Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37

1997 ◽  
Vol 273 (4) ◽  
pp. C1386-C1396 ◽  
Author(s):  
P. R. Brink ◽  
K. Cronin ◽  
K. Banach ◽  
E. Peterson ◽  
E. M. Westphale ◽  
...  
Keyword(s):  
Gap Junction ◽  
Large Range ◽  
Cell Types ◽  
Channel Activity ◽  
Fluorescent Marker ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Whole Cell Patch Clamp ◽  
A Cell ◽  
Heterotypic Channels

Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage ( G j/ V j) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The G j/ V jdata imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.

scholarly journals Asparagine 175 of Connexin32 Is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Connexin26

2011 ◽  
Vol 286 (22) ◽  
pp. 19672-19681 ◽  
Author(s):  
So Nakagawa ◽  
Xiang-Qun Gong ◽  
Shoji Maeda ◽  
Yuhua Dong ◽  
Yuko Misumi ◽  
...  
Keyword(s):  
Experimental Data ◽  
Hydrogen Bonds ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Cell Junctions ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Critical Residue ◽  
Heterotypic Channels ◽  
Cell Cell

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn175 of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

Cardiac gap junction channel activity in embryonic chick ventricle cells

1988 ◽  
Vol 254 (1) ◽  
pp. H170-H180 ◽  
Author(s):  
R. D. Veenstra ◽  
R. L. DeHaan
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Gap Junction Channel ◽  
Whole Cell Patch Clamp ◽  
Junctional Conductance ◽  
Low Levels ◽  
Active Channels ◽  
Channel Currents

We have recorded single-gap junction-channel currents from pairs of 7-day chick embryo ventricle cells, using the double whole cell patch-clamp technique. Junctional conductance (Gj) was variable from one preparation to the next, ranging from 0.15 to 35.0 nS. Single-channel conductance (gamma j) of the main junctional channel was 166 +/- 51 pS and was independent of Gj; a second conductance level of 60–80 pS was also seen in favorable records. The transition time from the closed to the open state was 285 +/- 153 microseconds, with some slow transitions lasting 1–5 ms. Channels opened and closed stochastically; Gj could be defined by the product of the number of active channels in the junction (N), the mean open-state probability (Po) of the channels, and gamma j. Channel activity was unaffected by cell membrane potential or by transjunctional potential. Po and Gj were reversibly reduced to low levels by 1-octanol or by elevated [Cai], whereas gamma j was unchanged by these agents. The 60–80 pS conductance mechanism was octanol- and Ca-resistant, but it is not clear whether this represents a subconductance level of the main channel or a separate class of smaller channels.

Heterotypic gap junction channel formation between heteromeric and homomeric Cx40 and Cx43 connexons

2001 ◽  
Vol 281 (5) ◽  
pp. C1559-C1567 ◽  
Author(s):  
G. Trevor Cottrell ◽  
Janis M. Burt
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Cell Voltage ◽  
Cell Types ◽  
Channel Formation ◽  
Gap Junction Channel ◽  
Heteromeric Channel ◽  
A7r5 Cells ◽  
Voltage Dependent ◽  
Heterotypic Channels

Recent evidence indicating formation of functional homomeric/heterotypic gap junction channels by connexin40 (Cx40) and connexin43 (Cx43) raises the question of whether data previously interpreted as support for heteromeric channel formation by these connexins might not instead reflect the activity of homomeric/heterotypic channels. To address this question and to further characterize the behavior of these channels, we used dual whole cell voltage-clamp techniques to examine the junctions formed between cells that express only Cx40 (Rin40) or Cx43 (Rin43) and compared the results with those obtained when either of these cell types was paired with cells that naturally express both connexins (A7r5 cells). Rin40/Rin43 cell pairs formed functional gap junctions that displayed a strongly asymmetric voltage-dependent gating response. Single-channel event amplitudes ranged between 34 and 150 pS, with 90- to 130-pS events predominating. A7r5/Rin43 and A7r5/Rin40 cell pairs had voltage-dependent gating responses that varied greatly, with most pairs demonstrating strong asymmetry. These cell pairs exhibited a variety of single-channel events that were not consistent with homomeric/homotypic Cx40 or Cx43 channels or homomeric/heterotypic Cx40/Cx43 channels. These data indicate that Cx40 and Cx43 form homomeric/heterotypic as well as heteromeric/heterotypic channels that display unique gating and conductance properties.

Opposing gates model for voltage gating of gap junction channels

2001 ◽  
Vol 281 (5) ◽  
pp. C1604-C1613 ◽  
Author(s):  
Ye Chen-Izu ◽  
Alonso P. Moreno ◽  
Robert A. Spangler
Keyword(s):  
Gap Junction ◽  
Present Model ◽  
Voltage Gating ◽  
Voltage Range ◽  
Gap Junction Channels ◽  
Gating Model ◽  
Gap Junction Channel ◽  
Intercellular Channels ◽  
Heterotypic Channels

Gap junctions are intercellular channels that link the cytoplasm of neighboring cells. Because a gap junction channel is composed of two connexons docking head-to-head with each other, the channel voltage-gating profile is symmetrical for homotypic channels made of two identical connexons (hemichannels) and asymmetric for the heterotypic channels made of two different connexons (i.e., different connexin composition). In this study we have developed a gating model that allows quantitative characterization of the voltage gating of homotypic and heterotypic channels. This model differs from the present model in use by integrating, rather than separating, the contributions of the voltage gates of the two member connexons. The gating profile can now be fitted over the entire voltage range, eliminating the previous need for data splicing and fusion of two hemichannel descriptions, which is problematic when dealing with heterotypic channels. This model also provides a practical formula to render quantitative several previously qualitative concepts, including a similarity principle for matching a voltage gate to its host connexon, assignment of gating polarity to a connexon, and the effect of docking interactions between two member connexons in an intact gap junction channel.

