Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node.
The sympathetic nervous system increases heart rate by activating beta adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current (I(f)) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. I(f) is produced by hyperpolarization-activated cyclic nucleotide-sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. beta Adrenergic regulation of I(f) in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of beta adrenergic agonists to shift the voltage dependence of I(f) in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of I(f). Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate.
Liao
Zhandi
Z
Department of Physiology and Biophysics, Division of Cardiology, University of Colorado, Anschutz Medical Campus, Aurora, CO 80045, USA.
Lockhead
Dean
D
Larson
Eric D
ED
Proenza
Catherine
C
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006339:Heart Rate / Q000187:drug effects; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating / Q000187:drug effects; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012849:Sinoatrial Node / Q000187:drug effects / Q000201:enzymology* / Q000294:innervation; D013564:Sympathetic Nervous System / Q000502:physiology*; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014162:Transfection
D000318:Adrenergic beta-Agonists; D054815:Cyclic Nucleotide-Gated Cation Channels; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases