studies. The physical characteristics of these isolated gap junction complexes were compared to those of native membrane-bound gap junctions in rat liver. These analyses indicate that the isolated complex (connexon) principally contains a hexameric arrangement of gap junction protein to form a single membrane hemi-channel.

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.

The effects of estrogen (E2) and progesterone (P) were examined on the expression levels of multiple gap junction (GJ) gene products (alpha 1 = Cx43, beta 1 = Cx32, beta 2 = Cx26) in the uterus and ovaries of immature rats by immunohistochemistry, electron microscopy and northern blot analysis. E2 induced the expression of alpha 1 connexin in the uterus (specifically in the myometrium and in endometrial stroma proximal to luminal epithelium) and ovaries. The E2-induced alpha 1 expression was completely suppressed by P in the uterus, but only partly in ovaries. Steroid hormones also modulated the quantity, size, and distribution of beta 1 and beta 2 containing junctional plaques along lateral cell borders in polarized luminal and glandular uterine epithelia. Small GJs were detected at basolateral regions in proliferative luminal epithelium following administration of E2. In contrast, large GJs were localized at subapical-lateral cell borders of the secretory epithelium following P-treatment. The co-administration of E2 + P had a synergistic effect on beta 1 and beta 2 expression in the luminal epithelium, but an inhibitory effect on beta 2 expression in glandular epithelium. Myometrial GJs were detected in freeze-fracture replicas as aggregates containing regularly arranged particles with particle free zones. In contrast, GJs in secretory epithelium contained particles which were arranged in a non-crystalline fashion. These GJs contained domains of mixed and segregated beta 1 and beta 2 antigens within a single plaque as revealed by laser scanning confocal microscopy analysis of immuno-double-labeled secretory epithelium. The demonstration of segregated antigens within a single GJ plaque indicates the possibility of multiple channel populations formed by homo-oligomeric connexons. These results suggest that different connexins can be differentially regulated by steroid hormones in different cell types, and that the same steroid hormone can have different effects on the same connexin in different cell types.

OBJECTIVE: The purpose of this study was to determine if gap junctions are a necessary component of the human laboring uterus and if their presence in myometrium is a prerequisite for both term and preterm labor. STUDY DESIGN: We obtained 27 human myometrial samples at cesarean section or nongravid hysterectomy. Gap junction formation was analyzed in a blind fashion by freeze fracture and indirect immunofluorescence. Six samples were obtained from term patients with no labor, six from term patients in labor, six from preterm patients with no labor, six from patients in preterm labor, and three from nongravid hysterectomy specimens. RESULTS: Gap junction structures were identified in the human myometrium of patients in term and in preterm labor but not in the other patient samples. In addition, evidence was obtained for the expression of (α 1 ) gap junction ribonucleic acid and (α 1 ) gap junction protein in term samples of human myometrium. CONCLUSION: Gap junctions are a necessary component of the human myometrium during term and preterm labor. The formation of gap junctions may be a final common event for the development of labor, and inhibition of gap junction activity could be a novel approach for the treatment of preterm labor.

The mRNA and protein expression of alpha 1 (connexin 43), beta 1 (connexin 32), and beta 2 (connexin 26) gap junction genes were examined in the regenerating rat liver after 70% partial hepatectomy (PH). Expression of beta 1 and beta 2 steady-state mRNA levels changed minimally until 12 h after PH when both transcripts decreased to approximately 15% of baseline values. A similar decrease in assembled connexin levels was detected by immunoblot and indirect immunofluorescence at 18 h after PH. Both transcripts simultaneously increased between 24 and 42 h and again rapidly decreased by 48 h post-PH. beta 1 and beta 2 assembled gap junction protein expression increased at 48 h post-PH and rapidly decreased by 56 h. By 72 to 84 h post-PH, beta 1 and beta 2 mRNA and assembled protein expression returned to near baseline levels and were maintained. Interestingly, inhibition of protein synthesis with cycloheximide completely inhibited disappearance of the beta 2 transcript, in contrast to beta 1 mRNA which was unaffected. Nuclear run-on assays showed no change in transcriptional rates for either gene during the regenerative period. However, both beta 1 and beta 2 transcripts exhibited significantly decreased mRNA half-lives at 12 h post-PH (3.8 and 3.7 h, respectively) relative to those at 0 h (10.9 and 6.1 h, respectively). Surprisingly, although the transcriptional rate for alpha 1 was similar to that observed for beta 2, no alpha 1 transcripts were detectable by northern or RNase protection analysis. The results suggest that in the regenerating rat liver, beta 1 and beta 2 gap junction genes are not regulated at the transcriptional level. Rather, the cyclical modulation of their steady-state transcripts is regulated primarily by posttranscriptional events of which mRNA stability is at least one critical factor in the control process.

