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First published online 1 August 2006
doi: 10.1242/jcs.03089

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

Paul D. Lampe^1,*, Cynthia D. Cooper¹, Timothy J. King^1, and Janis M. Burt²

¹ Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center and Department of Pathobiology, University of Washington, 1100 Fairview Avenue N., M5C800, P.O. Box 19024, Seattle, WA 98109, USA
² Department of Physiology, University of Arizona, Tucson, AZ

View larger version (73K): [in a new window]   Fig. 1. Increased electrophoretic mobility of Cx43 in the ischemic heart involves dephosphorylation at serine residues 325, 328 and/or 330. Western blot of total protein isolated from three control (lanes 1,3,5, respectively) or three globally ischemic hearts (30' at 37°C; lanes 2,4,6, respectively) simultaneously probed for total Cx43 (Cx43NT-1) and Cx43 phosphorylated at S325, S328 and/or S330 followed by GAPDH and vinculin antibodies. Notice the increased mobility of Cx43 and decreased pS325/328/330-Cx43 content in the ischemic samples. Molecular mass standards are 28, 49, 62 and 98 kDa (bottom to top); P, position of the Cx43 P isoform; P0, position of the Cx43 P0 isoform.

View larger version (134K): [in a new window]   Fig. 2. Differential phosphorylation of Cx43 localized at intercalated disks vs lateral edges of myocytes in the normal and ischemic heart. The localization of total Cx43 (Sigma C6219) and pS325/328/330-Cx43 in normal and ischemic heart is shown. Notice the increase in Cx43 at the lateral edges of the myocytes of the ischemic heart. Lateral edge Cx43 was not detectably phosphorylated at residues S325, S328 and/or S330. By contrast, Cx43 at the intercalated disks of ischemic heart retained phosphorylation at residues S325, S328 and/or S330 (indicated by arrowheads). Bar, 20 µm.

View larger version (48K): [in a new window]   Fig. 3. The pS325/328/330-Cx43 antibody is specific for the P2 isoform of Cx43 and pS328/330. (A) Equal amounts of protein lysate from fibroblasts expressing wild-type Cx43 (WT) and Cx43-TM (TM, clone A) were either treated with alkaline phosphatase (+AP) or untreated and probed in a western blot with Cx43NT1 (left panel, Total Cx43) and the pS325/328/330-Cx43 (right panel) antibodies on the same blot. Molecular mass standards in kDa are marked on the left side and the P2 isoform is marked with an asterisk. (B) The amount of antibody bound to the different peptides representing singly, doubly and triply phosphorylated Cx43 linked to an ELISA well (open bars) is shown together with the ability of these peptides to compete with Cx43 present in heart lysates in a western immunoblot format (filled bars). Error bars represent the mean ± s.d.

View larger version (61K): [in a new window]   Fig. 4. Cx43-TM-expressing cells do not phosphorylate Cx43 to yield the P2 isoform and contain less Triton-insoluble (TI) Cx43. Whole-cell (WC) and TI cellular lysates from cells expressing wild-type Cx43 (WT) or three Cx43-TM clones (TM-A, TM-B, and TM-C) were western blotted and probed for total Cx43 with the Cx43NT1 antibody. A darker exposure of lysates of the TM-C clone is shown. Migration positions of a 50- and 36-kDa marker are indicated with an asterisk and the vinculin loading controls are indicated with a V. The Triton-insoluble vinculin could be associated with the cytoskeleton or adhesive junctions.

View larger version (45K): [in a new window]   Fig. 5. Comparison of Cx43 localization in parental knockout (KO) cells, wild-type Cx43 (WT) cells and Cx43-TM (TM-A)-expressing cells. Apparent gap junctions are marked by arrowheads in the WT panel. Bar, 10 µm.

View larger version (19K): [in a new window]   Fig. 6. Cells expressing Cx43 S325/328/330A are inefficient at dye transfer. Wild-type (WT)-, parental knockout (KO)- and Cx43-TM (clones TM-A, TM-B and TM-C)-expressing cells were microinjected with Lucifer Yellow dye. After 3 minutes of transfer, digital images were taken and the number of recipient cells was determined (n=the number of injected cells). Bars show the mean and error bars ± s.e.m. The extent of dye transfer was significantly different (**P<0.02) for WT compared with all of the other cell types.

View larger version (24K): [in a new window]   Fig. 7. Histogram of channel events observed in Cx43-TM (gray) and Cx43-WT-expressing cells. (A) Event frequency as percent of total events in each bin. (B) Difference induced by mutation of serine residues 325, 328 and 330. Notice the reduced incidence of events corresponding to fully opened channels (106±1 pS) and increased incidence of 75±2 pS. The 54±1 pS population was not different from wild-type and mutant data sets. Difference data were fit using Origin software yielding Chi2 and R2 for fit are 5.677 and 0.94, respectively (-WT, 1851 events, n=13; TM, 418 events, n=7).

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