Characterization of the expression of episodic ataxia type I Kv1.1 potassium channel mutants
Potassium channels are present in organisms ranging from bacteria to animals, and constitute one of the largest and most diverse groups of ion channels. Due to the large repertoire of functions they serve, it is not surprising that mutations in the genes that encode them lead to a number of diseases, such as Episodic Ataxia type I (EA-I). To date studies on EA-I have focused on characterizing the effect that mutations associated with the disease have on channel function. Electrophysiology analysis has shown that, in most of the cases, EA-I mutants exhibit reduced currents which could be due to channel dysfunction, and/or to a decreased amount of protein on the cell surface. Knowledge is lacking on the effect that these mutations have on Kv1.1 cell surface protein levels, protein folding, and protein half-life. In this study, a series of constructs were designed corresponding to 14 known EAI mutations. These plasmids were expressed in cells and Kv1.1 total protein and Kv1.1 surface proteins were examined by immunofluorescence and quantified by western blot analysis. By comparing the level of Kv1.1 protein expressed on the cell surface to the total amount of Kv1.1 protein expressed in cells, ratios were obtained and used in predicting potential failures in targeting to the cell surface. Once this analysis was completed and mutants which exhibit possible intracellular retention were identified, assays were performed to determine the possible mechanism responsible for this retention. Protein half-life was ascertained by conducting pulse-chase experiments. In addition, a detergent solubility assay was performed to provide insight into potential protein misfolding. The effect that these mutations have on other Kv1 subfamily members was observed by co-transfecting some of the EA-I mutants with Kv1.2. Biochemical and cell biological data obtained from these experiments, as well as published data on functional expression, is fundamental in determining the full effect that different mutations have on Kv1.1 biology. In addition, results acquired from this work may prove to be beneficial in finding new therapies to fight the symptoms associated with EA-I.
Ribeiro-Hurley, Eugenia M, "Characterization of the expression of episodic ataxia type I Kv1.1 potassium channel mutants" (2006). ETD Collection for Fordham University. AAI3216923.