Abstract：
Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.
摘要：
SCN8A的致病变异，它编码电压门控钠（NaV）通道NaV1.6，与包括发育性和癫痫性脑病在内的神经发育障碍有关。先前确定SCN8A变体功能的方法可能通过使用新生儿表达的NaV1.6（NaV1.6N）的选择性剪接同种型而混淆，和工程突变使通道河豚毒素（TTX）具有抗性。我们通过比较两种发育调节的剪接同种型（NaV1.6N，NaV1.6A).我们采用自动膜片钳记录来提高吞吐量，并开发了具有低水平内源性NaV电流的新型神经元细胞系（ND7/LoNav），以消除对TTX抗性突变的需要。ND7/LoNav细胞中NaV1.6N或NaV1.6A的表达产生的NaV电流在激活和失活的电压依赖性方面具有小但显着的差异。与相应的野生型(WT)通道相比,两种同种型的TTX抗性版本表现出显著的功能差异。我们证明了所研究的15种疾病相关变体中的许多都表现出同工型依赖性功能效应，并且许多研究的SCN8A变体表现出不容易分类为功能增益或功能丧失的功能特性。我们的工作说明了在研究SCN8A变体时考虑分子和细胞背景的价值。