The membrane topology and quaternary structure of rat cardiac gap junction ion channels containing alpha 1 connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic "loop" domain of at least Mr = 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native Mr = 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in an Mr approximately 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252-271. Immunoelectron microscopy demonstrated that the Mr approximately 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to the Mr approximately 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the alpha 1 polypeptide. Therefore, the Mr approximately 13,000 carboxyterminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexins indicates conservation in the molecular design of the gap junction channels formed by alpha or beta connexins.

The expression of four different gap junction (GJ) transcripts, corresponding to the α1,α3,β1 and β2 gene products, has been examined in the ciliary epithelium of human and bovine eyes, and in cultures of ciliary epithelial cells. Northern blot analysis revealed that oq mRNA, 3.6-kb in size, was the predominant transcript expressed in intact tissue and in cultures of pigmented ciliary epithelial cells (PE). No transcripts from the α3, β1 or β2 gap junction genes were detected in intact tissue or ciliary epithelial cells as demonstrated by Northern blotting. When the levels of α gap junction mRNA were compared between PE and NPE in primary cultures, a striking difference was observed in the level of ai transcripts: there was about a 6 to 8-fold increase in α1 levels in PE cells, relative to the NPE cells. To verify the differential level of expression of ai GJ mRNA in the two cell types, indirect immunofluorescence localization studies were performed on semithin cryostat sections of ciliary processes. Thes...

The expression of products from three different gap junction genes (alpha 1, beta 1 and beta 2) was studied in pre- and postimplantation mouse embryos, during organogenesis, during differentiation of F9 teratocarcinoma cells, and in cultured embryonic stem (ES) cells. In this analysis, the following results were obtained. 1) Pre- and postimplantation mouse embryos. The alpha 1 transcript was the earliest gap junction RNA detected (in the 4 cell stage embryo) and its abundance increased significantly throughout subsequent development. 2) Organogenesis. Evidence was obtained for developmental expression of these three different gap junction genes during early embryogenesis and throughout the late stages of organogenesis. The expression patterns for these genes may be related to differences in gap junctional communication requirements for fetal organ development versus neonatal and adult organ function, or the utilization of different genes by different cell types during organogenesis. 3) During the differentiation of F9 cells in culture, expression of these three genes was modulated. Thus, this is the first evidence for modulation of gap junction gene expression during the differentiation of a single cell type in culture. 4) In an ES cell culture line, alpha 1 was the only gap junction gene product detected. This is consistent with the findings of alpha 1 expression in the embryonic inner cell mass region and in undifferentiated teratocarcinoma cells.

Gap junctions in the heart play an important functional role by electrically coupling cells, thereby organizing the pattern of current flow to allow co-ordinated muscle contraction. Cardiac gap junctions are therefore intimately involved in normal conduction as well as the genesis of potentially lethal arrhythmias. We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated 2D crystals of a recombinant, C-terminal truncated form of connexin 43 (Cx43; alpha 1), the principal cardiac gap junction protein. The projection map at 7 A resolution revealed that each 30 kDa connexin subunit has a transmembrane alpha-helix that lines the aqueous pore and a second alpha-helix in close contact with the membrane lipids. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by approximately 30 degrees. We are now recording images of tilted, frozen-hydrated 2D crystals, and a preliminary 3D map has been computed at an in-plane resolution of approximately 7.5 A and a vertical resolution of approximately 25 A. As predicted by our model, the two apposing connexons that form the channel are staggered with respect to each other for certain connexin molecular boundaries within the hexamer. Within the membrane interior each connexin subunit displays four rods of density, which are consistent with an alpha-helical conformation for the four transmembrane domains. Preliminary studies of BHK hamster cells that express the truncated Cx43 designated alpha 1 Cx263T demonstrate that oleamide, a sleep inducing lipid, blocks in vivo dye transfer, suggesting that oleamide causes closure of alpha 1 Cx263T channels. The comparison of the 3D structures in the presence and absence of oleamide may provide an opportunity to explore the conformational changes that are associated with oleamide-induced blockage of dye transfer. The structural details revealed by our analysis will be essential for delineating the molecular basis for intercellular current flow in the heart, as well as the general molecular design and functional properties of this important class of channel proteins.

