Transcription and Translation Effects of hERG Channel Gene Synonymous Variation

Mutations that alter protein sequence are a common cause of hereditary disease with examples in nearly every ion channel gene. Genetic nucleotide variation that does not change amino acid sequence may also contribute to changes in protein expression and function leading to disease by mechanisms less understood. We have examined synonymous nucleotide changes in KCNH2 (encoding the hERG channel) and their effects in channel expression and trafficking. A synthetic, codon-modified version of the channel (hERG-CM contrasted to the native sequence, hERG-NT) exhibits perturbations in all aspects regulating channel expression (transcription, mRNA stability, translation, channel trafficking, and protein half-life). Here, we sought to further explore contributions of 5’ sequence differences that affect transcription and translation. Using NanoLuc reporter fusion constructs in a mammalian cell line, we determined that the initial 51-nucleotides of coding sequence contributed less (2-fold change) than that of the initial 1191-nucleotides (7-fold change) towards enhanced expression of hERG-NT. Moreover, this difference appeared to be accounted for by changes in transcription. When we examined these regions in cell-free in vitro translation systems hERG-CM exhibited a 3-fold greater rate of translation. Additional differences in cellular and cell-free transcription and expression of hERG variants were elucidated by low concentrations of cycloheximide and ribosomal ‘toeprinting’. These results indicate a complex interplay between nucleotide variation and biosynthesis of the ERG channel. We conclude that synonymous variation within the coding sequence may affect some ion channel gene expression in unexpected ways that are unpredictable. Accordingly, not all synonymous variation in KCNH2 may be considered neutral