Protein kinase A-mediated phosphorylation of HERG potassium channels in a human cell line.
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 line.


METHODS
HERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody.


RESULTS
Elevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular 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 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated.


CONCLUSIONS
PKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.
Wei
Zhang
Z
Department of Cardiology, Medical University Hospital Heidelberg, Heidelberg, Germany.
Thomas
Dierk
D
Karle
Christoph A
CA
Kathöfer
Sven
S
Schenkel
Johannes
J
Kreye
Volker A W
VA
Ficker
Eckhard
E
Wible
Barbara A
BA
Kiehn
Johann
J
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D027682:Cation Transport Proteins; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D000072237:ERG1 Potassium Channel; D004789:Enzyme Activation / Q000187:drug effects; D051638:Ether-A-Go-Go Potassium Channels; D005260:Female; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008845:Microinjections; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010627:Phenethylamines / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D024642:Potassium Channels, Voltage-Gated; D018075:RNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D014982:Xenopus laevis
D000889:Anti-Arrhythmia Agents; D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; C072312:ERG protein, human; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; C092509:KCNH6 protein, human; D010627:Phenethylamines; D010726:Phosphodiesterase Inhibitors; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D018075:RNA, Complementary; D013449:Sulfonamides; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases; D000262:Adenylyl Cyclases; C063533:dofetilide; D015056:1-Methyl-3-isobutylxanthine