The C-terminus of connexin43 adopts different conformations in the Golgi and gap junction as detected with structure-specific antibodies

Abstract

The C-terminus of the most abundant and best-studied gap-junction protein, connexin43, contains multiple phosphorylation sites and protein-binding domains that are involved in regulation of connexin trafficking and channel gating. It is well-documented that SDS/PAGE of NRK (normal rat kidney) cell lysates reveals at least three connexin43-specific bands (P0, P1 and P2). P1 and P2 are phosphorylated on multiple, unidentified serine residues and are found primarily in gap-junction plaques. In the present study we prepared monoclonal antibodies against a peptide representing the last 23 residues at the C-terminus of connexin43. Immunofluorescence studies showed that one antibody (designated CT1) bound primarily to connexin43 present in the Golgi apparatus, whereas the other antibody (designated IF1) labelled predominately connexin43 present in gap junctions. CT1 immunoprecipitates predominantly the P0 form whereas IF1 recognized all three bands. Peptide mapping, mutational analysis and protein-protein interaction experiments revealed that unphosphorylated Ser364 and/or Ser365 are critical for CT1 binding. The IF1 paratope binds to residues Pro375-Asp379 and requires Pro375 and Pro377. These proline residues are also necessary for ZO-1 interaction. These studies indicate that the conformation of Ser364/Ser365 is important for intracellular localization, whereas the tertiary structure of Pro375-Asp379 is essential in targeting and regulation of gap junctional connexin43.

N-cadherin haploinsufficiency affects cardiac gap junctions and arrhythmic susceptibility

“…1). Similar results were obtained using an antibody that recognizes hypophosphorylated Cx43 at S364/S365 (25) indicating that multiple serine residues were affected in the heterozygous hearts (Supplemental data).…”

“…We previously demonstrated an increase in dephosphorylated Cx43 in N-cadherin-deficient hearts, which is associated with reduced Cx43 plaques, reduced epicardial conduction velocity and spontaneous ventricular tachycardia (16). In cultured noncardiac cells, dephosphorylated Cx43 at S364/S365 is associated with trafficking through the Golgi apparatus, hence nonfunctional gap junctions (25). Furthermore, a specific increase in dephosphorylated Cx43 (S364/S365) is observed in protein lysates from ischemic hearts (25).…”

“…In cultured noncardiac cells, dephosphorylated Cx43 at S364/S365 is associated with trafficking through the Golgi apparatus, hence nonfunctional gap junctions (25). Furthermore, a specific increase in dephosphorylated Cx43 (S364/S365) is observed in protein lysates from ischemic hearts (25). In the present study, an increase in hypophosphorylated Cx43 (S364/S365) was observed in N-cadherin heterozygous hearts, with even higher levels in the N-cad/Cx43 compound heterozygotes, which is consistent with less functional gap junctions and greater susceptibility to induced arrhythmias compared to the single mutants.…”

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

“…Given that phosphorylation of Cx43 by PKCa has been previously reported to affect the protein conformation and its interaction with other intracellular proteins [6,22,[43][44][45], taken together all these data implied that TRPC1-mediated m-calpain/PKCa pathway was critically involved in Cx43 remodeling at the plasma membrane. Table 1 Boltzmann Parameters for gap Junctions in untreated and SCR-, GdCb-TRPCl-siRNA-treated C2C12 cell pairs stimulated or not with SIP…”

“…This assumption is consistent Note that the active pool of membrane associated-PKCa progressively decreases with myoblast differentiation both in unstimulated and S1P-stimulated cells and that the treatment with TRPC1-siRNA, m-calpain-siRNA and GdCl 3 prevents the reduction of active PKCa with previous observations in the literature showing that Ca 2? / PKCa -dependent phosphorylation of connexins [44] can influence their interaction with other proteins at the plasma membrane [22,45].…”