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J. Biol. Chem., Vol. 281, Issue 17, 11769-11779, April 28, 2006

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Regulation of Surface Localization of the Small Conductance Ca²⁺-activated Potassium Channel, Sk2, through Direct Phosphorylation by cAMP-dependent Protein Kinase^*

Yajun Ren, Lyndon F. Barnwell, Jon C. Alexander^¶, Farah D. Lubin, John P. Adelman^||, Paul J. Pfaffinger^¶, Laura A. Schrader^¶, and Anne E. Anderson^¶1

From the Cain Foundation Laboratories, Department of Pediatrics, Department of Neurology, ^¶Department of Neuroscience, Baylor College of Medicine, Houston, Texas, 77030 and the ^||Vollum Institute, Oregon Health and Sciences University, Portland, Oregon 97239

Received for publication, December 8, 2005 , and in revised form, February 28, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 µM) 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The small conductance, Ca²⁺-activated K⁺ (SK)² channels are found in both neuronal and non-neuronal tissue (1). Functionally, the SK channels are best characterized in the central nervous system. Three genes encode the SK channel subunits (SK1, SK2, and SK3) in mammalian brain (2). SK channels are blocked by the bee venom toxin, apamin, although SK1 is slightly less sensitive than SK2 and SK3 (2-4). In neurons throughout the nervous system, the apamin-sensitive SK channels modulate firing frequency by contribution to the afterhyperpolarization (AHP) that follows a single or a train of action potentials (5-7). SK2 is thought to specifically underlie the medium AHP current (I_mAHP) in hippocampal CA1 pyramidal cells (8). The I_mAHP is Ca²⁺-dependent with a time constant of 100-250 ms and sensitivity to apamin (9-11). The apamin-sensitive I_mAHP modulates instantaneous firing rates and sets the interspike duration in action potential trains to produce spike frequency adaptation (12). In addition, SK channels are localized to the dendrites of pyramidal cells in hippocampal area CA1 and pyramidal neurons of the lateral amygdala where they function to shape synaptic potentials and limit Ca²⁺ influx through NMDA receptors (13, 14) as well as plateau potentials evoked by exogenous glutamate application (15). Other studies have linked overexpression of SK2 and enhancement of the I_mAHP in hippocampus with neuroprotection (16), attenuation of hippocampal LTP, and memory deficits (16, 17). These functions have major implications for a role for SK2 channels in the formation of synaptic plasticity and learning and memory (17, 18).

In addition to the important role in pyramidal neurons, SK channels are critical for regulation of firing frequency in neurons in other parts of the nervous system. In magnocellular neurons of the hypothalamus, the characteristic bursting pattern of oxytocinergic cells that precedes milk ejection is modulated by a Ca²⁺-dependent, apamin-sensitive I_mAHP that appears to be formed by SK3 channels (19, 20). Furthermore in immature cerebellum, SK2 channels are highly expressed and determine the spontaneous firing pattern of Purkinje cells (21). In retinal ganglion cells, the apamin-sensitive I_AHP that controls firing pattern and excitability is mediated by SK2 channel subunits (22). Hence, understanding the functional regulation of SK2 channel subunits would yield valuable information about the molecular regulation of the I_mAHP.

Currently, the molecular mechanism of modulating the I_mAHP and neuronal firing frequency is poorly understood. A number of studies have shown that another component of the AHP current, the slow AHP current is regulated by kinase cascades (23, 24). One intriguing possible molecular mechanism of regulating the I_mAHP is direct kinase phosphorylation of the SK channel subunits conducting the I_mAHP. Indeed, direct K⁺ channel phosphorylation is a well known post-translational modification regulating neuronal excitability. Phosphorylation of K⁺ channels is associated with modulation of channel kinetics and cellular localization changes (25). In addition, recent studies have suggested that activity-dependent potassium channel phosphorylation and functional modulation may be controlled by net kinase activity (26).

