Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes

TLDR

Connexin43, a key gap junction protein in neonatal cardiomyocytes, was studied in cultured cells to examine its turnover and post‑translational modifications. The study aimed to determine how rapid Cx43 turnover and its multiple phosphorylation sites regulate gap‑junction communication. Immunoprecipitation and phosphatase treatments revealed phosphorylated Cx43 forms at 42 and 44 kDa, with the 42‑kDa band confirmed by 32P incorporation, and pulse‑chase experiments showed a 1–2‑hour half‑life for Cx43, indicating that phosphate groups persist throughout the protein’s lifespan.

Abstract

Cultured cardiomyocytes were used to study the turnover and post-translational modification of connexin43 (Cx43), a major gap junction protein in neonatal cardiac myocytes. Immunoprecipitation of [35S]Met-labelled lysates with anti-Cx43 antibodies followed by analysis using SDS/PAGE and fluorography revealed two bands, one at 40 kDa and the other at 42 kDa. Alkaline phosphatase treatment of [35S]Met-labelled Cx43 eliminated the band at 42 kDa, suggesting that it represented a phosphorylated form of the protein. This was confirmed by [32P]P1 incorporation into the 42 kDa band, but not into the band at 40 kDa. In addition, another alkaline phosphatase-sensitive phosphorylated form of Cx43 was identified at 44 kDa. In pulse-chase experiments, the half-life of Cx43 in cardiomyocytes was determined to be 1-2 h. Furthermore, the turnover rate of phosphate groups on Cx43 was found to be experimentally defined by the half-life of the protein. The observation that phosphate groups can remain with the protein throughout its life is consistent with the finding that in isolated adult rat heart gap junction plaques, Cx43 is primarily phosphorylated. We postulate that the rapid turnover of Cx43 and its multiple sites of phosphorylation play important roles in the regulation of cell-cell communication via gap junctions.