hERG channel distribution and interacting proteins in the heart

Potassium channels encoded by the human ether-a-go-go related gene (hERG) underlie cardiac IKr, one of the main repolarizing currents in the heart. Defects in IKr cause long QT syndrome (LQT), which can lead to arrhythmias and sudden cardiac death. While mutations in hERG are linked to inherited LQT, mechanisms underlying heterogeneity of the disease and susceptibility to the acquired form of the disorder are not well understood. Data presented in this thesis demonstrate two hERG isoforms, 1a and 1b, which differ in their cytoplasmic amino termini, are expressed in cardiac tissue where they associate, and co-localize to transverse tubules and intercalated discs. Isoform-specific antibodies also revealed differential distribution in cardiomyocytes, suggesting selective targeting to membrane domains. A yeast 2-hybrid screen identified five hERG-interacting proteins in the heart, Tara, RUFY2, COG4, DOC1, and GM130, each of which represents a potential target underlying susceptibility to LQT. Interacting regions between each protein and hERG were identified, as were homo-oligomerization domains for Tara and RUFY2. We found COG4 and RUFY2 directly interact, suggesting the two may form a hERG-regulatory complex. GM130, a cis-Golgi matrix protein, negatively regulated hERG maturation. Binding with GM130 was minimally supported by hERG's distal carboxy terminus (dCT) but enhanced by the presence of the upstream cyclic nucleotide binding domain homology region. The failure of GM130 to interact with oligomerized C-terminal contructs indicated GM130 recognizes the non-oligomerized state of the C-terminus, and indeed competes with the C-terminal homotypic interaction. In addition, we found GM130 bound preferentially to hERG 1b expressed alone as compared to hERG 1a or 1a 1b when co-expressed. Thus, the C-terminus is more available for GM130 binding in 1b homomers compared with 1a and 1a/1b channels, where the 1a N-terminus may promote homotypic C-terminal oligomerization. This protein-protein interaction may also be regulated by phosphorylation, as mutating a PKA site within the dCT region to a phospho-null alanine increased 1b binding, while mutation to a phospho-mimetic aspartate decreased binding.