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141.
Using a new fluorescence imaging technique, LAMP, we recently reported that Ca2+ influx through store operated Ca2+ channels (SOCs) strongly inhibits cell coupling in primary human fibroblasts (HF) expressing Cx43. To understand the mechanism of inhibition, we studied the involvement of cytosolic pH (pHi) and Ca2+([Ca2+]i) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca2+ influx, there was a modest decline of pHi measured by BCECF. Decreasing pHi below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pHi drop with thioacetate and bulk [Ca2+i rise with ionomycin was much less effective in inhibiting cell coupling than Ca2+ influx. Moreover, clamping pHi with a weak acid and a weak base during Ca2+ influx largely suppressed bulk pHi drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca2+i with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca2+ influx. We concluded that local Ca2+ elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca2+ influx. To assess how Ca2+ influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca2+ influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca2+ influx may be a more general phenomenon. 相似文献
142.
Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C2C12 mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C2C12 cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo. 相似文献
143.
Alexandra Gellhaus Christoph Wotzlaw Teresa Otto Joachim Fandrey Elke Winterhager 《Journal of cellular biochemistry》2010,110(1):129-140
Connexin43 (Cx43) forms gap junction channels but also serves as a signaling center by binding to proteins via its C‐terminus. We have previously demonstrated that transfection of Cx43 leads to significantly reduced proliferation of placental tumor cells through upregulating and binding of the growth regulator CCN3 (NOV) at the C‐terminus of Cx43. Here, we combined fluorescence resonance energy transfer (FRET), co‐immunoprecipitation and proliferation and expression assays to characterize the interaction complex of Cx43 and CCN3. FRET measurements confirmed the interaction of CCN3 with wild‐type Cx43 (amino acids 1‐382) and with mutants of Cx43 truncated at the C‐terminus resulting in Cx43 proteins of amino acids 1‐374, 1‐273, 1‐264, 1‐257 in 293T cells. These results matched the co‐immunoprecipitation data. Interestingly, although FRET revealed distinct efficiencies in interaction of Cx43 with CCN3 for all deletion constructs only wild‐type Cx43 and one deletion construct (1‐374) led to increased CCN3 expression. Only these interactions which were associated with increased CCN3 expression resulted in a reduced cell proliferation. Our study provides evidence that only defined binding properties between Cx43 and CCN3 leading to an upregulation of CCN3 are needed for signaling. Furthermore, the data obtained by FRET analysis allowed us to model the 3D structure of the C‐terminus of Cx43 interacting with CCN3. J. Cell. Biochem. 110: 129–140, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
144.
Michael G. Kozoriz John Church Mark A. Ozog Christian C. Naus Claudia Krebs 《The Journal of biological chemistry》2010,285(41):31107-31119
Increases in extracellular potassium concentration ([K+]o), which can occur during neuronal activity and under pathological conditions such as ischemia, lead to a variety of potentially detrimental effects on neuronal function. Although astrocytes are known to contribute to the clearance of excess K+o, the mechanisms are not fully understood. We examined the potential role of mitochondria in sequestering K+ in astrocytes. Astrocytes were loaded with the fluorescent K+ indicator PBFI and release of K+ from mitochondria into the cytoplasm was examined after uncoupling the mitochondrial membrane potential with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Under the experimental conditions employed, transient applications of elevated [K+]o led to increases in K+ within mitochondria, as assessed by increases in the magnitudes of cytoplasmic [K+] ([K+]i) transients evoked by brief exposures to CCCP. When mitochondrial K+ sequestration was impaired by prolonged application of CCCP, there was a robust increase in [K+]i upon exposure to elevated [K+]o. Blockade of plasmalemmal K+ uptake routes by ouabain, Ba2+, or a mixture of voltage-activated K+ channel inhibitors reduced K+ uptake into mitochondria. Also, reductions in mitochondrial K+ uptake occurred in the presence of mito-KATP channel inhibitors. Rises in [K+]i evoked by brief applications of CCCP following exposure to high [K+]o were also reduced by gap junction blockers and in astrocytes isolated from connexin43-null mice, suggesting that connexins also play a role in K+ uptake into astrocyte mitochondria. We conclude that mitochondria play a key role in K+o handling by astrocytes. 相似文献
145.
