Editing of Kv1.1 RNA Transcripts is Altered by Human Mutations Respons‐ible for Episodic Ataxia Type‐1

Abstract

Transcripts encoding the Kv1.1‐subtype of voltage‐gated potassium channel un‐der‐go an adenosine‐to‐inosine (A‐to‐I) RNA editing event resulting in the con‐ver‐sion of a genomically‐encoded isoleucine (AUU) to a valine codon (IUU) at amino acid position 400 (I400V). Previous studies have shown that this single amino acid change alters the recovery rate following channel inactivation and has been pro‐posed to mod‐u‐late the kinetics of neuronal repolarization due to its location within the predicted ion pore of the channel. Numerous nonsense and mis‐sense muta‐tions in the Kv1.1 gene (kcna1) have been associated with the hu‐man po‐tas‐sium channelopathy, Episodic Ataxia type‐1 (EA1), which is char‐ac‐ter‐ized by epi‐sodes of stress‐induced ataxia and myokymia, and can be associated with epi‐leptic seizures. Three human EA1 mu‐ta‐tions (V404I, I407M, and V408A) are within close prox‐imity to the I400V editing site, and are contained within the RNA duplex structure required for A‐to‐I con‐ver‐sion. We have employed both in vitro and in vivo systems to determine the impact that these EA1 mutations might have on Kv1.1 RNA editing, as well as determined the electro‐phys‐iological changes in chan‐nel function derived from tran‐scripts containing EA1 mutations with either an edited or non‐edited status. In vitro tran‐scribed RNA substrates containing EA1 mutations were incubated with nuclear extracts isolated from cells overexpres‐sing the ADAR2 editing enzyme. Each of the EA1 mu‐ta‐tions dif‐fer‐en‐tial‐ly de‐creased A‐to‐I conversion of Kv1.1 RNAs in relation to their over‐all prox‐im‐ity to the editing site. Furthermore, a mouse model of EA1, encoding the human V408A mutation at the endogenous kcna1 locus, also re‐vealed a significant decrease in Kv1.1 editing in all tissues examined. Analysis of EA1 mutant chan‐nels using a Xenopus oocyte expression system, in a non‐edited (I400) or edited (V400) background, revealed significant changes in the elec‐‐tro‐physiological char‐ac‐teristics of the channel. These observations suggest that altered Kv1.1 function in EA1 could result from both the missense mutation and alterations in editing ef‐fici‐ency. These results also are con‐sis‐tent with a gen‐et‐ically‐modified mouse mod‐el of Kv1.1 editing, where mice sole‐ly expressing the non‐edited Kv1.1 iso‐form exhibit stress‐in‐duced ataxia and an increase in induced‐sei‐zure suscepti‐bil‐i‐ty.Support or Funding InformationJoel G. Hardman Chair in Pharmacology (RBE)Multidisciplinary Training in Molecular Endocrinology (METP) NIH/NIDDK – T32 DK007563 (EFK)Ruth L. Kirschstein National Research Service Award NINDS F31 NS087911 (EFK)