In the present study, we show that activation of PKA causes a decrease in SK2 surface expression. Furthermore, the PKA phosphorylation sites within the SK2 channel subunit were mapped, and we developed a phospho-selective antibody based on the identified sites. The phospho-selective antibody was used as a tool for further study of PKA modulation of the SK2 subunit. 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. These studies advance our understanding of the molecular regulation of SK channels by post-translational modification.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Molecular Biology—The original SK2 cDNA construct was kindly provided by Dr. J. P. Adelman. This construct represents the short isoform of SK2 (49 kDa). Recently a long isoform of SK2 (78 kDa) was defined with extended sequence in NT (27). For the purpose of this study, we focused on the 49-kDa isoform of the SK2 subunit. Plasmids containing the NT (amino acid residues 1-140), CT (residues 395-580), and SK2 subunit second intracellular loop (residues 278-306) domains were constructed using the modified glutathione S-transferase (GST) fusion vector pGEX-YR with NotI and EcoRI sites. Site-directed mutagenesis was performed on the PTC-200 Peltier Thermal Cycler (MJ Research, Waltham, MA) using Pfu DNA polymerase (Stratagene, La Jolla, CA). Point mutations were made using the site-directed mutagenesis kit (Stratagene). The following primers were used: S15A (forward, 5'-GGCGTCATGCGTCCGCTCGCCAACTTGAGCTCGTCCCGCCGG-3'; reverse, 5'-CCGGCGGGACGAGCTCAAGTTGGCGAGCGGACGCATGACGCC-3'), S136A (forward, 5'-CCTGTTTGAGAAGCGCAAGCGGCTCGCCGACTATGCGCTCATCTTCGGC-3'; reverse, 5'-GCCGAAGATGAGCGCATAGTCGGCGAGCCGCTTGCGCTTCTCAAACAGG-3'), S465A (forward, 5'-CAATTAAGAGCGGTGAAGATGGAACAGAGG-3'; reverse, 5'-CCTCTGTTCCATCTTCACCGCTCTTAATTG-3'), S561A (forward, 5'-GCTGAGCGTTCCCGGGCCTCGTCCAGGAGG-3'; reverse, 5'-CCTCCTGGACGAGGCCCGGGAACGCTCAGC-3'), S568A (forward, 5'-GTCCAGGAGGCGGCGGGCCTCCTCCACAGCGC-3'; reverse, 5'-GCGCTGTGGAGGAGGCCCGCCGCCTCCTGGAC-3'), S559stop (forward, 5'-TACAATGCTGAGCGTTAACGGTCCTCGTCCAGG-3'; reverse, 5'-CCTGGACGAGGACCGTTAACGCTCAGCATTGTA-3'), S568stop (forward, 5'-GTCCAGGAGGCGGCGGTAATCCTCCACAGCGC-3'; reverse, 5'-GCGCTGTGGAGGATTACCGCCGCCTCCTGGAC-3'), T575stop (forward, 5'-CCTCCACAGCGCCACCATAATCATCTGAGAGTAGCTAG-3'; reverse, 5'-CTAGCTACTCTCAGATGATTATGGTGGCGCTGTGGAGG-3'), SAAA mutant (forward, 5'-GTCCAGGAGGCGGCGGTCCGCCGCCGCAGCGCCACCAACTTCATC-3'; reverse, 5'-GATGAAGTTGGTGGCGCTGCGGCGGCGGACCGCCGCCTCCTGGAC-3'), ASAA mutant (forward, 5'-GTCCAGGAGGCGGCGGGCCTCCGCCGCAGCGCCACCAACTTCATC-3'; reverse, 5'-GATGAAGTTGGTGGCGCTGCGGCGGAGGCCCGCCGCCTCCTGGAC-3'), and AASA mutant (forward, 5'-GTCCAGGAGGCGGCGGGCCGCCTCCGCAGCGCCACCAACTTCATC-3'; reverse, 5'-GATGAAGTTGGTGGCGCTGCGGAGGCGGCCCGCCGCCTCCTGGAC-3'), AAAT mutant (forward, 5'-GTCCAGGAGGCGGCGGGCCGCCGCCACAGCGCCACCAACTTCATC-3'; reverse, 5'-GATGAAGTTGGTGGCGCTGTGGCGGCGGCCCGCCGCCTCCTGGAC-3'), AAAA mutant (forward, 5'-GTCCAGGAGGCGGCGGGCCGCCGCCGCAGCGCCACCAACTTCATC-3'; reverse, 5'-GATGAAGTTGGTGGCGCTGCGGCGGCGGCCCGCCGCCTCCTGGAC-3'), and AAA mutant (S568A/S569A/S570A) (forward, 5'-GTCCAGGAGGCGGCGGGCCGCCGCCACAGCGCCACCAACTTCATC-3'; reverse, 5'-GATGAAGTTGGTGGCGCTGTGGCGGCGGCCCGCCGCCTCCTGGAC-3'). Mutations were confirmed by DNA sequencing (SeqWright, Houston, TX).

