[Show abstract][Hide abstract] ABSTRACT: Voltage-gated potassium channel Kv3.3 is the causative gene of spinocerebellar ataxia type 13 (SCA13), an autosomal dominant neurological disorder. The four dominant mutations identified to date cause Kv3.3 channels to be nonfunctional or have altered gating properties in Xenopus oocytes. In the present study, we report that SCA13 mutations affect functional as well as protein expression of Kv3.3 channels in a mammalian cell line. The reduced protein level of SCA13 mutants is caused by a shorter protein half-life, and blocking the ubiquitin-proteasome pathway increases the total protein of SCA13 mutants more than wild type. SCA13 mutated amino acids are highly conserved, and the side chains of these residues play a critical role in the stable expression of Kv3.3 proteins. In addition, we show that mutant Kv3.3 protein levels could be partially rescued by treatment with the chemical chaperone TMAO and to a lesser extent with co-expression of Kv3.1b. Thus, our results suggest that amino acid side chains of SCA13 positions affect the protein half-life and/or function of Kv3.3, and the adverse effect on protein expression cannot be fully rescued.
Preview · Article · Jun 2013 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: Episodic ataxia type 1 (EA-1) is an autosomal dominant neurological disorder caused by mutations in the potassium channel Kv1.1. Two EA-1 mutations, I262T and S342I, have been identified with unique clinical phenotypes, but their functional and biochemical properties have not been fully investigated. Here we characterized these two mutations in transfected mammalian cells both electrophysiologically and biochemically. We found that the I262T mutation resulted in a ∼7-fold reduction in the K+ current amplitude compared with wild type channels, whereas the S342I mutation produced an apparent nonfunctional channel when expressed alone. Co-expression of wild type and mutant channels showed that both I262T and S342I exerted dominant-negative effects on wild type function. The protein expression analysis showed that I262T resulted in ∼2-fold decrease in surface protein levels of Kv1.1, which partially contributed to the decreased surface conductance density, whereas the S342I mutation showed no effects on surface protein expression. Conservative amino acid substitution experiments suggest that the wild type amino acids at these two positions are required for normal channel function. Our results broaden the knowledge of EA-1 mutations and the underlying mechanisms of the associated disorder.
No preview · Article · May 2012 · Archives of Biochemistry and Biophysics