scholarly journals Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function

2012 ◽  
Vol 23 (17) ◽  
pp. 3299-3311 ◽  
Author(s):  
Oscar Jara ◽  
Rodrigo Acuña ◽  
Isaac E. García ◽  
Jaime Maripillán ◽  
Vania Figueroa ◽  
...  
Keyword(s):  
Gap Junction ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Full Length ◽  
Intermediate Step ◽  
Wild Type ◽  
Gap Junction Channels ◽  
Junction Protein ◽  
And Function ◽  
Pore Domain

To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37–Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.

scholarly journals Asymmetry of an intracellular scaffold at vertebrate electrical synapses

2017 ◽  
Author(s):  
Audrey J Marsh ◽  
Jennifer Carlisle Michel ◽  
Anisha P Adke ◽  
Emily L Heckman ◽  
Adam C Miller
Keyword(s):  
Gap Junction ◽  
Synapse Formation ◽  
Neural Circuit ◽  
Electrical Synapse ◽  
Electrical Synapses ◽  
Gap Junction Channels ◽  
Junction Protein ◽  
Chemical Synapses ◽  
And Function

AbstractNeuronal synaptic connections are electrical or chemical and together are essential to dynamically defining neural circuit function. While chemical synapses are well known for their biochemical complexity, electrical synapses are often viewed as comprised solely of neuronal gap junction channels that allow direct ionic and metabolic communication. However, associated with the gap junction channels are structures observed by electron microscopy called the Electrical Synapse Density (ESD). The ESD has been suggested to be critical for the formation and function of the electrical synapse, yet the biochemical makeup of these structures is poorly understood. Here we find that electrical synapse formation in vivo requires an intracellular scaffold called Tight Junction Protein 1b (Tjp1b). Tjp1b is localized to electrical synapses where it is required for the stabilization of the gap junction channels and for electrical synapse function. Strikingly, we find that Tjp1b protein localizes and functions asymmetrically, exclusively on the postsynaptic side of the synapse. Our findings support a novel model in which there is molecular asymmetry at the level of the intracellular scaffold that is required for building the electrical synapse. ESD molecular asymmetries may be a fundamental motif of all nervous systems and could support functional asymmetry at the electrical synapse.

scholarly journals Decreased connexin43 expression in the mouse heart potentiates pacing-induced remodeling of repolarizing currents

2008 ◽  
Vol 295 (5) ◽  
pp. H1905-H1916 ◽  
Author(s):  
Andrianos Kontogeorgis ◽  
Xiaodong Li ◽  
Eunice Y. Kang ◽  
Jonathan E. Feig ◽  
Marc Ponzio ◽  
...  
Keyword(s):  
Gap Junction ◽  
Ventricular Myocytes ◽  
Critical Role ◽  
Mouse Heart ◽  
Wild Type ◽  
Short Term ◽  
Wild Type Littermate ◽  
Cx43 Expression ◽  
Significant Prolongation ◽  
Electrophysiological Effects

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose toward arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiological effects of GJR. We paced wild-type (normal Cx43 abundance) and heterozygous Cx43 knockout (Cx43+/−; 66% mean reduction in Cx43) mice for 6 h at 10–15% above their average sinus rate. We investigated the electrophysiological effects of pacing on the whole animal using programmed electrical stimulation and in isolated ventricular myocytes with patch-clamp studies. Cx43+/− myocytes had significantly shorter action potential durations (APD) and increased steady-state ( Iss) and inward rectifier ( IK1) potassium currents compared with those of wild-type littermate cells. In Cx43+/− hearts, pacing resulted in a significant prolongation of ventricular effective refractory period and APD and significant diminution of Iss compared with unpaced Cx43+/− hearts. However, these changes were not seen in paced wild-type mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiological changes, which may contribute to the worsened prognosis often associated with pacing in the failing heart.

scholarly journals GJA1 Depletion Causes Ciliary Defects by Affecting Rab11 Trafficking to the Ciliary Base

2020 ◽  
Author(s):  
Dong Gil Jang ◽  
Keun Yeong Kwon ◽  
Yeong Cheon Kweon ◽  
Byung-gyu Kim ◽  
Kyungjae Myung ◽  
...  
Keyword(s):  
Gap Junction ◽  
Primary Cilium ◽  
Transport Channel ◽  
Junction Protein ◽  
Complex Functions ◽  
Mother Centriole ◽  
Pericentriolar Material ◽  
Gap Junction Protein ◽  
And Function ◽  
Distinct Distribution