Protein Expression and Purification—GST fusion protein constructs of the SK2-NT and -CT (GST SK2-NT and GST SK2-CT, respectively) were expressed in Escherichia coli BL21 (DE3) using methods previously described (28). Briefly, single colonies of BL-21 (DE3) cells transformed with plasmids containing the NT, CT, or second intracellular loop domains were selected and grown in LB broth. After growing to an optical density of 0.6-0.8 (A₆₀₀), the bacteria were induced by incubation at room temperature with 200 µM isopropyl -D-thiogalactopyranoside (Sigma) for 4 h and were harvested by centrifugation at 3,000 x g for 10 min. The cells were resuspended and incubated in STE buffer (in mM): 10 Tris-HCl, pH 8.0, 1 EDTA, 150 NaCl, containing Protease Inhibitor Mixture (Sigma) and 100 µg/ml lysozyme (Sigma) for 15 min on ice. 10 mM dithiothreitol and 1.5% N-laurylsarcosine were added, and then the sample was sonicated for a total of 2 min. The lysates were then centrifuged (10,000 x g, 20 min, 4 °C) and adjusted to 0.7% N-laurylsarcosine and 2% Triton X-100. The GST fusion proteins were purified using glutathione affinity absorption. Glutathione-agarose beads (Amersham Biosciences) were washed, resuspended in phosphate-buffered saline (PBS, pH 7.4; in mM: 137 NaCl, 2.7 KCl, 4.3 Na₂HPO₄, 1.4 KH₂PO₄) and then incubated with the lysates for 1 h at room temperature or overnight at 4 °C. The beads were washed three times with PBS buffer by centrifugation (100 x g, 5 min, 4 °C). After the final wash, the bead preparation was resuspended in PBS buffer containing Protease Inhibitor Mixture. The recombinant proteins were left on the beads for subsequent experiments and stored at 4 °C.

PKA Phosphorylation of GST SK2 Fusion Proteins—GST SK2-NT or -CT fusion proteins were incubated for 30 min at 37 °C in reaction mixtures (50 µl) containing 70 ng of the catalytic subunit of PKA (Sigma), Tris buffer, and ATP mix buffer(100 µM ATP, 100 mM MgCl₂, and 10 µCi of [-³²P]ATP). For time-course studies of phosphorylation of the GST SK2-NT and -CT fusion proteins incubation periods of 5, 10, 30, 60, and 90 min were used. Reactions were stopped by boiling for 5 min at 95 °C with sample buffer (30 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 40% glycerol, 8% SDS, 0.04 mg/ml bromphenol blue). The GST fusion proteins were separated by 12.5% SDS-PAGE and visualized by Coomassie Blue staining. Phosphopeptides were identified by autoradiography. As a control, parallel reactions were performed for GST alone and PKA alone with ATP mix buffer. The optical density of autoradiography was normalized to the corresponding Coomassie bands (29).

Phosphopeptide Mapping—Identification of the specific amino acids phosphorylated by PKA within the SK2 CT was performed by a modified tandem mass spectroscopy method in the Protein Chemistry Core Facility at Baylor College of Medicine as previously described (30). The product of a 60-min preparative scale reaction (volume of 300-400 µl) of activated PKA, the GST SK2-CT, and ATP was separated by SDS-PAGE (12.5%) stained by Coomassie Blue and digested in-gel by Lys-C. The separated peptide fragments were desalted using a C₁₈ ZipTip column (Millipore) then analyzed by electrospray ionization mass spectroscopy (API 3000 LC/MS/MS System, PE Sciex, Thornhill, Ontario, Canada). Phosphopeptides were identified by a -79-dalton precursor ion scan and subsequently sequenced with tandem mass spectrometry.

Phospho-antibody Production—Synthetic peptides corresponding to SK2 residues 558-580, which included the identified phosphorylation sites (Ser⁵⁶⁸, Ser⁵⁶⁹, and Ser⁵⁷⁰, unphosphorylated and phosphorylated) were produced by the Protein Chemistry Core Facility at Baylor College of Medicine. The peptides were synthesized with a cysteine at the C-terminal end of the peptide to allow conjugation to the carrier protein keyhole limpet hemocyanin via m-maleimidobenzoyl-N-hydroxysuccinimide ester. The conjugates of the phosphopeptides were used for the production of custom antibodies (Cocalico, Inc., CA). After a preimmune blood sample was taken, the conjugates of the phosphopeptide were injected into two albino New Zealand rabbits with the adjuvant Titermax via intradermal injections. The animals were boosted with antigen several times. The phospho-SK2 antiserum was affinity-purified and characterized with Western blotting (28, 29).

