Alternative splicing at the last coding exon of Cav2.1 regulates the interaction with Cav4 and plays an essential role in preventing a complex neurological disease in mice. Mus musculus

Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Cav2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail. To gain insight into the functional role of the AS in vivo and its relevance in the pathogenesis of SCA6, here we created knockin mice that exclusively express MPc by inserting the splice-site mutation. The resultant Sca6CtmKO/CtmKO mice developed non-progressive neurological phenotypes resembling those of the Cav4 mutant lethargic (lh/lh), featuring reduced locomotor activity, ataxia and absence seizure without significant alterations in the basic properties of the channel. Interactions of Cav2.1 with Cav4 and Rimbp2 were significantly reduced while those with GABAB2 was enhanced in the cerebellum of Sca6CtmKO/CtmKO mice. Treatment with the GABAB antagonist CGP35348 partially rescued the motor impairments seen in Sca6CtmKO/CtmKO mice. These results suggest that the MPI and MPc-type CaV2.1 channels function in unique Cav2 nano-environments and the carboxyl-terminal domain plays an essential role in the prevention of a complex neurological phenotype. Overall design: We used MPI 118Q/118Q and CtmKO/CtmKO mice at six weeks old in order to investigate spinocerebellar ataxia type 6 (SCA6). Statistical analysis of differently expressed genes was performed using the Linear Models for Microarray Data (limma) package in R/Bioconductor.

可变剪接（Alternative Splicing, AS）发生于Cav2.1型电压门控钙通道（Voltage-gated Calcium Channel, VGCC）基因的最后一个编码外显子（第47号外显子），可在大脑中产生两种主要同工型：MPI与MPc。二者的剪接受体位点存在差异：人类MPI可翻译为与6型脊髓小脑共济失调（Spinocerebellar Ataxia Type 6, SCA6）相关的多聚谷氨酰胺束，而MPc则剪接至紧邻的终止密码子，最终形成较短的胞质尾区。
为深入探究该可变剪接在体内的功能作用及其与SCA6发病机制的相关性，本研究通过插入剪接位点突变，构建了仅表达MPc的敲入小鼠。所得Sca6^CtmKO/CtmKO小鼠出现了与Cavβ4突变型嗜睡症小鼠（lh/lh）相似的非进行性神经表型，具体表现为运动活性降低、共济失调及失神发作，且未对通道的基本特性造成显著改变。
在Sca6^CtmKO/CtmKO小鼠的小脑中，Cav2.1与Cavβ4及Rimbp2的相互作用显著减弱，而与GABAB受体2型（GABAB2）的相互作用则显著增强。使用GABAB受体拮抗剂CGP35348进行处理后，可部分挽救Sca6^CtmKO/CtmKO小鼠出现的运动功能损伤。
上述结果表明，MPI与MPc型CaV2.1通道在独特的Cav2纳米微环境中发挥功能，且羧基末端结构域在预防复杂神经表型中发挥关键作用。
总体实验设计：本研究选取6周龄的MPI 118Q/118Q及CtmKO/CtmKO小鼠，用于探究6型脊髓小脑共济失调（SCA6）的相关机制。采用R/Bioconductor平台中的微阵列数据线性模型（Linear Models for Microarray Data, limma）包，对差异表达基因进行统计学分析。