Lysine Versus Arginine: RNA Editing In The Eag Potassium Channel

Abstract

Four RNA editing sites in eag, a Drosophila voltage-gated potassium channel, result in point mutations. One of these mutations, K467R, involves a highly conserved basic residue at the top of the S6 segment. We characterized wild-type and mutant channels using two-microelectrode voltage clamp and patch clamp in Xenopus oocytes. The homologous mutation is lethal in Shaker and hERG. Position 467 plays an important role in inactivation; the K467R mutation causes a 54% decrease in the fraction of inactivated current at +80 mV. The fraction of inactivated current is reduced at higher (10 mM) extracellular Mg+2 concentrations; constructs with a lysine at 467 are more sensitive to changes in extracellular Mg+2 than those with an arginine. Mutating position 467 to alanine, glutamine or cysteine resulted in intermediate inactivation phenotypes and a leftward shift of the peak current-voltage relationship, normalized at +80 mV. Using instantaneous IV measurements from cell-attached oocyte patches, we constructed normalized Po curves for 467Q, 467R and 467K. The Po-V curves for these mutations are superimposable, suggesting little effect on activation gating. However, 467Q and 467R produce inward rectification in instantaneous IV measurements, suggesting a change in ion permeation. Single channel current amplitudes at +40 mV, estimated from non-stationary noise analysis, are comparable for these mutants, which affect instantaneous rectification at more depolarized potentials. Preliminary experiments show no change in rectification between cell-attached and inside-out patches suggesting the permeation change is not due to block by cytoplasmic cations. Intracellular TBA (tetrabutylammonium) blocks 467R significantly better than 467K. Block by intracellular, but not extracellular, TEA (tetraethylammonium) interferes with inactivation. These results show that even a minor residue change can have a dramatic impact on channel biophysics.