This paper presents recent results from this laboratory concerning the topological structure and function of the major 27 kDa gap junction protein. Immunological, biochemical and biophysical observations now provide evidence for the localization of the 27 kDa protein to gap junction structures, both in vivo and in vitro, and for the participation of the 27 kDa protein in channel conductance.

The antibody 38C2 efficiently catalyzed a retro-Michael reaction to convert a novel, cell-permeable fluorogenic substrate into fluorescein within living cells. In vitro, the antibody converted the substrate to fluorescein with a k(cat) of 1.7 x 10(-5) s(-1) and a catalytic proficiency (k(cat)/k(uncat)K(m)) of 1.4 x 10(10) m(-1) (K(m) = 7 microm). For hybridoma cells expressing antibody or Chinese Hamster Ovarian (CHO) cells injected with antibody, incubation of the substrate in the extracellular medium resulted in bright intracellular fluorescence distinguishable from autofluorescence or noncatalyzed conversion of substrate. CHO cells loaded with antibody were 12 times brighter than control cells, and more than 85% of injected cells became fluorescent. The fluorescein produced by the antibody traveled into neighboring cells through gap junctions, as demonstrated by blocking dye transfer using the gap junction inhibitor oleamide. The presence of functional gap junctions in CHO cells was confirmed through oleamide inhibition of lucifer yellow transfer. These studies demonstrate the utility of the intracellular antibody reaction, which could generate tracer dyes in specific cells within complex multicellular environments simply by bathing the system in substrate.

Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.

Publisher Summary This chapter discusses the development in gap junctions. A role for gap junctions in development is proposed as the early studies show the presence of low resistance junctions in nonexcitable cells and tissues. These studies demonstrated that cells of various adult tissues and those of the early embryo generally exhibited low resistance pathways through which ions and electrical current can pass from cell to cell (ionic coupling). Thus, microelectrode impalements into squid embryos revealed extensive coupling between the yolk sac and other cells throughout the embryo. Gap junctions are found in embryos from a diverse array of organisms, including rodents, chick, and frog. It is suggested that cell–cell interaction mediated by gap junctions may play a role in various physiological functions, such as in maintaining tissue homeostasis or in growth regulation. The chapter summarizes some of the results obtained from studies, and closes with some speculations as to the possible role of gap junctions in development.

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in alpha1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for alpha1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing alpha1 connexins, and from alpha1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative alpha1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and alpha1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous alpha1 connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and alpha1 connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the alpha1 connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium- an elevation in the CMV43 mice vs. a reduction in the alpha1 connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non-neural crest- derived tissues. Overall, these findings suggest that gap junction communication mediated by alpha1 connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of alpha1 connexin function.

We reported previously (Falk, M. M., Kumar, N. M., and Gilula, N. B. (1994) J. Cell Biol. 127, 343–355) that the membrane integration of polytopic connexin polypeptides can be accompanied by an inappropriate cleavage that generates amino-terminal truncated connexins. While this cleavage was not detected in vivo, translation in standard cell-free translation/translocation systems resulted in the complete cleavage of all newly integrated connexins. Partial cleavage occurred in heterologous expression systems that correlated with the expression level. Here we report that the transmembrane topology of connexins generated in microsomal membranes was identical to the topology of functional connexins in plasma membranes. Characterization of the cleavage site and reaction showed that the connexins were processed by signal peptidase immediately downstream of their first transmembrane domain in a reaction similar to the removal of signal peptides from pre-proteins. Increasing the length and hydrophobic character of the signal anchor sequence of connexins completely prevented the aberrant cleavage. This result indicates that their signal anchor sequence was falsely recognized and positioned as a cleavable signal peptide within the endoplasmic reticulum translocon, and that this mispositioning enabled signal peptidase to access the cleavage sites. The results provide direct evidence for the involvement of unknown cellular factors in the membrane integration process of connexins.

