Genetics of the cell surface: Assignment of genes coding for external membrane proteins to human chromosomes 10 and 14

Using somatic cell hybridization gene mapping methodology, genes coding for human cell-surface proteins have been assigned to specific chromosomes. Lactoperoxidase-catalyzed iodination was employed to label external membrane proteins in cell hybrids between mouse and human cultured cells. Mouse and human external membrane proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. After electrophoresis, external membrane proteins were identified by autoradiography. An external membrane protein of 130,000 molecular weight (EMP-130) segregated concordantly with glutamic oxaloacetic transaminase_s (GOT_s, EC 2.6.1.1), an enzyme marker encoded on chromosome 10. External membrane proteins of 195,000 and 175,000 molecular weight (EMP-195 and EMP-175) segregated concordantly with nucleoside phosphorylase (NP, EC 2.4.2.1), an enzyme marker encoded on chromosome 14. Limited proteolysis of the 195,000 and 175,000 molecular weight proteins suggests that these two proteins are modified forms of each other and are encoded by the same locus. These findings demonstrate the mapping of human genes coding for external proteins EMP-130 and EMP-195 to chromosomes 10 and 14, respectively. Chromosome analyses of cell hybrids supported these assignments.     

Modeling the calcium gate of cardiac gap junction channel

We addressed the question how Ca2+ transients affect gap junction conductance (Gj) during action potential (AP) propagation by constructing a dynamic gap junction model coupled with a cardiac cell model. The kinetics of the Ca2+ gate was determined based on published experimental findings that the Hill coefficient for the [Ca2+]i-Gj relationship ranges from 3 to 4, indicating multiple ion bindings. It is also suggested that the closure of the Ca2+ gate follows a single exponential time course. After adjusting the model parameters, a two-state (open-closed) model, assuming simultaneous ion bindings, well described both the single exponential decay and the [Ca2+]i-Gj relationship. Using this gap junction model, 30 cardiac cell models were electrically connected in a one-dimensional cable. However, Gj decreased in a cumulative manner by the repetitive Ca2+ transients, and a conduction block was observed. We found that a reopening of the Ca2+ gate is possible only by assuming a sequential ion binding with one rate limiting step in a multistate model. In this model, the gating time constant (T) has a bell-shaped dependence on [Ca2+]i, with a peak around the half-maximal concentration of [Ca2+]i. Here we propose a five-state model including four open states and one closed state, which allows normal AP propagation; namely, the Gj is decreased -15% by a single Ca2+ transient, but well recovers to the control level during diastole. Under the Ca(2+)-overload condition, however, the conduction velocity is indeed decreased as demonstrated experimentally. This new gap junction model may also be useful in simulations of the ventricular arrhythmia.     

High resolution map of Caenorhabditis elegans gap junction proteins

The innexin family of gap junction proteins has 25 members in Caenorhabditis elegans. Here, we describe the first high‐resolution expression map of all members through analysis of live worms transformed with green fluorescent protein under the control of entire promoter regions. Our analyses show that innexins have dynamic expression patterns throughout development and are found in virtually all cell types and tissues. Complex tissues, such as the pharynx, intestine, gonad, as well as scaffolding tissues and guidepost cells express a variety of innexins in overlapping or complementary patterns, suggesting they may form heteromeric and heterotypic channels. Innexin expression occurs in several types of cells that are not known to form gap junctions as well as in a pair of migrating cells, suggesting they may have hemichannel function. Therefore, innexins likely play roles in almost all body functions, including embryonic development, cell fate determination, oogenesis, egg laying, pharyngeal pumping, excretion, and locomotion. Developmental Dynamics 238:1936–1950, 2009. © 2009 Wiley‐Liss, Inc.     

Chromosomal assignments of mouse connexin genes,coding for gap junctional proteins,by somatic cell hybridization

The connexin genes Cx31 and Cx45 coding for proteins of gap junctional subunits have been assigned to mouse chromosomes 4 and 11 by Southern blot hybridization of specific gene probes to DNA from mouse × Chinese hamster somatic cell hybrids. In addition, our results confirm the recent assignment of mouse connexin genes Cx26, Cx32, Cx37, Cx40, Cx43, and Cx46 to mouse chromosomes 14, X, 4, 3, 10, and 14, respectively, by analysis of interspecific backcrosses and by somatic cell hybridization. Our assignment of the Cx31 gene to mouse chromosome 4 locates the fourth connexin gene on this mouse chromosome to which the genes for Cx31.1, Cx37, and Cx30.3 have previously been assigned. Interestingly three of them (coding for Cx31, Cx31.1, and Cx30.3) are preferentially expressed in skin. Possibly some of the connexin genes clustered on mouse chromosome 4 may be regulated coordinately.     

Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours

Gap junctional intercellular communication (GJIC) has been proposed as a cellular mechanism for tumour suppression and there is experimental evidence in support of this. If aberrant GJIC contributes to the formation of human breast tumours, one might expect that the connexins (gap junction proteins) expressed by epithelial cells in normal human breast would be down-regulated in tumour epithelial cells, or that tumour cells might show aberrant expression of other connexin family members. This study examines the immunocytochemical expression of connexins 26 (Cx26) and 43 (Cx43) in normal human breast, 11 benign breast lesions, two special-type carcinomas, and 27 invasive carcinomas of no special histological type (NST). Cx26 generally was not expressed at detectable levels in normal human breast, but punctate Cx43 immunostaining of the myoepithelial cells was found. Cx43 staining of the myoepithelium was also a feature of the benign lesions and ductal carcinoma in situ (DCIS). In general, the epithelial cells of benign lesions failed to stain for either connexin. Similarly, a lobular carcinoma did not express Cx26 or Cx43, but there was punctate Cx43 in the epithelial cells of a mucoid carcinoma. Cx26 was up-regulated in the carcinoma cells of 15 of the 27 invasive NST carcinomas, although the staining was usually cytoplasmic and heterogeneous. Cx43 was expressed by stromal cells, possibly myofibroblasts, in all NST carcinomas. Furthermore, there was heterogeneous Cx43 expression in the carcinoma cells of 14 of the 27 NST carcinomas and the staining was often intercellular and punctate, characteristic of functional connexins. Up-regulation of Cx26 and/or Cx43 in the carcinoma cells of over two-thirds of invasive lesions of NST is not necessarily inconsistent with a tumour suppressor role for GJIC. However, the role of gap junctions in the formation and progression of solid human tumours is likely to be more complex than indicated from experimental systems. © 1998 John Wiley & Sons, Ltd.     

Chimeric evidence for a role of the connexin cytoplasmic loop in gap junction channel gating

Gap junction channels are regulated by gates that close upon exposure to 100% CO₂, probably via an increase in intracellular Ca²⁺ concentration, [Ca²⁺]_i. For defining connexin (Cx) domain(s) involved in gating, we have studied chemical and voltage gating sensitivities of channels made of Cx38, Cx32 or chimeras of the above, expressed in Xenopus oocytes. Cx38 channels are more sensitive to CO₂ and voltage than those of Cx32. A 3-min exposure to 100% CO₂ reduces Cx38 junctional conductance (G _j) to 0% of initial values at a maximum rate of 25%/min, whereas even a 15-min exposure to 100% CO₂ reduces Cx32 G _j by approximately 50% at the slow rate of 9%/min. Of the various Cx32 mutants and Cx32/38 chimeras constructed, two chimeras (Cx32/38I and Cx32/38N) expressed functional channels. Upon exposure to CO₂, channels made of Cx32/38I (Cx32 inner loop replaced with that of Cx38) reproduced precisely the uncoupling behavior of Cx38 channels in uncoupling magnitude and in both uncoupling and recoupling rates, whereas channels made of Cx32/38N (N-terminus replaced) behaved closer to Cx32 than to Cx38 channels. Cx38 channels were more voltage sensitive than those of Cx32, with V _0, i.e., the transjunctional voltage at which voltage-sensitive conductance is half maximal = 35.3 and 59.5 mV, and n, i.e., equivalent gating charge = 3.3 and 2.1, respectively. Of the two chimeras, Cx32/38I channels were similar to Cx38 channels, with V ₀ = 40.6 mV, G _{j min,} i.e., the theoretical minimal normalized junctional conductance = 0.35 and n = 3.0, whereas Cx32/38 N channels displayed very low voltage sensitivity, with V ₀ = 84.8 mV, G _{j min} = 0.5 and n = 1.1. The data suggest that the inner loop plays a major role in pH and voltage gating sensitivity, but whether other domains also participate in the gating mechanism cannot be excluded. Received: 2 October 1995/Received after revision: 16 November 1995/Accepted: 16 November 1995     

Expression of gap junction genes in astrocytes and C6 glioma cells.