Qian Shi Eric A. Banks X. Sean Yu Sumin Gu Janelle Lauer Gregg B. Fields Jean X. Jiang 《The Journal of biological chemistry》2010,285(24):18415-18422
We have previously shown that connexin (Cx) 50, unlike the other two lens connexins, Cx43 and Cx46, promotes chicken lens epithelial-fiber differentiation in a channel-independent manner. Here, we show that deletion of the PEST motif at the C terminus (CT) domain of Cx50 attenuates the stimulatory effect of Cx50 on lens fiber differentiation. Valine 362, a residue located within the PEST domain, is functionally involved. The structure of the Cx50 CT predicted by molecular modeling revealed four α-helices and Val362 was found to be located in the middle of the 3rd helix. Replacement of Val362 with amino acid residues that disrupt the α-helical structure predicted by molecular modeling, such as arginine, glutamate, or phenylalanine, attenuated the stimulatory effects of Cx50 on lens differentiation, whereas replacement with threonine, isoleucine, leucine, or proline, which maintain the structure preserved the function of Cx50. Circular dichroism (CD) studies supported the structural predictions and showed that the substitution with Glu, but not Thr or Pro, disrupted the α-helix, which appears to be the structural feature important for lens epithelial-fiber differentiation. Together, our results suggest that Val362 is important for maintaining the helical structure and is crucial for the role of Cx50 in promoting lens epithelial-fiber differentiation. 相似文献
146.
Helene Knævelsrud 《FEBS letters》2010,584(12):2635-31696
Ubiquitinated protein aggregates are hallmarks of a range of human diseases, including neurodegenerative, liver and muscle disorders. These protein aggregates are typically positive for the autophagy receptor p62. Whereas the ubiquitin-proteasome system (UPS) degrades shortlived and misfolded ubiquitinated proteins that are small enough to enter the narrow pore of the barrel-shaped proteasome, the lysosomal pathway of autophagy can degrade larger structures including entire organelles or protein aggregates. This degradation requires autophagy receptors that link the cargo with the molecular machinery of autophagy and is enhanced by certain posttranslational modifications of the cargo. In this review we focus on how autophagy clears aggregate-prone proteins and the relevance of this process to protein aggregate associated diseases. 相似文献
147.
Cinzia Ambrosi Oliver Gassmann Jennifer N. Pranskevich Daniela Boassa Amy Smock Junjie Wang Gerhard Dahl Claudia Steinem Gina E. Sosinsky 《The Journal of biological chemistry》2010,285(32):24420-24431
Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other. 相似文献
148.
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150.
The distribution of the three nitric oxide synthase (NOS) isoforms was determined immunohistochemically in the human minor and major salivary glands with comparison to that of rat salivary glands. In contrast to rat glands, which contained a dense plexus of neuronal NOS-immunoreactive nerve fibers, only a minority of the nerve fibers in human glands showed neuronal NOS immunoreactivity. Human labial and submandibular glands contained sparse NOS-immunoreactive fibers, while only occasional nerve fibers in the parotid or sublingual glands were stained. Furthermore, in contrast to the animal glands, most duct epithelial cells in all human salivary glands were immunoreactive for neuronal NOS. No specific immunoreactivity for inducible or endothelial NOS were observed in the nerve fibers or duct epithelium. We provide evidence to suggest that the role of nitric oxide in the regulation of salivary gland function is different in human as compared to experimental animals. Nitricergic innervation in human tissue is very sparse and thus nitric oxide is probably of minor importance as a neural regulator of salivary glands. Instead, NOS localized in duct epithelial cells suggests that nitric oxide might directly regulate saliva secretion and it is a putative source of nitrates previously reportedly secreted into the saliva. 相似文献