The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.

中文翻译：

---

钙调蛋白直接与Cx43羧基末端和细胞质环相互作用，该环含3个ODDD连锁突变体（M147T，R148Q和T154A），这些突变体保留α螺旋结构，但表现出功能丧失和细胞运输缺陷。

常染色体显性多效性疾病称为眼突指畸形（ODDD）是由间隙连接蛋白Cx43的突变引起的。迄今鉴定出的73个突变中，超过三分之一位于细胞质环（Cx43CL）域中。在这里，我们确定了通过三种ODDD突变（M147T，R148Q和T154A）定位在Cx43CL的预测的1-5-10钙调蛋白结合基序内的机制，该机制可以表明该疾病。核磁共振（NMR）和圆二色性表明，三个ODDD突变对Cx43CL形成α螺旋结构以及结合钙调蛋白的能力几乎没有影响。显微镜和染料转移试验相结合发现这些突变增加了细胞内Cx43的水平，而那些贩运到质膜的突变则没有形成功能通道。NMR还确定CaM可以直接与Cx43CT域相互作用。参与CaM相互作用的Cx43CT残基与被Pyk2和Src磷酸化的酪氨酸重叠。体外和细胞数据提供了证据，证明CaM与Cx43CT相互作用的重要性可能在于限制Pyk2和Src磷酸化及其随后的下游作用。