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

Abstract

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.

Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis

“…2a, b). In particular, TRPC1-siRNA induced a robust decrease in the P0 and P1 forms, without substantially modifying the levels of P2 forms, which have been previously reported to correspond to Cx43 protein phosphorylated at specific serine residues (S325, S328, and/or S330) [40]. These data indicated that other GdCl 3 -sensitive channels, rather than TRPC1, specifically regulated P2 forms.…”

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

“…Consistent with Musil and Goodenough (1991), the P2 isoform was insoluble after extraction of cells with Triton X-100 as indicated in the middle lane (Ab: Total, Prep: Tx Ins) of Fig 1A. When that same lane is simultaneously probed with an antibody (pS325) that is specific for Cx43 when it is phosphorylated at S325, S328 and/or S330, we found that only the P2 isoform was present (third lane, Ab:pS325, Prep:Tx Ins). We had previously shown that this phosphospecific antibody labeled the intercalated disk region of cardiomyocytes (Lampe et al, 2006) and that S325/328/330 phosphorylation was important in gap junction assembly (Cooper & Lampe, 2002). In Fig.…”

“…Myocardial ischemia leads to Cx43 "dephosphorylation" (i.e., the loss of P1, P2, etc and gain of P0) and loss of localization from the intercalated disk, which likely contributes to contractile failure and arrhythmias (Beardslee et al, 2000;Schulz et al, 2003). We have shown that Cx43 localized to intercalated disks is phosphorylated at S325, S328 and/or S330 and that ischemia leads to loss of this phosphorylation and re-localization of the protein (Lampe et al, 2006).…”

“…Cells expressing site directed mutants, in which these serines were converted to alanines, do not assemble gap junctions efficiently nor did they make the P2 isoform of Cx43 (Lampe et al, 2006). These data indicate that phosphorylation on S325, S328 and/or S330 are required for formation of the gap junction plaque associated P2 isoform of Cx43 (see model in Fig.…”

“…We purchased a phosphospecific antibody to Cx43 at S262 (pS262) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and a phosphospecific activated MAPK antibody from Cell Signaling Technology (Beverly, MA). We made rabbit anti-pS262, pS279/282, pS368, pS325/328/330-Cx43 phosphospecific antibodies by custom commercial preparation (ProSci Inc., Poway, CA; 13 week schedule) against synthetic peptides phosphorylated at the specified residues that had been linked via the N-terminal cysteine to maleimide-activated KLH (Pierce Biotechnology, Rockford, IL), and phosphospecific antibodies were affinity purified as we have previously published (Lampe et al, 2006).…”