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

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.

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Calcium-Calmodulin Gating of Connexin43 Gap Junctions in the Absence of the pH Gating Domain

10.1101/311613 ◽

2018 ◽

Author(s):

Siyu Wei ◽

Christian Cassara ◽

Xianming Lin ◽

Richard D Veenstra

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Ventricular Myocytes ◽

External Solution ◽

Inhibitory Peptide ◽

Pipette Solution ◽

Calmodulin Binding ◽

Whole Cell Patch Clamp ◽

Gating Mechanism ◽

Mimetic Peptides

AbstractIntracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction channels and the synergism or antagonism between pH and Ca2+ has been questioned for decades. In this study, we assessed whether the calcium gating mechanism occurs independently of the pH gating mechanism by utilizing the Cx43-M257 (Cx43K258stop) mutant, a carboxyl-terminal (CT) truncated version of Cx43 lacking the pH gating domain. Dual whole cell patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full length Cx43 or Cx43-M257 proteins. Addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 macroscopic gap junction conductance (gj) to zero within 15 min of perfusion, while this response was prevented by omitting 1.8 mM CaCl2 from the external solution or adding 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. The ability of connexin calmodulin binding domain (Cx CaMBD) mimetic peptides and the Gap19 peptide to inhibit the Ca2+/CaM gating response of Cx43 gap junctions was also examined. Internal addition of a Cx50 cytoplasmic loop CaMBD peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had no effect. Lastly, the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions were investigated. We confirmed that the fast kinetic inactivation component was absent in Cx43-M257 gap junctions, but also observed that the previously reported facilitated recovery of gj from inactivating potentials was abolished by CT truncation of Cx43. We conclude that CT pH gating domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact. Sequence-specific Cx CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has no effect on this chemical gating mechanism.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1992.263.5.c959 ◽

1992 ◽

Vol 263 (5) ◽

pp. C959-C977 ◽

Cited By ~ 153

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.1.c321 ◽

1996 ◽

Vol 271 (1) ◽

pp. C321-C331 ◽

Cited By ~ 63

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.

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Cholesterol modulates function of connexin 43 gap junction channel via PKC pathway in H9c2 cells

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2014.04.016 ◽

2014 ◽

Vol 1838 (8) ◽

pp. 2019-2025 ◽

Cited By ~ 15

Author(s):

Jun Zou ◽

Xiao-Yang Yue ◽

Sheng-Chao Zheng ◽

Guangwei Zhang ◽

He Chang ◽

...

Keyword(s):

Gap Junction ◽

Connexin 43 ◽

H9c2 Cells ◽

Gap Junction Channel

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Analysis of distribution patterns of gap junctions during development of embryonic chick facial primordia and brain

Development ◽

10.1242/dev.111.2.509 ◽

1991 ◽

Vol 111 (2) ◽

pp. 509-522

Author(s):

R. Minkoff ◽

S.B. Parker ◽

E.L. Hertzberg

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Uniform Distribution ◽

Rat Liver ◽

Connexin 43 ◽

Cell Communication ◽

Exterior Surface ◽

Junction Protein ◽

Primary Palate ◽

Gap Junction Protein

Gap junction distribution in the facial primordia of chick embryos at the time of primary palate formation was studied employing indirect immunofluorescence localization with antibodies to gap junction proteins initially identified in rat liver (27 × 10(3) Mr, connexin 32) and heart (43 × 10(3) Mr, connexin 43). Immunolocalization with antibodies to the rat liver gap junction protein (27 × 10(3) Mr) demonstrated a ubiquitous and uniform distribution in all regions of the epithelium and mesenchyme except the nasal placode. In the placodal epithelium, a unique non-random distribution was found characterized by two zones: a very heavy concentration of signal in the superficial layer of cells adjacent to the exterior surface and a region devoid of detectable signal in the interior cell layer adjacent to the mesenchyme. This pattern was seen during all stages of placode invagination that were examined. The separation of gap junctions in distinct cell layers was unique to the nasal placode, and was not found in any other region of the developing primary palate. One other tissue was found that exhibited this pattern-the developing neural epithelium of the brain and retina. These observations suggest the presence of region-specific signaling mechanisms and, possibly, an impedance of cell communication among subpopulations of cells in these structures at critical stages of development. Immunolocalization with antibodies to the ‘heart’ 43 × 10(3) Mr gap junction protein also revealed the presence of gap junction protein in facial primordia and neural epithelium. A non-uniform distribution of immunoreactivity was also observed for connexin 43.

