scholarly journals Biophysical Properties of Connexin-45 Gap Junction Hemichannels Studied in Vertebrate Cells

2002 ◽  
Vol 119 (2) ◽  
pp. 147-164 ◽  
Author(s):  
Virginijus Valiunas
Keyword(s):  
Gap Junction ◽  
Open Channel ◽  
Lucifer Yellow ◽  
External Solution ◽  
Dye Molecules ◽  
Dye Uptake ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Cell Recording ◽  
Gap Junction Hemichannels

Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca2+, whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, Vm. These currents did not occur in nontransfected cells. The hemichannel currents were inhibited by raising extracellular Ca2+ or by acidification with CO2. The unitary conductance exhibited Vm dependence (i.e., γhc,main increased/decreased with hyperpolarization/depolarization). Extrapolation to Vm = 0 mV led to a γhc,main of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, Po, was Vm-dependent, declining at negative Vm (Po < 0.11, Vm < −50 mV), and increasing at positive Vm (Po ∼0.76, Vm > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca2+ level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in nontransfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca2+-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca2+. Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca2+ concentration is reduced.

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 Gap junction permeability: selectivity for anionic and cationic probes

2011 ◽  
Vol 300 (3) ◽  
pp. C600-C609 ◽  
Author(s):  
G. Kanaporis ◽  
P. R. Brink ◽  
V. Valiunas
Keyword(s):  
Gap Junction ◽  
Lucifer Yellow ◽  
Second Messengers ◽  
Small Interfering Rnas ◽  
Weak Charge ◽  
Axial Diameter ◽  
Gap Junction Channels ◽  
Charge Selectivity ◽  
Specific Permeability ◽  
A Charge

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 Å, charge −2), carboxyfluorescein (CF; 8.2 Å; −2), and Alexa Fluor350 (AF350, 5.4 Å; −1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N, N, N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 Å, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 Å, +1). Comparison of probe permeability relative to K+ revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.

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.

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 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.

Differences in gap junction channels between cardiac myocytes, fibroblasts, and heterologous pairs

1992 ◽  
Vol 263 (5) ◽  
pp. C959-C977 ◽  
Author(s):  
M. B. Rook ◽  
A. C. van Ginneken ◽  
B. de Jonge ◽  
A. el Aoumari ◽  
D. Gros ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rat Heart ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Channel Conductance ◽  
Open Probability ◽  
Gap Junction Channels ◽  
Gap Junction Channel ◽  
Junction Protein

Cultures of neonatal rat heart cells contain predominantly myocytes and fibroblastic cells. Most abundant are groups of synchronously contracting myocytes, which are electrically well coupled through large gap junctions. Cardiac fibroblasts may be electrically coupled to each other and to adjacent myocytes, be it with low intercellular conductances. Nevertheless, synchronously beating myocytes interconnected via a fibroblast were present, demonstrating that nonexcitable cardiac cells are capable of passive impulse conduction. In fibroblast pairs as well as in myocyte-fibroblast cell pairs, no sensitivity to junctional voltage could be detected when transjunctional conductance was > 1-2 nS. However, in pairs coupled by a conductance of < 1 nS, complex voltage-dependent gating was evident; gap junction channel open probability decreased with increasing junctional voltage but a nongated residual conductance remained at all voltages tested. Single gap junction channel conductance between fibroblasts was approximately 21 pS, very similar to an approximately 18-pS channel conductance that was found between myocytes next to the major conductance of 43 pS. Single-channel conductance in heterologous myocyte-fibroblast gap junctions was approximately 32 pS, which matches the theoretical value of 29 pS for gap junction channels composed of a fibroblast connexon and the major myocyte connexon. A site-directed antibody against rat heart gap junction protein connexin43 recognized gap junctions between neonatal cardiomyocytes, as demonstrated by immunocytochemical labeling. In contrast, junctions between fibroblasts showed no labeling, while in myocyte-fibroblast junctions labeling occasionally was present. Our results suggest the existence of two gap junction proteins between neonatal rat cardiocytes, connexin43 and another yet unidentified connexin. An alternative explanation (cell-specific regulation of the conductance of connexin43 channels) is discussed.

Modulation of gap-junction channel gating at zebrafish retinal electrical synapses

1994 ◽  
Vol 72 (5) ◽  
pp. 2257-2268 ◽  
Author(s):  
D. G. McMahon ◽  
D. R. Brown
Keyword(s):  
Gap Junction ◽  
Horizontal Cell ◽  
Channel Gating ◽  
Horizontal Cells ◽  
General Mechanism ◽  
Spatial Integration ◽  
Open Probability ◽  
Electrical Synapses ◽  
Gap Junction Channels ◽  
Gap Junction Channel

1. Transmission at electrical synapses is modulated by a variety of physiological signals, and this modulation is a potentially general mechanism for regulating signal integration in neural circuits and networks. In the outer plexiform layer of the retina, modulation of horizontal-cell electrical coupling by dopamine alters the extent of spatial integration in the horizontal-cell network. By analyzing the activity of individual gap-junction channels in low-conductance electrical synapses of zebrafish retinal horizontal cells, we have defined the properties of these synaptic ion channels and characterized the functional changes in them during modulation of horizontal-cell electrical synapses. 2. Zebrafish horizontal-cell gap-junction channels have a unitary conductance of 50–60 pS and exhibit open times of several tens of milliseconds. The kinetic process of channel closure is best described by the sum of two rate constants. 3. Dopamine, and its agonist, (+/-)-6,7-dihydroxy-2-amino-tetralin (ADTN), modulates electrical synaptic transmission between horizontal cells predominantly by affecting channel-gating kinetics. These agents reduced the open probability of gap-junction channels two- to threefold by reducing both the duration and frequency of channel openings. Both time constants for channel open duration were reduced, whereas the duration of shut periods was increased. Similar changes in open-time kinetics were observed in power spectra of higher conductance gap junctions. 4. These results provide a description of rapid electrical synaptic modulation at the single channel level. The description should be useful in understanding the mechanisms of plasticity at these synapses throughout the vertebrate central nervous system.

New Roles for Connexons

Physiology ◽  
2003 ◽  
Vol 18 (3) ◽  
pp. 100-103 ◽  
Author(s):  
Lisa Ebihara
Keyword(s):  
Plasma Membrane ◽  
Ion Channels ◽  
Gap Junction ◽  
Paracrine Signaling ◽  
Gap Junction Channels ◽  
Cellular Processes ◽  
Gap Junction Hemichannels

Connexons or gap junction hemichannels are large, nonselective ion channels that reside in the nonjunctional plasma membrane before their assembly into gap junction channels. Increasing evidence suggests that these channels can open under certain conditions and may participate in a number of cellular processes, including the release of small metabolites such as ATP and NAD+, which are involved in paracrine signaling.

scholarly journals Remodeling of Cardiac Gap Junctional Cell–Cell Coupling

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2422
Author(s):  
Stefan Dhein ◽  
Aida Salameh
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Conduction System ◽  
Cell Coupling ◽  
Connexin Expression ◽  
Gap Junction Channels ◽  
Developmental States ◽  
Gap Junction Channel ◽  
Arrhythmogenic Substrate ◽  
Cell Cell

The heart works as a functional syncytium, which is realized via cell-cell coupling maintained by gap junction channels. These channels connect two adjacent cells, so that action potentials can be transferred. Each cell contributes a hexameric hemichannel (=connexon), formed by protein subuntis named connexins. These hemichannels dock to each other and form the gap junction channel. This channel works as a low ohmic resistor also allowing the passage of small molecules up to 1000 Dalton. Connexins are a protein family comprising of 21 isoforms in humans. In the heart, the main isoforms are Cx43 (the 43 kDa connexin; ubiquitous), Cx40 (mostly in atrium and specific conduction system), and Cx45 (in early developmental states, in the conduction system, and between fibroblasts and cardiomyocytes). These gap junction channels are mainly located at the polar region of the cardiomyocytes and thus contribute to the anisotropic pattern of cardiac electrical conductivity. While in the beginning the cell–cell coupling was considered to be static, similar to an anatomically defined structure, we have learned in the past decades that gap junctions are also subject to cardiac remodeling processes in cardiac disease such as atrial fibrillation, myocardial infarction, or cardiomyopathy. The underlying remodeling processes include the modulation of connexin expression by e.g., angiotensin, endothelin, or catecholamines, as well as the modulation of the localization of the gap junctions e.g., by the direction and strength of local mechanical forces. A reduction in connexin expression can result in a reduced conduction velocity. The alteration of gap junction localization has been shown to result in altered pathways of conduction and altered anisotropy. In particular, it can produce or contribute to non-uniformity of anisotropy, and thereby can pre-form an arrhythmogenic substrate. Interestingly, these remodeling processes seem to be susceptible to certain pharmacological treatment.

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.

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