Cryptic prokaryotic promoters explain instability of recombinant neuronal sodium channels in bacteria

Mutations in genes encoding the human-brain-expressed voltage-gated sodium (Na_V) channels Na_V1.1, Na_V1.2, and Na_V1.6 are associated with a variety of human diseases including epilepsy, autism spectrum disorder, familial migraine, and other neurodevelopmental disorders. A major obstacle hindering investigations of the functional consequences of brain Na_V channel mutations is an unexplained instability of the corresponding recombinant complementary DNA (cDNA) when propagated in commonly used bacterial strains manifested by high spontaneous rates of mutation. Here, using a combination of in silico analysis, random and site-directed mutagenesis, we investigated the cause for instability of human Na_V1.1 cDNA. We identified nucleotide sequences within the Na_V1.1 coding region that resemble prokaryotic promoter-like elements, which are presumed to drive transcription of translationally toxic mRNAs in bacteria as the cause of the instability. We further demonstrated that mutations disrupting these elements mitigate the instability. Extending these observations, we generated full-length human Na_V1.1, Na_V1.2, and Na_V1.6 plasmids using one or two introns that interrupt the latent reading frames along with a minimum number of silent nucleotide changes that achieved stable propagation in bacteria. Expression of the stabilized sequences in cultured mammalian cells resulted in functional Na_V channels with properties that matched their parental constructs. Our findings explain a widely observed instability of recombinant neuronal human Na_V channels, and we describe re-engineered plasmids that attenuate this problem.