Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca²⁺ signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes encode four CaMKII homologs: CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ. All CaMKIIs consist of a kinase domain, a regulatory segment, a variable linker region, and a hub domain, which is responsible for oligomerization. The four proteins differ primarily in linker length and composition because of extensive alternative splicing. Here, we report the heterogeneity of CaMKII transcripts in three complex samples of human hippocampus using deep sequencing. We showed that hippocampal cells contain a diverse collection of over 70 CaMKII transcripts from all four CaMKII-encoding genes. We characterized the Ca²⁺/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. The effect of the variable linker on Ca²⁺/CaM sensitivity depended on the kinase and hub domains. Moreover, we revealed a previously uncharacterized role for the hub domain as an allosteric regulator of kinase activity, which may provide a pharmacological target for modulating CaMKII activity. Using small-angle x-ray scattering and single-particle cryo–electron microscopy (cryo-EM), we present evidence for extensive interactions between the kinase and the hub domains, even in the presence of a 30-residue linker. Together, these data suggest that Ca²⁺/CaM sensitivity in CaMKII is homolog dependent and includes substantial contributions from the hub domain. Our sequencing approach, combined with biochemistry, provides insights into understanding the complex pool of endogenous CaMKII splice variants.

钙/钙调蛋白依赖性蛋白激酶 II (CaMKII) 在整个身体的Ca ²⁺信号传导中起着核心作用。在海马体中，学习和记忆需要 CaMKII。脊椎动物基因组编码四种 CaMKII 同源物：CaMKIIα、CaMKIIβ、CaMKIIγ 和 CaMKIIδ。所有 CaMKII 均由激酶结构域、调节段、可变接头区和负责寡聚化的中枢结构域组成。由于广泛的可变剪接，这四种蛋白质主要在接头长度和组成上不同。在这里，我们使用深度测序报告了人类海马的三个复杂样本中 CaMKII 转录本的异质性。我们发现海马细胞包含来自所有四个 CaMKII 编码基因的超过 70 个 CaMKII 转录本的多样化集合。我们表征了 Ca ²⁺海马 CaMKII 变体的 /CaM 敏感性跨越广泛的接头长度和组成。可变接头对 Ca ²⁺ /CaM 敏感性的影响取决于激酶和中枢结构域。此外，我们揭示了中枢结构域作为激酶活性的变构调节剂的先前未表征的作用，这可能为调节 CaMKII 活性提供药理学靶点。使用小角度 X 射线散射和单粒子冷冻电子显微镜 (cryo-EM)，我们提供了激酶和中心结构域之间广泛相互作用的证据，即使存在 30 个残基的接头。总之，这些数据表明 Ca ²⁺CaMKII 中的 /CaM 敏感性是同源依赖的，包括来自中心域的大量贡献。我们的测序方法与生物化学相结合，为了解内源性 CaMKII 剪接变体的复杂库提供了见解。