scholarly journals Molecular characterization and functional expression of the human cardiac gap junction channel.

1990 ◽  
Vol 111 (2) ◽  
pp. 589-598 ◽  
Author(s):  
G I Fishman ◽  
D C Spray ◽  
L A Leinwand
Keyword(s):  
Gap Junction ◽  
Dna Analysis ◽  
Single Channel ◽  
Expression System ◽  
Molecular Genetic ◽  
Predicted Amino Acid Sequence ◽  
Gap Junction Channel ◽  
Junction Protein ◽  
Junctional Conductance ◽  
Single Channel Currents

Gap junctions permit the passage of ions and chemical mediators from cell to cell. To identify the molecular genetic basis for this coupling in the human heart, we have isolated clones from a human fetal cardiac cDNA library which encode the full-length human cardiac gap junction (HCGJ) mRNA. The predicted amino acid sequence is homologous to the rat cardiac gap junction protein, connexin43 (Beyer, E. D., D. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629), differing by 9 of 382 amino acids. HCGJ mRNA is detected as early as fetal week 15 and persists in adult human cardiac samples. Genomic DNA analysis suggests the presence of two highly homologous HCGJ loci, only one of which is functional. Stable transfection of the HCGJ cDNA into SKHep1 cells, a human hepatoma line which is communication deficient, leads to the formation of functional channels. Junctional conductance in pairs of transfectants containing 10 copies of the HCGJ sequence is high (approximately 20 nS). Single channel currents are detectable in this expression system and correspond to conductances of approximately 60 pS. These first measurements of the HCGJ channel are similar to the junctional conductance recorded between pairs of rat or guinea pig cardiocytes.

Distinctive gap junction channel types connect WB cells, a clonal cell line derived from rat liver

1991 ◽  
Vol 260 (3) ◽  
pp. C513-C527 ◽  
Author(s):  
D. C. Spray ◽  
M. Chanson ◽  
A. P. Moreno ◽  
R. Dermietzel ◽  
P. Meda
Keyword(s):  
Gap Junction ◽  
Cell Line ◽  
Rat Liver ◽  
Connexin 43 ◽  
Single Channel ◽  
Freeze Fracture ◽  
Particle Sizes ◽  
Frequency Distributions ◽  
Gap Junction Channel ◽  
Junctional Conductance

Gap junctions, dye coupling, and junctional conductance were studied in a cell line (WB) that is derived from rat liver and displays a phenotype similar to “oval” cells. In freeze-fracture replicas, two distinctive particle sizes were detected in gap junctional plaques. Immunocytochemical studies indicated punctate staining at membrane appositions using antibodies to connexin 43 and to a brain gap junction-associated antigen (34 kDa). No staining was observed using antibodies prepared against rat liver gap junction proteins (connexins 32 and 26). Pairs of WB cells were electrically and dye coupled. Junctional conductance (gj) between cell pairs averaged approximately 10 nS; occasionally, gj was low enough that unitary junctional conductances (gamma j) could be detected. Using a CsCl-containing electrode solution, distinctive gamma j values were recorded: approximately 20-30 pS, approximately 80-90 pS, and the sum of the other sizes. The largest gamma j events were apparently due to random coincident openings or closures of the smaller channels. Several treatments reduced gj. Frequency distributions of gamma j were unaltered by 2 mM halothane or 3.5 heptanol, but the sizes of intermediate and largest events were reduced slightly by 100 nM phorbol ester, and the relative frequency of the largest events was increased by 10 microM glutaraldehyde. We conclude that the distinctive gamma j values represent openings and closures of two distinct types of gap junction channels rather than substates of a single channel type; these unitary conductances may correspond to the dual immunoreactivity and to the two particle sizes seen in freeze fracture.

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.

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium

2000 ◽  
Vol 278 (5) ◽  
pp. H1662-H1670 ◽  
Author(s):  
Jeffrey E. Saffitz ◽  
Karen G. Green ◽  
William J. Kraft ◽  
Kenneth B. Schechtman ◽  
Kathryn A. Yamada
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ventricular Myocytes ◽  
Intercellular Junctions ◽  
Ventricular Myocardium ◽  
Electron Microscopic ◽  
Confocal Immunofluorescence Microscopy ◽  
Normal Number ◽  
Gap Junction Channel ◽  
Junction Protein

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/− mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/− mice was due almost entirely to a reduction in the number of individual gap junctions (226 ± 52 vs. 150 ± 32 individual gap junctions/field in Cx43 +/+ and +/− ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.

scholarly journals Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model

2021 ◽  
Vol 22 (23) ◽  
pp. 13055
Author(s):  
Camillo Peracchia ◽  
Lillian Mae Leverone Peracchia
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Wild Type ◽  
X Ray Diffraction ◽  
The Past ◽  
Channel Regulation ◽  
Gating Model ◽  
Gap Junction Channel ◽  
Chemical Gating ◽  
Nanomolar Range

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca2+i]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.