Retinoids and phorbol esters have profound effects on proliferation and differentiation of epidermal keratinocytes when applied topically on rodent skin. Since both agents also modulate gap junction (GJ)-mediated cell-cell communication, we have examined the effects of all-trans retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of alpha1 (Cx43) and beta2 (Cx26) connexins, the two major gap junction gene products in mature rat epidermis. In fully differentiated, mature epidermis, alpha1 is expressed in the lower, less differentiated portion, while beta2 is localized in upper, more differentiated layers. Dorsal skin of 21-day old rats was treated topically with a single dose of RA, TPA or vehicle alone and used for histological and molecular analyses at different time points. Keratinocytes in interfollicular epidermis were examined for proliferation and differentiation using specific antibodies for keratins (K10, K14) and proliferating cell nuclear antigen (PCNA). An increase in epidermal thickness was noticed within 4 hours after the application of RA or TPA. This increase, however, appeared to be primarily due to hypertrophy, since no substantial changes were observed in the proliferative index of epidermal keratinocytes. PCNA immunoreactivity significantly increased after 8 hours treatment of RA or TPA, suggesting a hyperproliferative growth response. Epidermal hyperplasia was confirmed by monitoring the expression patterns of K10 and K14 in RA- or TPA-treated skin. RA-induced hyperplasia lasted longer as compared to TPA induction. Changes in keratin phenotypes were paralleled by an increase in alpha1 and beta2 connexin expression as well as their colocalization in same epidermal layers. Differences in hyperplastic growth response kinetics were also confirmed at the connexin level, with beta2 antigen sustained for longer and at higher levels in suprabasal layers of RA-treated skin. Overall, this type of connexin expression resembled that observed in the non-differentiated rat epidermis during embryonic development. An increase in alpha1 and beta2 connexin abundance was also observed at the protein and RNA levels. At 96 hours after RA or TPA treatment, expression of both connexins was similar to that of the control epidermis. Taken together, these findings suggest that a higher level of GJ-mediated cell-cell communication, is required for the maintenance of homeostasis during periods of rapid epidermal growth and differentiation.

Gap junction channels formed by alpha3 (Cx46) and alpha8 (Cx50) connexin provide pathways for communication between the fiber cells in the normal transparent lens. To determine the specific role of alpha3 connexin in vivo, the alpha3 connexin gene was disrupted in mice. Although the absence of alpha3 connexin had no obvious influence on the early stages of lens formation and the differentiation of lens fibers, mice homozygous for the disrupted alpha3 gene developed nuclear cataracts that were associated with the proteolysis of crystallins. This study establishes the importance of gap junctions in maintaining normal lens transparency by providing a cell-cell signaling pathway or structural component for the proper organization of lens membrane and cytoplasmic proteins.

Several different gap junction channel subunit isotypes, known as connexins, were synthesized in a cell-free translation system supplemented with microsomal membranes to study the mechanisms involved in gap junction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembrane topology. An integrated biochemical and biophysical analysis indicated that the connexins assembled specifically with other connexin subunits. No interactions were detected between connexin subunits and other co-translated transmembrane proteins. The connexins that were integrated into microsomal vesicles assembled into homo- and hetero-oligomeric structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons derived from gap junctions in vivo. Further, cell-free assembled homo-oligomeric connexons composed of beta1 or beta2 connexin were reconstituted into synthetic lipid bilayers. Single channel conductances were recorded from these bilayers that were similar to those measured for these connexons produced in vivo. Thus, this is the first direct evidence that the synthesis and assembly of a gap junction connexon can take place in microsomal membranes. Finally, the cell-free system has been used to investigate the properties of alpha1, beta1 and beta2 connexin to assemble into hetero-oligomers. Evidence has been obtained for a selective interaction between individual connexin isotypes and that a signal determining the potential hetero-oligomeric combinations of connexin isotypes may be located in the N-terminal sequence of the connexins.

