The long term goals are to relate the electrical activity of the heart to the genesis and processing of the ion channels responsible for this activity.
The specific aims are to: 1) study processing of HERG using misprocessed mutants linked to hLQT2 as probes; 2) test for native chaperones that may interact with HERG during processing and characterize a novel ER resident protein recently discovered by us to interact directly with HERG; 3) expand upon our recent discovery of a novel cytoplasmic protein member of a family of K+ channel chaperones that enhances HERG currents and may be useful as a chaperone to rescue misprocessed mutants; and 4) study the interactions between KvLQT1 and minK during processing using misprocessed mutants linked to hereditary long QT syndrome (hLQTS) as probes. The mutations in KvLQT1/minK and HERG are responsible for most cases of hLQTS and HERG is the target in the majority of cases of the more common acquired LQTS produced by cardiac and non-cardiac anti-arrhythmic drugs. An important outcome of our studies will be the identification of trafficking mutants in hLQTS and the discovery of agents which may enhance trafficking of mutant channels. Likewise we may identify drugs which alter trafficking of normal channels to reduce or increase currents thereby mimicking channel blockers or channel activators. The research uses misprocessed mutant HERG and KvLQT1/minK channels to identify sites along the processing pathway are critical for maturation and trafficking and conversely searches for processing proteins that interact with wild type and mutant channels. The methods include patch clamp electrophysiology, immunostaining, immunoblotting, immunopurification, pulse-chase labeling, mutagenesis, and yeast two hybrid screens. Our strategy has already led to the discovery of a novel ER-resident protein that interacts with HERG and a novel cytoplasmic protein that enhances HERG currents. Their mechanisms of action together with characterizing the processing of HERG and KvLQT1/minK are the goals of this proposal.
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