title: Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated.
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 of 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 the 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 the 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).
authors: Department of Pediatrics, University of Chicago, Chicago, IL 60637, USA.
authors: Joanna
authors: J
authors: Gemel
authors: Department of Pediatrics, University of Chicago, Chicago, IL 60637, USA.
authors: Adria R
authors: AR
authors: Simon
authors: Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
authors: Dakshesh
authors: D
authors: Patel
authors: Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
authors: Qin
authors: Q
authors: Xu
authors: Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
authors: Arvydas
authors: A
authors: Matiukas
authors: Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
authors: Richard D
authors: RD
authors: Veenstra
authors: Department of Pediatrics, University of Chicago, Chicago, IL 60637, USA. Electronic address: ecbeyer@uchicago.edu.
authors: Eric C
authors: EC
authors: Beyer
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D001281:Atrial Fibrillation / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D017630:Connexins / Q000235:genetics* / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D006325:Heart Atria / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D051379:Mice; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009842:Oligopeptides / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D055550:Protein Stability; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D059748:Proteolysis; D015398:Signal Transduction; D019076:Transgenes; D054875:Ubiquitination; D000097003:Gap Junction alpha-5 Protein
chemical_list: D017630:Connexins; D003513:Cycloheximide; D009842:Oligopeptides; D046988:Proteasome Endopeptidase Complex; D061988:Proteasome Inhibitors; D011500:Protein Synthesis Inhibitors; D000097003:Gap Junction alpha-5 Protein; C078846:epoxomicin
keywords: Atrial fibrillation; Connexin40; Gap junctions; Ion channels; Proteasome; Protein degradation