The Ca²⁺/calmodulin (CaM)-dependent kinase II (CaMKII) is well known for transmitting Ca²⁺-signals, which leads to a multitude of physiological responses. Its functionality is believed to involve CaMKII holoenzyme dynamics where trans-autophosphorylation of the crucial phosphorylation site, T286 occurs. Phosphorylation of this site does not occur when stimulated exclusively with the arrhythmia associated D130G mutant form of CaM in vitro. Here, we present evidence that the loss-of-CaMKII function correlates with premature phosphorylation of its inhibitory phosphosite T306 in CaMKIIα and T307 in CaMKIIδ as this site was up to 20-fold more phosphorylated in the presence of D130G CaM compared to wildtype CaM. Indeed, changing this phosphosite to a non-phosphorylatable alanine reversed the inhibitory effect of D130G both in vitro and in live cell experiments. In addition, several phosphosites with so far undescribed functions directing the Ca²⁺-sensitivity of the CaMKII sensor were also affected by the presence of the D130G mutation implicating a role of several additional autophosphosites (besides T286 and T306/T307) so far not known to regulate CaMKII Ca²⁺ sensitivity. Furthermore, we show that introducing a D130G mutation in the CALM2 gene of the P19CL6 pluripotent mouse embryonic carcinoma cell line using CRISPR/Cas9 decreased the spontaneous beat frequency compared to wildtype cells when differentiated into cardiomyocytes supporting an alteration of cardiomyocyte physiology caused by this point mutation. In conclusion, our observations shed for the first time light on how the D130G CaM mutation interferes with the function of CaMKII and how it affects the beating frequency of cardiomyocyte-like cells.

Ca ²⁺ /钙调蛋白 (CaM) 依赖性激酶 II (CaMKII) 以传递 Ca ²⁺信号而闻名，这会导致多种生理反应。其功能被认为涉及 CaMKII 全酶动力学，其中发生关键磷酸化位点 T286 的反式自磷酸化。在体外仅用与心律失常相关的 D130G 突变形式的 CaM 刺激时，不会发生该位点的磷酸化. 在这里，我们提供的证据表明，CaMKII 功能的丧失与其 CaMKIIα 中的抑制性磷酸化位点 T306 和 CaMKIIδ 中的 T307 的过早磷酸化相关，因为与野生型 CaM 相比，在 D130G CaM 存在下，该位点磷酸化高达 20 倍。事实上，在体外和活细胞实验中，将该磷酸位点改变为不可磷酸化的丙氨酸逆转了 D130G的抑制作用。此外，迄今为止尚未描述的几个具有指导CaMKII 传感器Ca ²⁺敏感性的功能的磷位点也受到 D130G 突变的影响，这意味着几个额外的自身磷位点（除了 T286 和 T306/T307 之外）的作用到目前为止尚不清楚调节 CaMKII Ca ²⁺灵敏度。此外，我们表明，与野生型细胞相比，当分化为心肌细胞时，使用 CRISPR/Cas9 在 P19CL6 多能小鼠胚胎癌细胞系的 CALM2 基因中引入 D130G 突变可降低自发搏动频率，支持由该点突变引起的心肌细胞生理学改变. 总之，我们的观察首次阐明了 D130G CaM 突变如何干扰 CaMKII 的功能以及它如何影响心肌细胞样细胞的搏动频率。