Abstract
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.
Highlights
The protein kinase (PKA) phosphorylation sites within the SK2 channel subunit were mapped, and we developed a phospho-selective antibody based on the identified sites
We showed that SK2 was phosphorylated in the COS7 cell expression system and that direct PKA phosphorylation of SK2 subunits in COS7 cells leads to altered cellular localization of the channel subunits, suggesting that the SK2 channel is a downstream target of the PKA cascade
Immunostaining for SK2 was visualized on the cell-surface membrane and concentrated in presumptive endoplasmic reticulum (ER) and Golgi apparatus in the vehicle control (Me2SO)
Summary
This was based on two observations: 1) Ser136 within the NT domain of SK2 resides at the beginning of the first transmembrane domain, suggesting that this residue may not be accessible to intracellular kinases in the full-length channel, and 2) Ser465 appears to be a minor phosphorylation site based on our studies using the GST SK2-CT construct (see Fig. 5B). No immunoreactivity was seen in lanes containing membranes from sham-transfected COS7 cells treated with vehicle (Sham) or forskolin (Sham plus FSK) that were probed with the phospho-SK2 or commercial SK2 antibodies (Fig. 9A).
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