Structure of a Recombinant Gap Junction Channel at 7Å Resolution

1998 ◽  
Vol 4 (S2) ◽  
pp. 464-465
Author(s):  
V.M. Unger ◽  
D. W. Entrikin ◽  
X. Guan ◽  
N.M. Kumar ◽  
N.B. Gilula ◽  
...  
Keyword(s):  
Gap Junction ◽  
Membrane Lipids ◽  
Close Contact ◽  
Cardiac Contraction ◽  
Metabolic Coupling ◽  
Gap Junction Channel ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
Α Helix ◽  
2D Crystals

Gap junction membrane channels mediate the electrical and metabolic coupling between cells. The channels are formed by the end-to-end docking of two hemichannels (connexons), each of which is formed by a hexameric cluster of protein subunits (connexins). The principal gap junction protein in the heart, α1 connexin (also designated Cx43), mediates action potential propagation between cells in order to synchronize cardiac contraction.We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated two-dimensional (2D) crystals of a recombinant, truncated α1- connexin (α1Cx263T). The projection map at 7Å resolution revealed that each 30kD connexin subunit has a transmembrane α-helix that lines the aqueous pore and a second α-helix in close contact with the membrane lipids [Nature Struct. Biol. 4: 39-43 (1997)]. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by ∼30°. Furthermore, apparent non-crystallographic twofold axes predicted that the two apposing connexons adopt identical conformations.

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.

Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances

2006 ◽  
Vol 291 (6) ◽  
pp. C1366-C1376 ◽  
Author(s):  
H.-Z. Wang ◽  
Peter R. Brink ◽  
George J. Christ
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Single Channel ◽  
Short Term ◽  
Bladder Tissue ◽  
Western Blots ◽  
Gap Junction Channel ◽  
Junctional Communication ◽  
Subconductance States ◽  
Human Detrusor

Several independent lines of investigation indicate that intercellular communication through gap junctions modulates bladder physiology and, moreover, that altered junctional communication may contribute to detrusor overactivity. However, as far as we are aware, there are still no direct recordings of gap junction-mediated intercellular currents between human or rat detrusor myocytes. Northern and Western blots were used to identify connexin expression in frozen human bladder tissue and short-term cultured human detrusor myocytes. Double whole cell patch (DWCP) recording revealed that human detrusor myocyte cell pairs were well coupled with an average junctional conductance of 6.5 ± 4.6 nS (ranging from 0.1 to 15 nS, n = 22 cell pairs). Macroscopic gap junction channel currents in human detrusor myocytes exhibited voltage dependence similar to homotypic connexin43. The normalized transjunctional conductance-voltage ( Gj- Vj) relationship was symmetrical and well described by a two-state Boltzmann relation ( Gmin≈ 0.33, V0= 63.6 mV, Z = 0.117 or equal to 2.95 gating charges), suggestive of a bilateral voltage-gated mechanism. In symmetric 165 mM CsCl, the measured single-channel slope conductance was ∼120 pS for the fully open channel and ∼26 pS for the major substate. Occasionally, other subconductance states were also observed. The single-channel mean open time declined with increasing Vj, accounting for the Vj-dependent decline of macroscopic junctional current. Qualitatively similar electrophysiological characteristics were observed in DWCP of freshly isolated rat detrusor myocytes. These data confirm and extend previous observations and are consistent with reports in other smooth muscle cells types in which Cx43-mediated intercellular communication has been identified.

scholarly journals Gap junction channels formed by coexpressed connexin40 and connexin43

2001 ◽  
Vol 281 (4) ◽  
pp. H1675-H1689 ◽  
Author(s):  
Virginijus Valiunas ◽  
Joanna Gemel ◽  
Peter R. Brink ◽  
Eric C. Beyer
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Single Channel ◽  
Potential Interaction ◽  
Whole Cell Patch Clamp ◽  
Junctional Conductance ◽  
Voltage Dependent ◽  
Potential Formation ◽  
Cardiovascular Cells ◽  
Gap Junction Hemichannels

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)6-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance ( g j,ss) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells ( V j) compared with homotypic gap junctions and/or an asymmetrical V j dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.

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