Protein kinase A inhibits intermediate conductance Ca2+-activated K+ channels expressed in Xenopus oocytes.
Intermediate-conductance (IK) Ca(2+)-activated K(+) channels are expressed in many different cell types where they perform a variety of functions including cell volume regulation, transepithelial secretion, lymphocyte activation and cell cycle progression. IK channels are thought to be regulated by phosphorylation; however, whether kinases act directly on the channel is unclear. Using IK channels heterologously expressed in Xenopus oocytes, we demonstrate that IK channels are potently inhibited (60%) by the catalytic subunit of protein kinase A (PKA). Inhibition of IK channel current by PKA is abolished by mutation of four phosphorylation residues (S312, T327, S332, and T348) in the putative calmodulin-binding region of the channel. Evidence for direct modulation of the IK channel by PKA was further demonstrated using GST fusion proteins. The major site of phosphorylation was found to be serine 332; however, other residues were also phosphorylated. We conclude that IK channels can be directly regulated by the cAMP second-messenger system. The mechanism appears to involve direct phosphorylation by PKA of a modulatory locus in the cytoplasmic region of the channel, the site at which calmodulin is thought to interact. Modulation of IK channels by protein kinases may be an important mechanism regulating cell function.
Neylon
Craig B
CB
Department of Neurobiology, Duke University Medical Center, P.O. Box 3209, Durham, NC, USA.
D'Souza
Theresa
T
Reinhart
Peter H
PH
D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology*; D004553:Electric Conductivity; D015870:Gene Expression; D005978:Glutathione / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D024681:Potassium Channels, Calcium-Activated / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D024681:Potassium Channels, Calcium-Activated; D011993:Recombinant Fusion Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D005978:Glutathione