Electron cryo-microscopy and image analysis of frozen-hydrated, two-dimensional crystals of gap junction membrane channels formed by recombinant alpha 1 connexin (Cx43) reveal a ring of transmembrane alpha-helices that lines the aqueous pore and a second ring of alpha-helices in close contact with the membrane lipids.

Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.

We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution.

A molecule isolated from the cerebrospinal fluid of sleep-deprived cats has been chemically characterized and identified as cis-9,10-octadecenoamide. Other fatty acid primary amides in addition to cis-9,10-octadecenoamide were identified as natural constituents of the cerebrospinal fluid of cat, rat, and human, indicating that these compounds compose a distinct family of brain lipids. Synthetic cis-9,10-octadecenoamide induced physiological sleep when injected into rats. Together, these results suggest that fatty acid primary amides may represent a previously unrecognized class of biological signaling molecules.

The biosynthetic membrane integration and intracellular transport of gap junction (GJ) proteins (connexins) was studied using in vitro and in vivo systems. Connexins were co-translationally integrated into endoplasmic reticulum (ER) membranes. However, their proper membrane topology was not achieved in in vitro translocation assays. Analysis of connexin expression in vitro and in vivo suggests that a novel membrane integration mechanism exists for GJ proteins that is dependent on an unknown integration factor.

Connexins, the proteins that form gap junction channels, are polytopic plasma membrane (PM) proteins that traverse the plasma membrane bilayer four times. The insertion of five different connexins into the membrane of the ER was studied by synthesizing connexins in translation-competent cell lysates supplemented with pancreatic ER-derived microsomes, and by expressing connexins in vivo in several eucaryotic cell types. In addition, the subcellular distribution of the connexins was determined. In vitro-synthesis in the presence of microsomes resulted in the signal recognition particle-dependent membrane insertion of the connexins. The membrane insertion of all connexins was accompanied by an efficient proteolytic processing that was dependent on the microsome concentration. Endogenous unprocessed connexins were detectable in the microsomes used, indicating that the pancreatic microsomes serve as a competent recipient in vivo for unprocessed full length connexins. Although oriented with their amino terminus in the cytoplasm, the analysis of the cleavage reaction indicated that an unprecedented processing by signal peptidase resulted in the removal of an amino-terminal portion of the connexins. Variable amounts of similar connexin cleavage products were also identified in the ER membranes of connexin overexpressing cells. The amount generated correlated with the level of protein expression. These results demonstrate that the connexins contain a cryptic signal peptidase cleavage site that can be processed by this enzyme in vitro and in vivo in association with their membrane insertion. Consequently, a specific factor or condition must be required to prevent this aberrant processing of connexins under normal conditions in the cell.

Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of alpha 1 (connexin 43) and beta 2 (connexin 26) gap junction proteins is down-modulated, whereas that of beta 3 (connexin 31) and beta 4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process.

The expression of four different gap junction gene products (alpha 1, beta 1, beta 2, and beta 3) has been analysed during rat skin development and the hair growth cycle. Both alpha 1 (Cx43) and beta 2 (Cx26) connexins were coexpressed in the undifferentiated epidermis. A specific, developmentally regulated elimination of beta 2 expression was observed in the periderm at E16. Coinciding with the differentiation of the epidermis, differential expression of alpha 1 and beta 2 connexins was observed in the newly formed epidermal layers. alpha 1 connexin was expressed in the basal and spinous layers, while beta 2 was confined to the differentiated spinous and granular layers. Large gap junctions were present in the basal layer, while small gap junctions, associated with many desmosomes, were typical for the differentiated layers. Although the distribution pattern for alpha 1 and beta 2 expression remained the same in the neonatal and postnatal epidermis, the RNA and protein levels decreased markedly following birth. Hair follicle development was marked by expression of alpha 1 connexin in hair germs at E16. Following beta 2 detection at E20, the expression increased for both alpha 1 and beta 2 in developing follicles. A cell-type-specific expression was detected in the outer root sheath, in the matrix, in the matrix-derived cells (inner root sheath, cortex and medulla) and in the dermal papilla. In addition, alpha 1 was specifically expressed in the arrector pili muscle, while sebocytes expressed both alpha 1 and beta 3 (Cx31) connexin. beta 1 connexin (Cx32) was not detected at any stage analysed. The results indicate that multiple gap junction genes contribute to epidermal and follicular morphogenesis. Moreover, based on the utilization of gap junctions in all living cells of the surface epidermis, it appears that the epidermis may behave as a large communication compartment that may be coupled functionally to epidermal appendages (hair follicles and sebaceous glands) via gap junctional pathways.