AbstractThe gap junction complex functions as a transport channel across the membrane. Among gap junction subunits, gap junction protein alpha 1 (GJA1) is the most commonly expressed subunit. However, the roles of GJA1 in the formation and function of cilia remain unknown. Here, we examined GJA1 functions during ciliogenesis in vertebrates. GJA1 was localized to the motile ciliary axonemes or pericentriolar material (PCM) around the primary cilium. GJA1 depletion caused the severe malformation of both primary cilium and motile cilia. Interestingly, GJA1 depletion caused strong delocalization of BBS4 from the PCM and basal body and distinct distribution as cytosolic puncta. Further, CP110 removal from the mother centriole was significantly reduced by GJA1 depletion. Importantly, Rab11, key regulator during ciliogenesis, was immunoprecipitated with GJA1 and GJA1 knockdown caused the mis-localization and mis-accumulation of Rab11. These findings suggest that GJA1 is necessary for proper ciliogenesis by regulating the Rab11 pathway.

scholarly journals Involvement of the Gap Junction Protein, Connexin43, in the Formation and Function of Invadopodia in the Human U251 Glioblastoma Cell Line

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 117 ◽  
Author(s):  
Amandine Chepied ◽  
Zeinaba Daoud-Omar ◽  
Annie-Claire Meunier-Balandre ◽  
Dale W. Laird ◽  
Marc Mesnil ◽  
...  
Keyword(s):  
Gap Junction ◽  
Molecular Mechanisms ◽  
Glioblastoma Cells ◽  
Dynamic Interactions ◽  
Cx43 Expression ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
And Function ◽  
Low Cell Density ◽  
Invadopodia Formation

The resistance of glioblastomas to treatments is mainly the consequence of their invasive capacities. Therefore, in order to better treat these tumors, it is important to understand the molecular mechanisms which are responsible for this behavior. Previous work suggested that gap junction proteins, the connexins, facilitate the aggressive nature of glioma cells. Here, we show that one of them—connexin43 (Cx43)—is implicated in the formation and function of invadopodia responsible for invasion capacity of U251 human glioblastoma cells. Immunofluorescent approaches—combined with confocal analyses—revealed that Cx43 was detected in all the formation stages of invadopodia exhibiting proteolytic activity. Clearly, Cx43 appeared to be localized in invadopodia at low cell density and less associated with the establishment of gap junctions. Accordingly, lower extracellular matrix degradation correlated with less mature invadopodia and MMP2 activity when Cx43 expression was decreased by shRNA strategies. Moreover, the kinetics of invadopodia formation could be dependent on Cx43 dynamic interactions with partners including Src and cortactin. Interestingly, it also appeared that invadopodia formation and MMP2 activity are dependent on Cx43 hemichannel activity. In conclusion, these results reveal that Cx43 might be involved in the formation and function of the invadopodia of U251 glioblastoma cells.

scholarly journals An Alternatively Translated Connexin 43 Isoform, GJA1-11k, Localizes to the Nucleus and Can Inhibit Cell Cycle Progression

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 473
Author(s):  
Irina Epifantseva ◽  
Shaohua Xiao ◽  
Rachel E. Baum ◽  
André G. Kléber ◽  
TingTing Hong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Gap Junction ◽  
Connexin 43 ◽  
Cell Cycle Progression ◽  
Tumor Formation ◽  
Full Length ◽  
Cycle Progression ◽  
Junction Protein ◽  
Truncated Isoforms

Connexin 43 (Cx43) is a gap junction protein that assembles at the cell border to form intercellular gap junction (GJ) channels which allow for cell–cell communication by facilitating the rapid transmission of ions and other small molecules between adjacent cells. Non-canonical roles of Cx43, and specifically its C-terminal domain, have been identified in the regulation of Cx43 trafficking, mitochondrial preconditioning, cell proliferation, and tumor formation, yet the mechanisms are still being explored. It was recently identified that up to six truncated isoforms of Cx43 are endogenously produced via alternative translation from internal start codons in addition to full length Cx43, all from the same mRNA produced by the gene GJA1. GJA1-11k, the 11kDa alternatively translated isoform of Cx43, does not have a known role in the formation of gap junction channels, and little is known about its function. Here, we report that over expressed GJA1-11k, unlike the other five truncated isoforms, preferentially localizes to the nucleus in HEK293FT cells and suppresses cell growth by limiting cell cycle progression from the G0/G1 phase to the S phase. Furthermore, these functions are independent of the channel-forming full-length Cx43 isoform. Understanding the apparently unique role of GJA1-11k and its generation in cell cycle regulation may uncover a new target for affecting cell growth in multiple disease models.

