Identification of the modulators of cardiac ion channel function

[摘要] The human ether-à-go-go-related gene (HERG) encodes the protein underlying thecardiac potassium current IKr. Mutations in HERG may produce defective channels andcause Long QT Syndrome (LQTS), a cardiac disease affecting 1 in 2500 people. Thedisease is characterised by a prolonged QT interval on a surface electrocardiogram andhas a symptomatic variability of sudden cardiac death in childhood to asymptomaticlongevity. We hypothesised that genetic variation in the proteins that interact with HERGmight modify the clinical expression of LQTS. Yeast two-hybrid methodology was usedto screen a human cardiac cDNA library in order to identify putative HERG N-terminusligands. Successive selection stages reduced the number of putative HERG ligandcontainingcolonies (preys) from 268 to 8. Putative prey ligands were sequenced andidentified by BLAST-search. False positive ligands were excluded based on theirfunction and subcellular location. Three strong candidate ligands were identified: Rhoassociatedcoiled-coil containing kinase 1 (ROCK1), γ-sarcoglycan (SGCG) andmicrotubule-associated protein 1A (MAP1A). In vitro co-immunoprecipitation (Co-IP)and mammalian two-hybrid (M2H) analyses were used to validate these proposedinteractions, but failed to do so. This should be further investigated. Analysis ofconfirmed interactions will shed light on their functional role and might contribute tounderstanding the symptomatic variability seen in LQTS.

[发布机构] Stellenbosch University