Characterization of readthrough protein products using HEK293 cell line expressing hSCN1A, hSCN1B and hSCN2B
Date Issued
2016
Date
2016
Author(s)
You, Jhih-Yi
Abstract
Mutations in sodium channels are one of clinically relevant genetic epilepsies with a various range of severity. Especially, the most predominant target of mutation is the NaV1.1 channel encoded by the SCN1A gene. Truncation mutations in the SCN1A gene account for nearly 50% of SMEI patients, and most of them have intractable epilepsy after current epilepsy treatment. Due to the poor prognosis, patient with SCN1A nonsense mutation address an unmet medical need. Nonsense suppression therapy is a newly developed therapeutic approach for nonsense mutation diseases, which incorporates an amino acid at premature termination codons (PTCs). This process may lead to a missense mutation in a functional or non-functional readthrough protein. Recently, a study in S. cerevisiae identified that some specific sets of amino acids are inserted at PTCs after gentamicin treatment, which narrows the possible sets of proteins and its functionality after readthrough. Based on the above, the potential of nonsense suppression therapy for patients with SCN1A nonsense mutation is worthy of investigation. To investigate the electrophysiological properties of readthrough-induced SCN1A proteins in vitro, we generated chimeric constructs pCMV-EGFP-hSCN1A (N-terminal tagging of SCN1A) harboring wild-type (SCN1A-WT), nonsense mutation (SCN1A-E1099X) or three possible readthrough-induced proteins (SCN1A-E1099Q, SCN1A-E1099K, SCN1A-E1099Y). Meanwhile, we generated HEK293 cells stably expressing SCN1B and SCN2B proteins for modulating the kinetics of SCN1A protein. Our results showed that SCN1B and SCN2B proteins in stable cell lines were successfully cleaved by 2A peptides. By whole cell recordings, we characterized the electrophysiological properties of SCN1A-WT, SCN1AE1099X and SCN1AE1099Q. SCN1AE1099X predominantly lost the capacity of generating currents predominantly, decreasing Na+ current to <100 pA. SCN1AE1099Q retained the same Na+ currents and biophysical functions of wild-type channels, which indicated SCN1AE1099Q can restore the expression of Na+ channel with the normal function. Our analyses not only provide a new way to establish multi-protein expression of SCN1B and SCN2B proteins in HEK293 cell , but also validate the functionality of readthrough SCN1A protein.
Subjects
readthrough
nonsense mutation
electrophysiology
Type
thesis
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