Understanding the regulation ion channel is critical to understanding basic mechanisms of cellular excitability. Defective ion channel loci, and misprocessing of their gene products, have been linked to a rapidly expanding list of disorders, including cystic fibrosis, cardiac arrhythmias, and certain forms of epilepsy. RNA editing is an important regulatory mechanism for ion channels at the synapse. This proposal draws extensively from biophysical analysis of voltage-dependent K+ challenge go answer a question of fundamental biological importance: is RNA editing also important at the level of the action potential? The focus of these studies is the delayed rectifier K+ channel of the squid giant axon (SqKv1A). Thus far, 12 codons which are edited have been identified. These sites are scattered in a variety of functionally important channel domains, and preliminary data suggest that they modify physiological properties.
The aim of this work is to define the functional effects of editing in the channel's score and tetramerization (T1) domain. An unknown combination of 5 core editing sites effect deactivation and steady-state voltage sensitivity. The relevant sites will be identified, and the biophysical basis for their actions will be examined on a single channel level. Position 87 in the T1 domain, which is edited from arginine to glycine (R87G), has a tremendous effect on regulating functional expression in Xenopus oocytes. R87 G homotetramers express at 100 fold lower levels than their edited counterparts. Because this position is partially edited in the giant axon, it's effect on heterotetramer expression will be examined. To accomplish this, monomer specific tags will be developed. In the future it is intended that these studies will be extended to the native system. For this reason the giant axon is ideal: it is identifiable, extensively studied, and its dimensions permit high resolution voltage clamp on the macroscopic, gating, and single channel levels.
