Voltage-gated potassium channels play an important role in shaping electrical signals in the brain. The Kv1.1- subtype of voltage-gated potassium channel has been implicated in modulating action potential propagation and mutations within the Kv1.1 gene are associated with episodic ataxia type-1 (EA1), a dominant human neurological disorder with widely variable clinical manifestations, including stress-induced ataxia, myokymia, neuromyotonia, and epilepsy. RNA transcripts encoding the Kv1.1 channel subunit are subject to an adenosine-to- inosine RNA editing event in which a genomically-encoded isoleucine codon (AUU) is converted to a valine codon (IUU) in the mature mRNA to alter the amino acid identity at position 400 of the encoded channel protein. This amino acid residue lies in the highly conserved ion-conducting pore of the channel and RNA editing is known to alter the rate of potassium channel inactivation in heterologous expression systems. To determine the physiological importance of this non-synonymous amino acid alteration, we have developed genetically- modified mouse lines that solely express either the non-edited (I) or edited (V) channel isoforms. In preliminary analyses, non-edited Kv1.1 (I)-expressing mice displayed phenotypic characteristics consistent with EA1, suggesting that modulation of Kv1.1 activity through editing is an important regulator of motor control and seizure susceptibility. The proposed studies will focus upon molecular, behavioral and neurophysiological characterization of Kv1.1 mutant animals with a particular focus on EA1-like phenotypes that have been observed in preliminary studies for non-edited Kv1.1 (I) mice. These studies will also determine the relationship between Kv1.1 editing compared to an established mouse model of EA1, engineered to bear a human mutation associated with this disorder. The initial characterization will quantify potential compensatory changes in gene expression for proteins involved in Kv1.1 signaling, as well as determine the time-frame in which homozygous Kv1.1 (I) animals die of an incompletely penetrant lethality, providing insights into the developmental importance of Kv1.1 editing. Behavioral analyses of various aspects of locomotor coordination will be performed under both control and stressed conditions since the motor dysfunction observed in EA1 is exacerbated by stress. In order to assess alterations in brain electrical activity for mutant Kv1.1 mice, electroencephalography (EEG) studies will monitor spontaneous seizures and chemical convulsants will be used to determine induced-seizure thresh- olds. To assess whether previously characterized EA1 mutations alter Kv1.1 function by missense amino acid incorporation or disruption of editing, we will use both in vitro and in vivo model systems to quantify editing profiles for EA1 mutants. Finally, we will employ electrophysiological techniques on Purkinje neurons in cerebellar slices to examine potential alterations in Kv1.1 channel kinetics. It is anticipated that the proposed studies will not only provide critical insights into he importance of RNA editing for the regulation of Kv1.1 function, but also into how altered editing for Kv1.1 transcripts may result in locomotor and neurological dysfunction.
The proposed studies will increase our understanding of cellular processes governing the voltage-gated potassium channel subunit, Kv1.1, an important regulator of brain activity affecting motor coordination and normal neuronal signaling. RNA transcripts encoding Kv1.1 are modified by an RNA editing event that changes the amino acid sequence of the encoded protein to alter its electrical properties. Utilizing novel mouse models that restrict expression of the Kv1.1 channel to either the edited or non-edited isoform will provide a unique opportunity to advance our understanding of the functional effects of this RNA processing event in vivo and to aid investigators in the development of therapeutic strategies for treating disorders related to Kv1.1 function, including locomotor discoordination and increased seizure susceptibility.