Reconstitution of beta-adrenergic modulation of large conductance, calcium-activated potassium (maxi-K) channels in Xenopus oocytes. Identification of the camp-dependent protein kinase phosphorylation site.
The human large conductance, calcium-activated potassium (maxi-K) channel (alpha and beta subunits) and beta2-adrenergic receptor genes were coexpressed in Xenopus oocytes in order to study the mechanism of beta-adrenergic modulation of channel function. Isoproterenol and forskolin increased maxi-K potassium channel currents in voltage-clamped oocytes expressing the receptor and both channel subunits by 33 +/- 5% and 35 +/- 8%, respectively, without affecting current activation or inactivation. The percentage of stimulation by isoproterenol and forskolin was not different in oocytes coexpressing the alpha and beta subunits versus those expressing the only the alpha subunit, suggesting that the alpha subunit is the target for regulation. The stimulatory effect of isoproterenol was almost completely blocked by intracellular injection of the cyclic AMP dependent protein kinase (cAMP-PK) regulatory subunit, whereas injection of a cyclic GMP dependent protein kinase inhibitory peptide had little effect, indicating that cellular coupling of beta2-adrenergic receptors to maxi-K channels involves endogenous cAMP-PK. Mutation of one of several potential consensus cAMP-PK phosphorylation sites (serine 869) on the alpha subunit almost completely inhibited beta-adrenergic receptor/channel stimulatory coupling, whereas forskolin still stimulated currents moderately (16 +/- 4%). These data demonstrate that physiological coupling between beta2 receptors and maxi-K channels occurs by the cAMP-PK mediated phosphorylation of serine 869 on the alpha subunit on the channel.
Nara
M
M
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046, USA.
Dhulipala
P D
PD
Wang
Y X
YX
Kotlikoff
M I
MI
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004594:Electrophysiology; D015870:Gene Expression / Q000235:genetics; D007545:Isoproterenol / Q000494:pharmacology; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008845:Microinjections; D016296:Mutagenesis / Q000235:genetics; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D015221:Potassium Channels / Q000502:physiology*; D024681:Potassium Channels, Calcium-Activated; D018343:Receptors, Adrenergic, beta-2 / Q000502:physiology; D014981:Xenopus
D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D010455:Peptides; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D018343:Receptors, Adrenergic, beta-2; D010768:Phosphoserine; D005576:Colforsin; C064719:iberiotoxin; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D007545:Isoproterenol