Functional Consequences of RNA Editing of the eag Potassium Channel

Abstract

RNA editing is an enzymatic process that selectively alters one or more specific bases in a transcript. This change can potentially affect the function of a protein by introducing a point mutation. Four RNA editing sites in eag, a Drosophila voltage-gated potassium channel, result in such recoding. These sites and the editing mutations are K467R, Y548C, N567D and K699R. They are located at the top of the S6 segment (site 1, residue 467), and the cytoplasmic C-terminal domain including the putative cyclic nucleotide binding domain (sites 2-4). We examined the consequences of editing at these sites by using mutant constructs, each containing one edited site on the background of an all-genomic (unedited) channel. We characterized these channels using two-microelectrode voltage clamp in Xenopus oocytes. The fully edited construct (all four sites) had the slowest activation kinetics and a paucity of inactivation at depolarized voltages, whereas the fully unedited channel exhibited the fastest activation and most dramatic inactivation. Editing the first two sites slowed activation kinetics more so than at the other two sites. Site 1 plays an important role in inactivation; mutating this site from lysine (unedited) to arginine (edited) causes a 54% decrease in the steady-state (700 ms) fraction of inactivated current at +80 mV. Mutating this residue to alanine, glutamine, glutamate or cysteine resulted in intermediate inactivation phenotypes. These neutral or acidic side-chains also caused a leftward shift of the peak current-voltage relationship. Exposure of the cysteine mutant to a variety of methanethiosulfonate reagents had little effect on channel biophysics. These results show that the position of the editing site and the identity of the amino acid at that site are important for fine-tuning the channel's function.