Biology subjects

Abramova, T. V.

Publications and source records attributed to Abramova, T. V..

3 recordsLinked to original sources

bioRxiv2024

Differential roles of NaV1.2 and NaV1.6 in neocortical pyramidal cell excitability

Mature neocortical pyramidal cells functionally express two sodium channel (NaV) isoforms: NaV1.2 and NaV1.6. These isoforms are differentially localized to pyramidal cell compartments, and as such are thought to contribute to different aspects of neuronal excitability. But determining their precise roles in pyramidal cell excitability has been hampered by a lack of tools that allow for selective, acute block of each isoform individually. Here, we leveraged aryl sulfonamide-based molecule (ASC) inhibitors of NaV channels that exhibit state-dependent block of both NaV1.2 and NaV1.6, along with knock-in mice with changes in NaV1.2 or NaV1.6 structure that prevents ASC binding. This allowed for acute, potent, and reversible block of individual isoforms that permitted dissection of the unique contributions of NaV1.2 and NaV1.6 in pyramidal cell excitability. Remarkably, block of each isoform had contrasting--and in some situations, opposing--effects on neuronal action potential output, with NaV1.6 block decreasing and NaV1.2 block increasing output. Thus, NaV isoforms have unique roles in regulating different aspects of pyramidal cell excitability, and our work may help guide development of therapeutics designed to temper hyperexcitability through selective NaV isoform blockade.

neuroscience↗

bioRxiv2023

Molecular and Cellular Context Influences SCN8A Variant Function

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of NaV1.6 (NaV1.6N), and engineered mutations to render the channel tetrodotoxin (TTX) resistant. In this study, we investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in two developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a novel 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 that differed significantly in voltage-dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared to the corresponding wild-type (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.

neuroscience↗

bioRxiv2023

Epilepsy-associated SCN2A (Nav1.2) Variants Exhibit Diverse and Complex Functional Properties

Pathogenic variants in neuronal voltage-gated sodium (NaV) channel genes including SCN2A, which encodes NaV1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. SCN2A is also a high confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function (GoF) variants cause epilepsy whereas loss-of-function (LoF) variants are associated with ASD and ID. However, this framework is based on a limited number of functional studies conducted under heterogenous experimental conditions whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of more than 30 SCN2A variants using automated patch clamp recording to assess the analytical validity of this approach and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common population variants using two distinct alternatively spliced forms of NaV1.2 that were heterologously expressed in HEK293T cells. Multiple biophysical parameters were assessed on 5,858 individual cells. We found that automated patch clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for a subset of variants that were previously studied using manual patch clamp. Additionally, many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-function properties that are difficult to classify overall by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of a larger number of variants, greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor valuable for accurate assessment of NaV channel variant dysfunction. Together, this approach will enhance our ability to discern relationships between variant channel dysfunction and neurodevelopmental disorders.

neuroscience↗