Joviano-Santos J V, Santos-Miranda A, Neri E A, Fonseca-Alaniz M H, Krieger J E, Pereira A C, Roman-Campos D
Laboratory of CardioBiology, Department of Biophysics, Federal University of São Paulo, Brazil.
Laboratory of Genetics and Molecular Cardiology, Heart Institute, University of São Paulo Medical School, São Paulo, Brazil.
Life Sci. 2021 Aug 1;278:119646. doi: 10.1016/j.lfs.2021.119646. Epub 2021 May 26.
SCN5A gene encodes the α-subunit of Na1.5, mainly found in the human heart. SCN5A variants are the most common genetic alterations associated with Brugada syndrome (BrS). In rare cases, compound heterozygosity is observed; however, its functional consequences are poorly understood. We aimed to analyze the functional impact of de novo Na1.5 mutations in compound heterozygosity in distinct alleles (G400R and T1461S positions) previously found in a patient with BrS. Moreover, we evaluated the potential benefits of quinidine to improve the phenotype of mutant Na channels in vitro.
The functional properties of human wild-type and Na1.5 variants were evaluated using whole-cell patch-clamp and immunofluorescence techniques in transiently expressed human embryonic kidney (HEK293) cells.
Both variants occur in the highly conservative positions of SCN5A. Although all variants were expressed in the cell membrane, a significant reduction in the Na current density (except for G400R alone, which was undetected) was observed along with abnormal biophysical properties, once the variants were expressed in homozygosis and heterozygosis. Interestingly, the incubation of transfected cells with quinidine partially rescued the biophysical properties of the mutant Na channel.
De novo compound heterozygosis mutations in SNC5A disrupt the Na macroscopic current. Quinidine could partially reverse the in vitro loss-of-function phenotype of Na current. Thus, our data provide, for the first time, a detailed biophysical characterization of dysfunctional Na channels linked to compound heterozygosity in BrS as well as the benefits of the pharmacological treatment using quinidine on the biophysical properties of Na1.5.
SCN5A基因编码Na1.5的α亚基，主要存在于人体心脏中。SCN5A变异是与Brugada综合征（BrS）相关的最常见基因改变。在罕见情况下，会观察到复合杂合性；然而，其功能后果却知之甚少。我们旨在分析先前在一名BrS患者中发现的不同等位基因（G400R和T1461S位点）的新生Na1.5突变在复合杂合性中的功能影响。此外，我们评估了奎尼丁在体外改善突变型Na通道表型的潜在益处。
在瞬时转染的人胚肾（HEK293）细胞中，使用全细胞膜片钳和免疫荧光技术评估人野生型和Na1.5变异体的功能特性。
两种变异均发生在SCN5A的高度保守位置。尽管所有变异体都在细胞膜中表达，但一旦变异体以纯合子和杂合子形式表达，就会观察到Na电流密度显著降低（单独的G400R除外，未检测到）以及异常的生物物理特性。有趣的是，用奎尼丁孵育转染细胞可部分挽救突变型Na通道的生物物理特性。
SNC5A中的新生复合杂合性突变破坏了Na宏观电流。奎尼丁可部分逆转Na电流的体外功能丧失表型。因此，我们的数据首次提供了与BrS中复合杂合性相关的功能失调Na通道的详细生物物理特征，以及使用奎尼丁进行药物治疗对Na1.5生物物理特性的益处。
