Cell Commun Signal. 2014 Sep 28;12:58. doi: 10.1186/s12964-014-0058-6.
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.
肌醇 1,4,5-三磷酸受体（IP3R）在多种细胞类型的 Ca2+释放过程中发挥关键作用。此外，IP3R 分布在心室闰盘，但它在该特定部位的功能尚不清楚。缝隙连接（GJ）蛋白 Cx43 是心室心肌中主要的缝隙连接蛋白，通过在多个残基上磷酸化/去磷酸化，与调节 Cx43 性质的几种信号通路相连。在这里，我们研究了 IP3R 在细胞间通讯中的调节作用及其潜在机制。
在新生大鼠和成年小鼠心室肌细胞中，免疫染色和 Western blot 检测到的 GJ 斑中，IP3R 与 Cx43 共定位和共免疫沉淀。用拮抗剂阻断 IP3R 或用 shRNA 沉默全 IP3R 表达会阻碍 6-羧基荧光素（6-CFDA）在培养的汇合新生心肌细胞中的 GJ 扩散，并使 Ca2+瞬变不同步，而用 IP3 酯或 ATP 刺激 IP3R 则产生相反的效果。同样，在分离的成年心肌细胞的细胞对中，IP3R 的激活或抑制调节 6-CFDA 通过 GJ 的传播。此外，IP3R 的激活或抑制分别促进或抑制 Cx43 在 S279/282 上的磷酸化。定点突变表明，表达 Cx43-S282A（丙氨酸）突变体抑制了 S279/282 的磷酸化和 GJ 通透性，而 S279A 突变体在心室肌细胞中则表现出相反的效果。在 HEK293 细胞中表达这些突变体表明，具有双重 S279/282 突变的细胞无法表达外源性 Cx43，而具有单个 S279 或 S282 突变的细胞表现出 Cx43 过表达，同时 S279/282 的磷酸化增加，并促进细胞间通讯。
这些结果首次表明，IP3R 与 Cx43 物理相互作用，并参与调节 Cx43 在 S279/282 上的磷酸化，从而影响心室肌细胞的 GJ 细胞间通讯。
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