N-terminal residues in Cx43 and Cx40 determine physiological properties of gap junction channels, but do not influence heteromeric assembly with each other or with Cx26

Gemel, Joanna; Lin, Xianming; Veenstra, Richard D.; Beyer, Eric C.

doi:10.1242/jcs.02953

Cited by 42 publications

(48 citation statements)

References 44 publications

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“…1, A and B). Cx40 hemichannels were affinitypurified using a strategy similar to the one we utilized previously to isolate connexons formed of various connexins (19,20). The affinity purification of Cx40 was facilitated by incorporation of an HA tag after its C terminus that allowed binding to and elution from beads coupled to anti-HA antibodies.…”

Section: Resultsmentioning

confidence: 99%

“…Affinity Purification of HA-tagged Cx40-Solubilization of connexons with detergent was performed as described previously (17)(18)(19)(20) with a few modifications. Briefly, cultured cells were harvested in PBS (pH 7.4) containing mini EDTA-free protease inhibitors (Roche) (one tablet per 5 ml of PBS), pelleted, and resuspended in the same buffer.…”

Section: Methodsmentioning

confidence: 99%

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Atomic Force Microscopy of Connexin40 Gap Junction Hemichannels Reveals Calcium-dependent Three-dimensional Molecular Topography and Open-Closed Conformations of Both the Extracellular and Cytoplasmic Faces

Allen

Gemel

Beyer

et al. 2011

Journal of Biological Chemistry

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Atomic force microscopy was used to study the three-dimensional molecular topography and calcium-sensitive conformational changes of Connexin40 hemichannels (connexons) reconstituted in 1,2-dioeloyl-sn-glycero-3-phosphatidylcholine lipid bilayers. Two classes of objects were observed that differed in their protrusion heights above the bilayer (2.6 versus 4.2 nm). Comparison to reconstituted connexons containing Connexin40 truncated to eliminate most of its C-terminal cytoplasmic domain showed that the two height classes corresponded to the shorter extracellular and taller cytoplasmic aspects of the hemichannels and that the C-terminal tail of Connexin40 contributes ϳ1.6 nm in thickness. Hemichannels imaged in solutions containing < 10 M Ca 2؉ showed 3.1-3.2 nm depressions (openings) in 30% of the cytoplasmic faces and 65% of the extracellular faces, and high-resolution three-dimensional topography of extracellular or cytoplasmic aspects of some connexons was observed. After addition of 3.6 mM Ca 2؉ , > 75% of the connexons in either orientation adopted closed conformations. In contrast, hemichannels imaged in the presence of 0.1 mM EDTA showed large (5.6-to 5.8-nm diameter) openings in nearly all hemichannels regardless of orientation, and detailed topography was visible in many connexons. Real-time imaging following the addition of 3.6 mM Ca 2؉ showed transitions of both extracellular and cytoplasmic orientations from "open" into "closed" conformations within several minutes. These studies provide the first high-resolution topographic information regarding a connexin with a large cytoplasmic domain and suggest that the extramembranous portions of Connexin40 contribute to a channel entrance that is relaxed by chelation of residual divalent cations.Gap junctions are specialized plasma membrane regions containing intercellular channels. They are made of apposing hemichannels (connexons) containing hexameric subunits from a family of proteins called connexins (Cx), 4 which has 21 members in the human and mouse genomes (1, 2). There is substantial similarity among the connexin sequences, especially in their transmembrane and extracellular regions. However, the major intracellular domains (cytoplasmic loop and carboxyl-terminal tail) are connexin-specific.Our understanding of the structures of these molecules (particularly the conserved regions) has recently been substantially advanced. An atomic resolution structure was determined from crystals of Cx26, a connexin with a very short C-terminal domain (3). These data confirmed and extended many of the observations that had previously been made in studies of a C-terminally truncated Cx43 (4). There has been substantially less information regarding the structures of connexins containing large cytoplasmic domains or connexons in dispersed (noncrystalline) arrangements. Cx40 and Cx43 are among such connexins. They are found in cardiac gap junctions that have been noted to have a "fuzzy layer" on their cytoplasmic surfaces in electron micrographs (5-7). Atomic force micros...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Atomic Force Microscopy of Connexin40 Gap Junction Hemichannels Reveals Calcium-dependent Three-dimensional Molecular Topography and Open-Closed Conformations of Both the Extracellular and Cytoplasmic Faces

Allen

Gemel

Beyer

et al. 2011

Journal of Biological Chemistry

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“…The contribution of Cx43 to the dysregulation of renin secretion implies either a cross-talk with Cx40 or the mediation of an extracellular signal generated in the endothelial and/or smooth muscle cells of the afferent arteriole. Both mechanisms are conceivable, inasmuch as Cx43 and Cx40 can form heteromeric channels, at least under certain conditions (160,551,554,555), and that the close apposition of renin, endothelial and smooth muscle cells provides an ideal setting for paracrine interactions within the afferent arteriole.…”

