Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart

Paul D Lampe; Cynthia D Cooper; Timothy J King; Janis M Burt

doi:10.1242/jcs.03089

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Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart

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Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart

Paul D Lampe, Cynthia D Cooper, Timothy J King and Janis M Burt

Journal of cell science, Vol.119(Pt 16), pp.3435-3442

08/15/2006

DOI: https://doi.org/10.1242/jcs.03089

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https://hdl.handle.net/2376/115897

PMID: 16882687

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Abstract

Gap Junctions - metabolism

Myocardial Ischemia - metabolism

Rabbits

Phosphorylation

Connexin 43 - metabolism

Mice, Inbred C57BL

Cells, Cultured

Rats

Cell Communication - physiology

Serine - metabolism

Mice, Knockout

Myocardial Ischemia - pathology

Myocardium - cytology

Animals

Immunoglobulin G - immunology

Myocardium - metabolism

Fibroblasts - cytology

Heart Ventricles - metabolism

Mice

Ion Channel Gating

Fibroblasts - metabolism

The functional consequences of Connexin43 (Cx43) phosphorylation remain largely unexplored. Using an antibody that specifically recognizes Cx43 phosphorylated at serine residues 325, 328 and/or 330 (pS325/328/330-Cx43), we show that labeling of this form of Cx43 as well as of total Cx43 is restricted to the intercalated disk region of normal ventricular tissue. In ischemic heart, significant relocalization of total Cx43 to the lateral edges of myocytes was evident; however pS325/328/330-Cx43 remained predominately at the intercalated disk. Western blots indicated a eightfold decrease in pS325/328/330-Cx43 in ischemic tissue. Peptide-binding- and competition-experiments indicated that our antibody mainly detected Cx43 phosphorylated at S328 and/or S330 in heart tissue. To evaluate how this change in Cx43 phosphorylation contributes to ischemia-induced downregulation of intercellular communication, we stably transfected Cx43(-/-) cells with a Cx43 construct in which serine residues 325, 328 and 330 had been mutated to alanine (Cx43-TM). Cx43-TM was not efficiently processed to isoforms that have been correlated with gap junction assembly. Nevertheless, Cx43-TM cells were electrically coupled, although development of coupling was delayed. Fully opened channels were only rarely observed in Cx43-TM cells, and Lucifer-Yellow-dye-coupling was significantly reduced compared with wild-type cells. These data suggest that phosphorylation of Cx43 at serine residues 325, 328 and/or 330 influences channel permselectivity and regulates the efficiency of gap junction assembly.

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Title: Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart
Creators: Paul D Lampe - Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center, Department of Pathobiology, University of Washington, 1100 Fairview Avenue N., M5C800, PO Box 19024, Seattle, WA 98109, USA. plampe@fhcrc.org
Cynthia D Cooper
Timothy J King
Janis M Burt
Publication Details: Journal of cell science, Vol.119(Pt 16), pp.3435-3442
Academic Unit: Molecular Biosciences, School of
Publisher: England
Grant note: R01 GM055632 / NIGMS NIH HHS HL058732 / NHLBI NIH HHS GM055632 / NIGMS NIH HHS R01 HL058732 / NHLBI NIH HHS
Identifiers: 99900548225201842
Language: English
Resource Type: Journal article