Connexin40 and connexin43 determine gating properties of atrial gap junction channels

Department of Pharmacology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
Journal of Molecular and Cellular Cardiology (Impact Factor: 5.22). 05/2009; 48(1):238-45. DOI: 10.1016/j.yjmcc.2009.05.014
Source: PubMed

ABSTRACT While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance (g(j)). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g(j). Slightly more than half of atrial g(j) and <or=20% of ventricular g(j) were inhibited. In myocytes from Cx40 null mice, the inhibition of ventricular g(j) was completely abolished, and the block of atrial g(j) was reduced to <20%. Compared to ventricular gap junctions, the transjunctional voltage (V(j))-dependent inactivation of atrial g(j) was reduced and kinetically slowed, while the V(j)-dependence of fast and slow inactivation was unchanged. We conclude that Cx40 and Cx43 are equally abundant in atrium and make similar contributions to atrial g(j). Co-expression of Cx40 accounts for most, but not all, of the differences in the V(j)-dependent gating properties between atrium and ventricle that may play a role in the genesis of slow myocardial conduction and arrhythmias.

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Available from: Eric C Beyer, Sep 11, 2014
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    • "Cell homogenates were prepared 48 h after transfection with connexin DNA as described by Gong et al. [15]. Immunoblots were performed as described earlier [5]. The protein concentrations of homogenates were determined using the method of Bradford (Bio-Rad, Richmond, CA) [16]. "
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    ABSTRACT: Several Cx40 mutants have been identified in patients with atrial fibrillation (AF). We have been working to identify physiological or cell biological abnormalities of several these human mutants that might explain how they contribute to disease pathogenesis. Wild type (wt) Cx40 or four different mutants (P88S, G38D, V85I, and L229M) were expressed by transfection of communication-deficient HeLa cells or HL-1 cardiomyocytes. Biophysical channel properties and the sub-cellular localization and protein levels of Cx40 were characterized. Wild type Cx40 and all mutants except P88S formed gap junction plaques and induced significant gap junctional conductances. The functional mutants showed only modest alterations of single channel conductances or gating by trans-junctional voltage as compared to wtCx40. However, immunoblotting indicated that the steady state levels of G38D, V85I, and L229M were reduced relative to wtCx40; most strikingly, G38D was only 20 - 31% of wild type levels. After inhibition of protein synthesis with cycloheximide, G38D (and to a lesser extent the other mutants) disappeared much faster than wtCx40. Treatment with the proteasomal inhibitor, epoxomicin, greatly increased levels of G38D and restored the abundance of gap junctions and the extent of intercellular dye transfer. Thus, G38D, V85I, and L229M are functional mutants of Cx40 with small alterations of physiological properties, but accelerated degradation by the proteasome. These findings suggest a novel mechanism (protein instability) for the pathogenesis of AF due to a connexin mutation and a novel approach to therapy (protease inhibition).
    Journal of Molecular and Cellular Cardiology 06/2014; 74. DOI:10.1016/j.yjmcc.2014.06.010 · 5.22 Impact Factor
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    • "For double-labeling experiments, cells were incubated simultaneously with both mouse anti-mCherry monoclonal and rabbit anti-GFP antibodies (Abcam). Immunoblots were performed as described earlier [10] using cell homogenates prepared 48 h after transfection with connexin DNA as described by Gong et al. [11]. For immunofluorescence microscopy, cultured cells were fixed in methanol/acetone (1:1), stained as previously described [8], and imaged using a Zeiss Axioplan 2 microscope or Leica TCS SP2 laser-scanning confocal microscope. "
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    ABSTRACT: Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (Vj) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of Vj gating properties and increased the gap junction channel conductance (γj) by 20-30 pS. While co-expression of the two mutations had similar effects on Vj gating, it synergistically increased γj by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel.
    FEBS Letters 01/2014; DOI:10.1016/j.febslet.2014.01.010 · 3.34 Impact Factor
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    • "Cx37, Cx40, and Cx43 are abundant components of the gap junctions in various cells of the cardiovascular system, and they are coexpressed in some of these cells. In ventricular myocytes , Cx43 is the predominant connexin; however, in atrial myocytes, the abundances of Cx40 and Cx43 are approximately equal (Lin et al. 2010). In diseased myocardium, gap junctions may undergo remodeling, and the levels of Cx40 and Cx43 may be altered (reviewed by Severs et al. 2008). "
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    ABSTRACT: Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4 μM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro-2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43-expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others.
    Journal of Membrane Biology 06/2012; 245(5-6):231-41. DOI:10.1007/s00232-012-9444-4 · 2.17 Impact Factor
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