Regulation of surface localization of the small conductance Ca2+-activated potassium channel, Sk2, through direct phosphorylation by cAMP-dependent protein kinase.
Small conductance, Ca2+-activated voltage-independent potassium channels (SK channels) are widely expressed in diverse tissues; however, little is known about the molecular regulation of SK channel subunits. Direct alteration of ion channel subunits by kinases is a candidate mechanism for functional modulation of these channels. We find that activation of cyclic AMP-dependent protein kinase (PKA) with forskolin (50 microm) causes a dramatic decrease in surface localization of the SK2 channel subunit expressed in COS7 cells due to direct phosphorylation of the SK2 channel subunit. PKA phosphorylation studies using the intracellular domains of the SK2 channel subunit expressed as glutathione S-transferase fusion protein constructs showed that both the amino-terminal and carboxyl-terminal regions are PKA substrates in vitro. Mutational analysis identified a single PKA phosphorylation site within the amino-terminal of the SK2 subunit at serine 136. Mutagenesis and mass spectrometry studies identified four PKA phosphorylation sites: Ser465 (minor site) and three amino acid residues Ser568, Ser569, and Ser570 (major sites) within the carboxyl-terminal region. A mutated SK2 channel subunit, with the three contiguous serines mutated to alanines to block phosphorylation at these sites, shows no decrease in surface expression after PKA stimulation. Thus, our findings suggest that PKA phosphorylation of these three sites is necessary for PKA-mediated reorganization of SK2 surface expression.
Ren
Yajun
Y
Cain Foundation Laboratories, Department of Pediatrics, Department of Neurology, Department of Neuroscience, Baylor College of Medicine, Houston, Texas, 77030, USA.
Barnwell
Lyndon F
LF
Alexander
Jon C
JC
Lubin
Farah D
FD
Adelman
John P
JP
Pfaffinger
Paul J
PJ
Schrader
Laura A
LA
Anderson
Anne E
AE
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D051640:Small-Conductance Calcium-Activated Potassium Channels; D005576:Colforsin; D000242:Cyclic AMP; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases