Regulation of HERG channel bio-processing

Abstract

The long QT syndrome (LQTS) is a common cardiac disorder with potentially lethal consequences. While mostly asymptomatic, LQTS patients may develop Torsade de Pointes (TdP) arrhythmia, which can proceed into ventricular fibrillation causing death. Most forms of LQTS are caused by aberrant ion channel function. In particular, human ether-a-go-go-related gene product (HERG), a potassium channel responsible for cardiac repolarization, has been implicated in many congenital and most acquired forms of LQTS. Patients with HERG dysfunction develop TdP only when faced with certain emotional stimuli, suggesting TdP pathogenesis depends on channel regulation. Previously, we have shown that chronic beta-adrenergic stimulation augmented total HERG protein levels in Hek293 cells.;Here we developed an ER surface-targeted PKA inhibitor, whose specificity was confirmed using targeted FRET-based biosensors, radiolabeling and phospho-specific antibodies. The construct inhibited PKA-dependent HERG augmentation. This was in contrast to PKI constructs targeted to the outer mitochondrial membrane, which inhibited HERG augmentation only partially. Next, we assembled a synonymous but highly modified HERG mRNA sequence (HERG-CM); its protein product responded to PKA just as well as native HERG (HERG-NT). These results suggest that PKA regulates HERG co-translationally by phosphorylating the nascent polypeptide.;We also set out to determine whether primary mRNA sequence can regulate HERG protein biology. Using velocity gradient centrifugation and immunofluorescence microscopy, we show that when expressed in Hek293 cells, HERG-NT is mostly retained in the ER, but HERG-CM reaches the plasma membrane more efficiently. Fluorescent in situ hybridization and co-immunoprecipitation studies suggest that this disparity is not due to differential HERG mRNA distribution and translation. Limited proteolytic digestion studies suggest difference in folding. Chimeric studies show that the 5' region of the mRNA is primarily responsible for the observed effects.;Finally, we report developing novel tools for studying HERG regulation. We immunized 15 mice with mixtures of eight ∼100 amino acid-long fragments of HERG and producing one monoclonal antibody recognizing a proximal C-terminal HERG epitope and three monoclonal antibodies to a distal C-terminal epitope. Isolation of persistently secreting hybridomas was only possible when Bcl-2-expressing fusion partners were used.