The expression of the gap junction genes coding for the liver-type connexin32 and the heart-type connexin43 was examined in primary cultures of astrocytes and in cultures of C6 glioma cells. In both cell types, only connexin43 mRNA was detectable. However, the level of this mRNA was greatly reduced in C6 glioma cells compared to astrocytes. This was consistent with the further observation that astrocytes in primary culture were extensively dye-coupled, whereas such coupling was very restricted in cultures of C6 glioma cells. Connexin43 was immunocytochemically localized in astrocytes, but was not readily detected in C6 cells.     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.     

pH sensitivity of the cardiac gap junction proteins,connexin 45 and 43

Intercellular communication through gap junction channels can be regulated by changes in intracellular pH (pH_i). This regulation may play an important role in ischemic heart tissue. Using the dual voltage-clamp technique, we compared the pH_i sensitivity of gap junction channels composed of connexin 43 (Cx43) and Cx45, two of the gap junction proteins that are expressed in heart. We made use of SKHep1 cells, endogenously expressing low levels of Cx45 and SKHep1 cells stably transfected with rat Cx43. To manipulate the pH_i we applied the NH₃/NH₄ ⁺ pH-clamp method. At pH_i 6.7 the g_j of Cx45 channels was reduced to 20% of control values (pH_i 7.0) and at pH_i 6.3 all channels closed. The g_j of Cx43 channels was 70% of control values at pH_i 6.7 and 40% at pH_i 6.3. Cx43 channels closed at pH_i 5.8. Single channel conductances were 17.8 pS for Cx45 and 40.8 pS for Cx43 at pH_i 7.0 and did not change significantly at lower pH_i. This suggests that the decrease in macroscopic conductance observed at low pH_i results from the decrease in open probability of gap junctional channels rather than from a decrease in single channel conductance. Our results demonstrate that gap junction channels built of Cx45 are far more pH sensitive than channels built of Cx43.     

Connexin expression and gap junction communication compartments in the developing mouse limb.

Fundamental to the understanding of mouse limb morphogenesis and pattern formation is the need to elucidate the spatial and temporal distribution of gap junction proteins (connexins, Cx) and cell-cell communication compartments. To this end, we used immunofluorescence and confocal microscopy together with 3-dimensional reconstruction software to map the distribution of Cx43 and Cx32 in 11-14.5 days postcoitum (dpc) mouse limbs. Cx43 was strictly localized to the apical ectodermal ridge (AER) and nonridge ectoderm throughout all stages of mouse limb development studied. Cx32, on the other hand, was abundant in the mesenchyme with only low levels of expression in the 11-13.5 dpc ectoderm. However, at 14-14.5 dpc there was a clear increase in Cx32 expression in the ectoderm. Double labeling for connexins and confocal microscopy revealed Cx43 and Cx32 in the same optical section of the basal cells of the ectoderm but in separate plaques. Lucifer yellow dye injections showed that the cells of the AER were in direct communication with the nonridge ectoderm but dye was never observed to spread to the mesenchyme. Cells of the mesenchyme were coupled to each other but to a much lesser extent than cells of the ectoderm. Finally, although there was an increase in Cx32 expression in the ectoderm at 14-14.5 dpc, this was not correlated with any detectable change in communication compartments. Thus, the lack of dye transfer between the ectoderm and underlying mesenchyme from the peak of AER height through its decline suggests that bulk transfer of morphogens between these two layers is not necessary for mouse limb development.     

Specificity in the participation of connexin proteins in flow-induced endothelial gap junction communication

Endothelial cell (EC) dysfunction and atherosclerotic plaque formation coincide with human circulatory regions where blood flow is altered (disturbed). In areas of undisturbed uniform blood flow, including the majority of the vasculature, the vessel wall is relatively atherosclerotic lesion-resistant with normal endothelium. The molecular mechanisms of blood flow regulation of EC function and atherogenesis are unclear. We hypothesize that EC dysfunction potentiating atherosclerosis is related to disturbed flow (DF)-induced EC gap junctional intercellular communication (GJIC) changes via the gap junction connexin (Cx) 37, 40, and 43 proteins, which are involved in EC proliferation and vasoactivity that are known to be altered in atherosclerosis. We investigated human EC GJIC using an in vitro model of the hemodynamic features found in atherosclerotic-prone DF regions in vivo. Using dye transfer assays, Cx-specific mimetic peptide inhibitors, proliferation assays, and immunocytochemistry, we correlated functional GJIC via gap junction channels formed by hemichannels composed of the two most abundant endothelial Cx—Cx40 and Cx43—to EC proliferation and expression of vasoactive endothelial-type nitric oxide synthase (eNOS). We found that, in uniform flow conditions, substantial GJIC was conducted through gap junctions containing Cx40 hemichannels and correlated to a nonproliferative EC phenotype and membrane localization of eNOS, similar to physiological conditions. In DF, GJIC was largely attained through Cx43 hemichannel-containing gap junctions, EC phenotype was proliferative (attributed to loss of contact inhibition), and intracellular eNOS was more abundant than membrane eNOS, typical of atherosclerotic sites in vivo. This is the first in vitro study to demonstrate local hemodynamically defined Cx protein specificity in human EC GJIC with a potential role in endothelial dysfunction characteristic of early atherosclerosis.