Section: Cx40 In Renin Secretionmentioning

confidence: 99%

Connexins: Key Mediators of Endocrine Function

Bosco

Haefliger

Meda

2011

Physiological Reviews

158

185

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The appearance of multicellular organisms imposed the development of several mechanisms for cell-to-cell communication, whereby different types of cells coordinate their function. Some of these mechanisms depend on the intercellular diffusion of signal molecules in the extracellular spaces, whereas others require cell-to-cell contact. Among the latter mechanisms, those provided by the proteins of the connexin family are widespread in most tissues. Connexin signaling is achieved via direct exchanges of cytosolic molecules between adjacent cells at gap junctions, for cell-to-cell coupling, and possibly also involves the formation of membrane “hemi-channels,” for the extracellular release of cytosolic signals, direct interactions between connexins and other cell proteins, and coordinated influence on the expression of multiple genes. Connexin signaling appears to be an obligatory attribute of all multicellular exocrine and endocrine glands. Specifically, the experimental evidence we review here points to a direct participation of the Cx36 isoform in the function of the insulin-producing β-cells of the endocrine pancreas, and of the Cx40 isoform in the function of the renin-producing juxtaglomerular epithelioid cells of the kidney cortex.

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Section: Electrophysiological Recordingsmentioning

confidence: 99%

“…Double whole cell patch clamp recordings of junctional currents (I j ) were performed as described previously [20,21]. All transjunctional voltage (V j ) errors resulting from the patch electrode series resistance (R el ) were corrected during the junctional conductance (g j ) calculations according to the expression: [21] The maximum g j (g j,max ) was determined from the linear regression fit of the steady state I j -V j curve from each experiment between ±5 and ±20 mV.…”

Section: Electrophysiological Recordingsmentioning

confidence: 99%

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins

Gemel

Lin

Collins

et al. 2008

Biochemical and Biophysical Research Communications

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Since most cells in the heart co-express multiple connexins, we studied the possible heteromeric interactions between connexin30.2 and connexin40, connexin43 or connexin45 in transfected cells. Double label immunofluorescence microscopy showed that connexin30.2 extensively co-localized with each co-expressed connexin at appositional membranes. When Triton X-100 solubilized connexons were affinity purified from co-expressing cells, connexin30.2 was isolated together with connexin40, connexin43, or connexin45. Co-expression of connexin30.2 with connexin40, connexin43, or connexin45 did not significantly reduce total junctional conductance. Gap junction channels in cells co-expressing connexin30.2 with connexin43 or connexin45 exhibited voltagedependent gating intermediate between that of either connexin alone. In contrast, connexin30.2 dominated the voltage dependence when co-expressed with connexin40. Our data suggest that connexin30.2 can form heteromers with the other cardiac connexins and that mixed channel formation will influence the gating properties of gap junctions in cardiac regions that co-express these connexins. Keywords Gap Junction; Intercellular Communication; ConnexinCurrent passage between cells in the heart is facilitated by intercellular channels clustered in gap junctions. These channels are formed by docking of two hemichannels (connexons) provided by the opposing cells. Each connexon is a hexamer of subunit proteins called connexins (Cx). As demonstrated in cells expressing individual connexins, each connexin can form channels by itself (homomeric/homotypic channels), and different connexins form channels with different conductance, permeability and gating properties [1]. In cells coexpressing different connexins, heteromeric channels (containing different connexins in the same connexon) can potentially be formed [2].Many studies have shown the presence of three well characterized connexin proteins in the heart, Cx40, Cx43, and Cx45. Cx43 is most abundant in atrial and ventricular myocytes and in Purkinje fibers [3,4]. Cx40 is expressed in atrial myocardium, atrioventricular bundle and ✉Address correspondence to: Eric C. Beyer, MD PhD, University of Chicago, Department of Pediatrics MC4060, 5841 S. Maryland Ave, Chicago, IL 60637-1470, TEL: 773-834-1498.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [5,6]. Cx45 is detected in sinoatrial and atrioventricular nodes, atrioventricular bundle and branches [7,8]. NIH Public AccessRecently, another cardiac connexin, mouse Cx30.2 (human Cx31.9) was discovered [9][10][11]. Cx30.2 expression was ...

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N-terminal residues in Cx43 and Cx40 determine physiological properties of gap junction channels, but do not influence heteromeric assembly with each other or with Cx26

Cited by 42 publications

References 44 publications

Atomic Force Microscopy of Connexin40 Gap Junction Hemichannels Reveals Calcium-dependent Three-dimensional Molecular Topography and Open-Closed Conformations of Both the Extracellular and Cytoplasmic Faces

Atomic Force Microscopy of Connexin40 Gap Junction Hemichannels Reveals Calcium-dependent Three-dimensional Molecular Topography and Open-Closed Conformations of Both the Extracellular and Cytoplasmic Faces

Connexins: Key Mediators of Endocrine Function

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins

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