Aberrant IP3 receptor activities revealed by comprehensive analysis of pathological mutations causing spinocerebellar ataxia 29
Aberrant IP3 receptor activities revealed by comprehensive analysis of pathological mutations causing spinocerebellar ataxia 29
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DOI：
10.1073/pnas.1811129115
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发表时间：
2018-11-27
影响因子：
11.1
通讯作者：
Mikoshiba, Katsuhiko
中科院分区：
文献类型：
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作者：
Ando, Hideaki;Hirose, Matsumi;Mikoshiba, Katsuhiko
脊髓小脑性共济失调29型（SCA 29）是一种常染色体显性遗传的先天性共济失调，以早发性运动延迟、肌张力减退和步态共济失调为特征。最近，细胞内Ca 2+通道，肌醇1，4，5-三磷酸（IP 3）受体1型（IP（3）R1）中的杂合错义突变被确定为SCA 29的原因。然而，这些突变的功能影响在很大程度上仍然未知。在这里，我们确定了病理性突变影响IP（3）R1活性和Ca 2+动力学的分子机制。使用基因组编辑产生的IP 3R无效HeLa细胞的Ca 2+成像显示，在IP（3）R1的IP 3结合结构域内或附近鉴定的所有SCA 29突变完全消除了通道活性。在这些突变中，R241 K、T267 M、T267 R、R269 G、R269 W、S277 I、K279 E、A280 D和E497 K损害了IP 3与IP（3）R1的结合，而T579 I和N587 D突变破坏了通道活性而不影响IP 3结合，这表明T579 I和N587 D损害了通道门控机制。碳酸酐酶相关蛋白VIII（CA 8）是一种在小脑浦肯野细胞中大量表达的IP（3）R1调节蛋白，是先天性共济失调的致病基因。IP（3）R1的CA 8结合位点内的SCA 29突变V1538 M完全消除了其与CA 8的相互作用和CA 8介导的IP（3）R1抑制。此外，CA 8的病理性突变通过降低蛋白质稳定性和与IP（3）R1的相互作用来降低CA 8介导的IP（3）R1抑制。这些结果证明了病理性突变导致IP（3）R1功能障碍的机制，即，IP 3结合的破坏、IP 3介导的门控和通过IP 3R调节蛋白的调节。由此产生的异常Ca 2+稳态可能有助于小脑共济失调的发病机制。
Spinocerebellar ataxia type 29 (SCA29) is autosomal dominant congenital ataxia characterized by early-onset motor delay, hypotonia, and gait ataxia. Recently, heterozygous missense mutations in an intracellular Ca2+ channel, inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP(3)R1), were identified as a cause of SCA29. However, the functional impacts of these mutations remain largely unknown. Here, we determined the molecular mechanisms by which pathological mutations affect IP(3)R1 activity and Ca2+ dynamics. Ca2+ imaging using IP3R-null HeLa cells generated by genome editing revealed that all SCA29 mutations identified within or near the IP3-binding domain of IP(3)R1 completely abolished channel activity. Among these mutations, R241K, T267M, T267R, R269G, R269W, S277I, K279E, A280D, and E497K impaired IP3 binding to IP(3)R1, whereas the T579I and N587D mutations disrupted channel activity without affecting IP3 binding, suggesting that T579I and N587D compromise channel gating mechanisms. Carbonic anhydrase-related protein VIII (CA8) is an IP(3)R1-regulating protein abundantly expressed in cerebellar Purkinje cells and is a causative gene of congenital ataxia. The SCA29 mutation V1538M within the CA8-binding site of IP(3)R1 completely eliminated its interaction with CA8 and CA8-mediated IP(3)R1 inhibition. Furthermore, pathological mutations in CA8 decreased CA8-mediated suppression of IP(3)R1 by reducing protein stability and the interaction with IP(3)R1. These results demonstrated the mechanisms by which pathological mutations cause IP(3)R1 dysfunction, i.e., the disruption of IP3 binding, IP3-mediated gating, and regulation via the IP3R-modulatory protein. The resulting aberrant Ca2+ homeostasis may contribute to the pathogenesis of cerebellar ataxia.