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Heart gap junction preparations reveal hemiplaques by atomic force microscopy

AJP Cell Physiology ◽

10.1152/ajpcell.1995.268.4.c968 ◽

1995 ◽

Vol 268 (4) ◽

pp. C968-C977 ◽

Cited By ~ 52

Author(s):

R. Lal ◽

S. A. John ◽

D. W. Laird ◽

M. F. Arnsdorf

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Long Range ◽

Connexin 43 ◽

Plasma Membranes ◽

Aqueous Media ◽

Freeze Fracture ◽

Isolated Rat Heart ◽

Hexagonal Packing ◽

Atomic Force

Current structural models of gap junctions indicate two apposed plasma membranes with hexagonally packed hemichannels in each membrane aligning end to end. These channels connect the cytoplasms of contacting cells. Images of isolated rat heart gap junctions have been made with the atomic force microscope in aqueous media. We show that native cardiac gap junctions have a thickness of 25 +/- 0.6 nm. This decreases to 17 nm when they are treated with trypsin, which is known to remove some cytoplasmic components of connexin 43. Imaging shows subunits with a center to center spacing of approximately 9-10 nm and long range hexagonal packing, measurements in agreement with studies using freeze-fracture and negative-stain electron microscopy. In addition to gap junctions, we imaged structures that had all the characteristics of native gap junctions except their thickness was limited to 9-11 nm. They also show long range hexagonal packing and center to center spacing of 9-10 nm. These structures decrease in thickness, to 6-9 nm, when treated with trypsin. We have called these structures hemiplaques. They appear to be present endogenously in the preparation, as we have ruled out their being an artifact of imaging by AFM. However, it remains to be determined if they are a consequence of the procedure used in isolating gap junctions or a possible intermediary in gap junction formation.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1991.260.3.c513 ◽

1991 ◽

Vol 260 (3) ◽

pp. C513-C527 ◽

Cited By ~ 41

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.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.5.h1662 ◽

2000 ◽

Vol 278 (5) ◽

pp. H1662-H1670 ◽

Cited By ~ 65

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.

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Gap junction internalization and processing in vivo: a 3D immuno-electron microscopy study

Journal of Cell Science ◽

10.1242/jcs.252726 ◽

2020 ◽

pp. jcs.252726

Author(s):

Rachael P. Norris ◽

Mark Terasaki

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Connexin 43 ◽

New Technology ◽

Cell Communication ◽

Membrane Vesicles ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Annular Gap

Gap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. Less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell, called connexosomes or annular gap junctions. Here, we systematically investigated the fate of connexosomes in intact ovarian follicles. High pressure frozen, serial sectioned tissue was immunogold labeled for Connexin 43. Within a volume corresponding to ∼35 cells, every labeled structure was categorized and its surface area was measured. Measurements support the concept that multiple connexosomes form from larger invaginated gap junctions. Subsequently, the inner and outer membranes separate, Cx43 immunogenicity is lost from the outer membrane, and the inner membrane appears to undergo fission. One pathway for processing involves lysosomes, based on localization of Cathespin B to some processed connexosomes. In summary, this study demonstrates new technology for high-resolution analyses of gap junction processing.

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Fibroblast Growth Factor 4 Directs Gap Junction Expression in the Mesenchyme of the Vertebrate Limb Bud

The Journal of Cell Biology ◽

10.1083/jcb.138.5.1125 ◽

1997 ◽

Vol 138 (5) ◽

pp. 1125-1137 ◽

Cited By ~ 29

Author(s):

H. Makarenkova ◽

D.L. Becker ◽

C. Tickle ◽

A.E. Warner

Keyword(s):

Growth Factor ◽

Gap Junctions ◽

Gap Junction ◽

Fibroblast Growth Factor ◽

Connexin 43 ◽

Limb Bud ◽

Functional Coupling ◽

Fibroblast Growth ◽

Mesenchyme Cells

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell–cell communication and polarizing signaling are intimately connected.

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