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

J Biol Chem. 2021 Jan-Jun;296:100298. doi: 10.1016/j.jbc.2021.100298. Epub 2021 Jan 15.

Abstract

Mutations in genes encoding the human-brain-expressed voltage-gated sodium (NaV) channels NaV1.1, NaV1.2, and NaV1.6 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 NaV 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 NaV1.1 cDNA. We identified nucleotide sequences within the NaV1.1 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 NaV1.1, NaV1.2, and NaV1.6 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 NaV channels with properties that matched their parental constructs. Our findings explain a widely observed instability of recombinant neuronal human NaV channels, and we describe re-engineered plasmids that attenuate this problem.

Keywords: autism; brain; electrophysiology; epilepsy; molecular biology; neurological disease; plasmid; sodium channel.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Sequence
  • Cloning, Molecular / methods
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Gene Expression
  • HEK293 Cells
  • Humans
  • Membrane Potentials / physiology
  • Mutagenesis, Site-Directed / methods
  • NAV1.1 Voltage-Gated Sodium Channel / genetics*
  • NAV1.1 Voltage-Gated Sodium Channel / metabolism
  • NAV1.2 Voltage-Gated Sodium Channel / genetics*
  • NAV1.2 Voltage-Gated Sodium Channel / metabolism
  • NAV1.6 Voltage-Gated Sodium Channel / genetics*
  • NAV1.6 Voltage-Gated Sodium Channel / metabolism
  • Patch-Clamp Techniques
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Promoter Regions, Genetic*
  • Protein Engineering / methods*
  • Protein Stability
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • DNA, Complementary
  • NAV1.1 Voltage-Gated Sodium Channel
  • NAV1.2 Voltage-Gated Sodium Channel
  • NAV1.6 Voltage-Gated Sodium Channel
  • Recombinant Proteins
  • SCN1A protein, human
  • SCN8A protein, human