Characterization of five RNA editing sites in Shab potassium channels

RNA editing revises the genetic code at precise locations, creating single base changes in mRNA. These changes can result in altered coding potential and modifications to protein function. Sequence analysis of the Shab potassium channel of Drosophila melanogaster revealed five such RNA editing sites. Four are constitutively edited (I583V, T643A, Y660C and I681V) and one undergoes developmentally regulated editing (T671A). These sites are located in the S4, S5-S6 loop and the S6 segments of the channel. We examined the biophysical consequences of editing at these sites by creating point mutations, each containing the genomic (unedited) base at one of the five sites in the background of a channel in which all other sites are edited. We also created a completely unedited construct. The function of these constructs was characterized using two-microelectrode voltage clamp in Xenopus oocytes. Each individual 'unediting' mutation slowed the time course of deactivation and the rise time during channel activation. Two of the mutants exhibited significant hyperpolarized shifts in their midpoints of activation. Constructs that deactivated slowly also inactivated slowly, supporting a mechanism of closed-state inactivation. One of the editing sites, position 660, aligns with the Shaker 449 residue, which is known to be important in tetraethylammonium (TEA) block. The aromatic, genomically-encoded residue tyrosine at this position in Shab enhances TEA block 14 fold compared to the edited residue, cysteine. These results show that both the position of the RNA editing site and the identity of the substituted amino acid are important for channel function.