Gap junctional communication between cells provides a mechanism for the movement of molecular information between cells via the unit of gap junction structure and function, the gap junctional channel. In the past five years, there has been rapid progress in identifying and characterizing a multigene family that is responsible for producing the gap junction polypeptides that are responsible for generating gap junctional channel oligomers between cells. The products of these genes have been referred to as connexins, and the multigene family can be categorized into two classes at present, the alpha class and the beta class. Members of these two classes can be distinguished on the basis of their primary sequence and overall predicted topological organization. The gap junction genes map to different chromosomes in both mice and humans, and these genes are utilized on a cell specific basis. Furthermore, these genes can be developmentally regulated, and multiple genes can be co-expressed simultaneously by the same cell type. Efforts to understand the precise structure-function relationship of the products of these different genes is now being approached by utilizing various expression systems. Criteria that can be used as a basis for determining membership in the multigene family is presented and discussed, as well as the rationale for using a nomenclature system for the gap junction multigene family that is based on genetic and structural relationships rather than the molecular size of the deduced protein products.

Publisher Summary Gap junctional communication among cells is an important mechanism for regulating events during early embryogenesis and subsequent organogenesis in vertebrate embryos. This chapter discusses the potential role and expression of gap junctional communication during mouse development. Gap junctions are specialized regions of the cell surface membrane that connect cells in most multicellular animal organisms. The membranes at this specialized site of cell–cell contact are characterized by a close apposition of the two membranes of adjacent cells that are separated by a narrow “gap.” The internal membrane differentiation that is associated with this site of junctional contact is characterized by a polygonal arrangement of membrane particles or oligomers, which have been referred to as “connexons.” All vertebrate embryos utilize gap junctional communication pathways by the blastula stage of development. In most populations of differentiated cells, gap junctional communication is normally associated with the final differentiation phenotype, with the exception of circulating blood and lymph cells and mature skeletal muscle fibers. Gap junctional communication plays an important role in the process of patterning that is required for producing proper structures during the development of this lineage of cells.

Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes expression the alpha 1 (connexin 43) and beta 2 (connexin 26) proteins. Within 5 min of TPA treatment, the alpha 1 protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The beta 2 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The alpha 1 protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while beta 2 expression was greatly reduced. The effects of TPA and ras on alpha 1 expression were additive; treatment of ras-transformed cells with TPA resulted in increased alpha 1 phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.

Gap junctions are widely distributed structures that mediate communication between cells. The channels that allow passage of small molecules between adjacent cells are made up of oligomeric proteins (connexins) that are encoded by a family of related genes. By probing somatic cell hybrid DNA on Southern filters with rat or human cDNAs or human genomic fragments, we have mapped four functioning gap junction genes, (alpha 1, beta 1, beta 2, and alpha 3), to different sites on human chromosomes: GJA1 (connexin43) to 6p21.1-q24.1; GJB1 (connexin32) to Xcen-q22; GJB2 (connexin26) to 13; and GJA3 (connexin46) also to 13, probably near GJB2. The GJA3 probe also hybridized to a restriction fragment that was mapped to chromosome 1. A GJA1-related pseudogene GJA1P was assigned to chromosome 5. The homologous loci in mouse were assigned to regions of known conserved syntenic groups: Gja-1 to chromosome 10; Gjb-1 to XD-F4 and Gjb-2 to 14. Of two sites of hybridization with the GJA3 probe, on mouse 14 and 5, we assume that the site on 14 corresponds to the GJA3 locus on human 13. Based on these data, additional members of this family of related genes can be isolated and characterized, and possible human and mouse mutations can be identified.