Deletion of protein kinase A phosphorylation sites in the HERG potassium channel inhibits activation shift by protein kinase A.
We investigated the role of protein kinase A (PKA) in regulation of the human ether-a-go-go-related gene (HERG) potassium channel activation. HERG clones with single mutations destroying one of four consensus PKA phosphorylation sites (S283A, S890A, T895A, S1137A), as well as one clone carrying all mutations with no PKA phosphorylation sites (HERG 4M) were constructed. These clones were expressed heterologously in Xenopus oocytes, and HERG potassium currents were measured with the two microelectrode voltage clamp technique. Application of the cAMP-specific phosphodiesterase (PDE IV) inhibitor Ro-20-1724 (100 microM), which results in an increased cAMP level and PKA stimulation, induced a reduction of HERG wild type outward currents by 19.1% due to a shift in the activation curve of 12.4 mV. When 100 microM Ro-20-1724 was applied to the HERG 4M channel, missing all PKA sites, there was no significant shift in the activation curve, and the current amplitude was not reduced. Furthermore, the adenylate cyclase activator forskolin that leads to PKA activation (400 microM, 60 min), shifted HERG wild type channel activation by 14.1 mV and reduced currents by 39.9%, whereas HERG 4M channels showed only a small shift of 4.3 mV and a weaker current reduction of 22.3%. We conclude that PKA regulates HERG channel activation, and direct phosphorylation of the HERG channel protein has a functional role that may be important in regulation of cardiac repolarization.
Thomas
D
D
Department of Cardiology, Medical University Hospital Heidelberg, Bergheimerstrasse 58, D-69115 Heidelberg, Germany.
Zhang
W
W
Karle
C A
CA
Kathöfer
S
S
Schöls
W
W
Kübler
W
W
Kiehn
J
J
D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D005260:Female; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010766:Phosphorylation; D015221:Potassium Channels / Q000737:chemistry / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D014982:Xenopus laevis
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; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D005576:Colforsin; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; D017868:Cyclic AMP-Dependent Protein Kinases