Human connexin 43 gap junction channel gating: evidence for mode shifts and/or heterogeneity

1996 ◽  
Vol 271 (1) ◽  
pp. C321-C331 ◽  
Author(s):  
P. R. Brink ◽  
S. V. Ramanan ◽  
G. J. Christ
Keyword(s):  
Gap Junction ◽  
Connexin 43 ◽  
Time Course ◽  
Homogeneous Population ◽  
Mode Shift ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Whole Cell Patch Clamp ◽  
Open Time ◽  
Multichannel Recordings

The gating parameters of human connexin 43 (Cx43) gap junction channels were determined using dual whole cell patch clamp and methods designed for analysis of multichannel recordings. Under steady-state conditions, the mean open time (MOT) of Cx43 gap junction channels was computed and it ranged from 0.43 to 5.25 s. The computed mean closed times (MCT) varied from 0.21 to 1.49 s. Analysis showed that, while the MOT declined with increasing transjunctional voltage (Vj), the apparent decline in the MCT with Vj was not statistically significant. The calculated open probabilities ranged from 0.50 to 0.95. Inspection of the data showed that there was a prolonged decay in junctional current, which had a time course of 60-150 s. The analysis excluded the possibility of a homogeneous voltage inactivated/deactivated population of independent and identical Cx43 gap junction channels. The analysis provided evidence for a homogeneous population of Cx43 channels, which can mode shift under the influence of voltage. The latter case cannot be distinguished from a heterogeneous population of Cx43 channels in which one population is voltage inactivated/deactivated and another is unaffected or weakly inactivated/deactivated by voltage.

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 Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain

2012 ◽  
Vol 302 (10) ◽  
pp. C1548-C1556 ◽  
Author(s):  
Qin Xu ◽  
Richard F. Kopp ◽  
Yanyi Chen ◽  
Jenny J. Yang ◽  
Michael W. Roe ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Connexin 43 ◽  
Binding Domain ◽  
Cytoplasmic Loop ◽  
Inhibitory Peptide ◽  
Open Probability ◽  
Calmodulin Binding ◽  
Gap Junction Channel ◽  
Whole Cell Patch Clamp

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca2+-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca2+ influx reduced Cx43 gap junction conductance (Gj) by 95%, while increasing cytosolic Ca2+ concentration threefold. By contrast, Cx40 Gj declined by <20%. The Ca2+-induced decline in Cx43 Gj was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca2+-free extracellular solution, if Ca2+ chelation was commenced before complete uncoupling, after which gj was only 60% recoverable. The Cx43 CL136–158 mimetic peptide, but not the scrambled control peptide, or Ca2+/CaM-dependent kinase II 290–309 inhibitory peptide also prevented the Ca2+/CaM-dependent decline of Cx43 Gj. Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca2+/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca2+/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca2+ regulatory properties of Cx43 and Cx40.

scholarly journals Role of Gap Junctions and Hemichannels in Parasitic Infections

2013 ◽  
Vol 2013 ◽  
pp. 1-17 ◽  
Author(s):  
José Luis Vega ◽  
Mario Subiabre ◽  
Felipe Figueroa ◽  
Kurt Alex Schalper ◽  
Luis Osorio ◽  
...  
Keyword(s):  
Gap Junction ◽  
Viral Infections ◽  
Cellular Communication ◽  
Parasitic Infections ◽  
Pathological State ◽  
Channel Changes ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Relevant Role

In vertebrates, connexins (Cxs) and pannexins (Panxs) are proteins that form gap junction channels and/or hemichannels located at cell-cell interfaces and cell surface, respectively. Similar channel types are formed by innexins in invertebrate cells. These channels serve as pathways for cellular communication that coordinate diverse physiologic processes. However, it is known that many acquired and inherited diseases deregulate Cx and/or Panx channels, condition that frequently worsens the pathological state of vertebrates. Recent evidences suggest that Cx and/or Panx hemichannels play a relevant role in bacterial and viral infections. Nonetheless, little is known about the role of Cx- and Panx-based channels in parasitic infections of vertebrates. In this review, available data on changes in Cx and gap junction channel changes induced by parasitic infections are summarized. Additionally, we describe recent findings that suggest possible roles of hemichannels in parasitic infections. Finally, the possibility of new therapeutic designs based on hemichannel blokers is presented.

scholarly journals Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions.

1995 ◽  
Vol 6 (12) ◽  
pp. 1707-1719 ◽  
Author(s):  
B R Kwak ◽  
M M Hermans ◽  
H R De Jonge ◽  
S M Lohmann ◽  
H J Jongsma ◽  
...  
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Cell Communication ◽  
Differential Regulation ◽  
Cell Coupling ◽  
Gap Junction Channels ◽  
Physiological Importance ◽  
Gap Junction Channel ◽  
Conductance States ◽  
Channel Properties

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.

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