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Objective To investigate the molecular mechanism of human ether-a-go-go-related gene ( HERG) potassium channels regulated by protein kinase A (PKA) in a human cell lin e. Methods HERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, a nd currents were measured with the patch clamp technique. The direct phosphoryl ation of HERG channel proteins expressed heterologously in Xenopus laevis oo cytes was examined by 32P labeling and immunoprecipitation with an a nti-HERG antibody. Results Elevation of the intracellular cAMP-concentration by incubation with the adenyl ate cyclase activator, forskolin (10μmol/L), and the broad range phosphodiest erase inhibitor, IBMX (100μmol/L), caused a HERG tail current reduction of 83 .2%. In addition, direct application of the membrane permeable cAMP analog, 8 -Br-cAMP (500μmol/L), reduced the tail current amplitude by 29.3%. Intrac ellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 m V towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of ≈155 kDa and a lower band with a molecular mass of ≈135 kDa, indicating that both the core- and the fully glyco sylated forms of the protein were phosphorylated.Conclusions PKA-mediated phosphorylation of HERG channels causes current reduction in a hum an cell line. The coupling between the repolarizing cardiac HERG potassium curr ent and the protein kinase A system could contribute to arrhythmogenesis under p athophysiological conditions.
