Abstract
Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect. In neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication. These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.
Highlights
Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types
Of interest is phosphorylation of S262, S368, S279, and S282, which has been identified to link with the protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and PKA pathways, the precise role of a single kinase or Cx43 protein partner in the regulation of Cx43 phosphorylation is far from clear, because a kinase can phosphorylate more than one site and one site can be phosphorylated by various kinases and signaling pathways at the same time [12,13,14,15]
Structural association of IP3 receptor (IP3R) with Cx43 in gap junctions of ventricular myocytes To detect the three IP3R isoforms that are expressed in neonatal and adult ventricles [16,17,18], anti-pan-IP3R antibodies and anti-Cx43 antibodies were used to co-immunolabel samples
Summary
Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Gap junctions (GJs) serve as intercellular communication channels to confer direct ion exchange and synchronization of electrical excitation between adjacent myocytes, allowing rhythmic coordinated myocardium contraction They permit intercellular exchange of metabolites and small signaling process that regulates its properties including assembling, trafficking, degradation, and electrical and metabolic coupling [1,2,9,10,11,12]. Studies have demonstrated that IP3R plays a critical role in regulating the local Ca2+ activity, including the nuclear Ca2+ signaling and affects gene transcription by the nuclear envelop-tethered IP3R-2 [17,18] This receptor protein is found in GJs in the ventricular myocardium [20], myoendothelium and sciatic nerve nodes [21,22], but its function in this particular site is not clear, yet. IP3R localizes to myoendothelium GJs on the endothelial cell side, but not on the vascular smooth-muscle cell side, leading to selective modulation of GJ coupling on the endothelial cell side by IP3 [21,27], suggesting that local IP3R is necessary for IP3-mediated GJ coupling
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