Slow intercellular Ca2+ signaling in wild-type and Cx43-null neonatal mouse cardiac myocytes

2000 ◽  
Vol 279 (6) ◽  
pp. H3076-H3088 ◽  
Author(s):  
Sylvia O. Suadicani ◽  
Monique J. Vink ◽  
David C. Spray
Keyword(s):  
Gap Junction ◽  
Cardiac Myocytes ◽  
Channel Blocker ◽  
Atp Release ◽  
Neonatal Mouse ◽  
Wild Type ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Main Pathway ◽  
Stimulation Of

Focal mechanical stimulation of single neonatal mouse cardiac myocytes in culture induced intercellular Ca2+ waves that propagated with mean velocities of ∼14 μm/s, reaching ∼80% of the cells in the field. Deletion of connexin43 (Cx43), the main cardiac gap junction channel protein, did not prevent communication of mechanically induced Ca2+ waves, although the velocity and number of cells communicated by the Ca2+ signal were significantly reduced. Similar effects were observed in wild-type cardiac myocytes treated with heptanol, a gap junction channel blocker. Fewer cells were involved in intercellular Ca2+ signaling in both wild-type and Cx43-null cultures in the presence of suramin, a P2-receptor blocker; blockage was more effective in Cx43-null than in wild-type cells. Thus gap junction channels provide the main pathway for communication of slow intercellular Ca2+ signals in wild-type neonatal mouse cardiac myocytes. Activation of P2-receptors induced by ATP release contributes a secondary, extracellular pathway for transmission of Ca2+ signals. The importance of such ATP-mediated Ca2+ signaling would be expected to be enhanced under ischemic conditions, when release of ATP is increased and gap junction channels conductance is significantly reduced.

scholarly journals Interactions between Brucella suis VirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems

2009 ◽  
Vol 191 (9) ◽  
pp. 2985-2992 ◽  
Author(s):  
Gisèle Bourg ◽  
Romain Sube ◽  
David O'Callaghan ◽  
Gilles Patey
Keyword(s):  
Critical Role ◽  
Type Iv Secretion ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iv ◽  
Brucella Suis ◽  
Periplasmic Domain ◽  
Tm Domain ◽  
And Function

ABSTRACT The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.

The Role of Gap Junction Proteins in Infertility

2010 ◽  
Vol 1 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Piyush Tripathi ◽  
Manorama Tripathi
Keyword(s):  
Gap Junction ◽  
Critical Role ◽  
Growth Control ◽  
Successful Outcome ◽  
Basic Unit ◽  
Junction Protein ◽  
Gap Junction Proteins ◽  
Infertility Patients ◽  
Junction Proteins

ABSTRACT Testis and ovary serve an important role of producing male and female gametes. Their normal functioning is very important for the proper formation of sperm and ovum and thus has a critical role in the successful fertility outcome. Synchronized activity of various cells in the gonads is needed to provide favorable niche for the growth and development of the germ cells. Among various ways of cellular communication, intercellular communication is mediated by gap junctions, which provides open but selective exchange of ion and molecules of restricted size between two adjoining cells. The basic unit of gap junction is connexins. Their important role has been speculated in the maintenance of homeostasis, morphogenesis, cell differentiation, and growth control in higher organisms. The expression of gap junction proteins in reproductive tissues has drawn the attention and interest of researcher to investigate their role in the reproductive outcome. The reports about the correlation of gap junction protein expression pattern in infertility patients and in animal models have suggested their implication in fertility. Some of these gap junction proteins seem to have redundant functions, whereas some could be very critical in the normal fertility and could not be dispensable for the successful outcome of the reproduction.

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