COS7 Cell Expression Systems and Membrane Preparation—COS7 cells were cultured and transfected with FuGene6 (Roche Diagnostics). In brief, cells were grown in poly-D-lysine-coated 6-well plates and maintained in Dulbecco's modified eagle's medium (Invitrogen) containing fetal bovine serum and penicillin/streptomycin. 1 µg of SK2 cDNA was used for transfection in each well with FuGene6 according to manufacturer's instructions. To optimize PKA phosphorylation of full-length SK2 expressed in the cells, we determined basal PKA phosphorylation of SK2 with medium that contained either 1% or 10% serum. Western blotting using a PKA-substrate phospho-antibody revealed basal phosphorylation in both 1 and 10% serum media. However, 10% serum demonstrated higher basal phosphorylation (data not shown). After PKA stimulation, there was a marked increase in the phosphorylation of PKA substrates with 1% serum relative to basal levels (data not shown). Therefore, to obtain a robust response to PKA activation, 1% serum was used in all subsequent PKA manipulation experiments. Transfection of the SK2 channel was performed on COS7 cells with FuGENE 6 and grown for 36-48 h. The media was switched from 10% serum to 1% serum 12 h before PKA manipulation. Sham transfections were performed using a blank vector (pDs-Red, Clontech, Palo Alto, CA) or a Kv4.2 construct. COS7 cells were treated with PKA modulators dissolved in Me₂SO. Me₂SO alone served as the vehicle control. In all conditions the final concentration of Me₂SO was 0.2%. The PKA pathway manipulations were are follows: 1) Me₂SO alone; 2) 15-min incubation with 50 µM forskolin (FSK, PKA activator) plus 100 µM Ro-201724 (phosphodiesterase inhibitor) (28); 3) 30-min preincubation with 10 µM H89 (PKA inhibitor) (31); or 4) preincubated with 10 µM H89 for 30 min followed by a 15-min incubation with 50 µM FSK/100 µM Ro-201724. Cell membrane preparation was performed; briefly, plates were put on ice to stop the reaction and washed three times by cold PBS with phenylmethylsulfonyl fluoride (PMSF, 100 µM). After harvesting by scraping cells off the plates in BHB buffer (in mM: 20 Tris, pH 7.5, 1 EGTA, 1 EDTA, 1 Na₄P₂O₇, 1 Na₃VO₄, 0.1 PMSF), membranes were prepared by centrifuging at 92,500 x g at 4 °C for 60 min. The cell pellet was resuspended in 5% SDS with 100 mM dithiothreitol, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 µM PMSF. Sample buffer was then added, and the samples were loaded on a SDS-PAGE gel for Western blotting.

Immunostaining of COS7 Cells—Transfected COS7 cells were treated with the PKA activators or inhibitors and then fixed with 4% paraformaldehyde (Polysciences, Inc., Warrington, PA) for 40 min at room temperature. Cells were rinsed twice with PBS and treated with 0.3% Triton X-100 for 15 min at room temperature. The cells were blocked with 10% fetal bovine serum for 1 h at room temperature followed by SK2 antibody diluted 1:500 in PBS overnight at 4 °C. The cells were then washed in 0.1% Triton X-100 or Tween 20 three times for 5 min each. Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody (Molecular Probes, Eugene, OR) was diluted to 1:5000 and incubated with cells for 30-40 min at room temperature. The cells were visualized on the Nikon TE-200 inverted fluorescence microscope. All immunohistochemistry studies were performed in three independent experiments.

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Modulation of SK2 channel surface expression by the PKA cascade. COS7 cells transfected with the full-length wild-type SK2 subunit were treated with Me2SO (vehicle control, 0.2%); forskolin (FSK, 50 µM, a PKA activator together with 100 µM Ro-201724, a phosphodiesterase inhibitor); H89 (10 µM, a PKA inhibitor); or FSK with H89 preincubation (H89+FSK). A, representative micrographs show the effects of the PKA pathway manipulations on localization of SK2 in COS7 cells by immunostaining with the commercial SK2 antibody (SK2). PKA cascade activation (FSK) decreased SK2 surface staining and increased perinuclear (endoplasmic reticulum and Golgi apparatus) SK2 staining compared with control (Me2SO). PKA cascade inhibition (H89) increased surface expression of the SK2 channel subunits compared with control. Transfected COS7 cells treated with H89+FSK showed no change in SK2 staining when compared with control (Me2SO (DMSO)). No SK2 staining was seen in the sham-transfected COS7 cells (supplemental Fig. S1). Scale bars, 20 µm. B, Western blotting with the SK2 antibody was performed on whole cell lysates and on extracted biotinylated surface proteins following the application of PKA pathway modulators. Representative blots of surface and total SK2 subunits are shown. The top panel (Surface) demonstrates that PKA activation (FSK) decreased, while PKA inhibition (H89) increased SK2 surface expression. The bottom panel (Total lysates) shows that there was no difference in total SK2 channel expression. All conditions were normalized to actin (Actin). C, densitometry of the Western blots shows a significant decrease in surface expression of SK2 following PKA activation (FSK, 65 ± 4% of control; *, p < 0.05) and an increase in SK2 surface expression with PKA inhibition (H89, 168 ± 13% of control; **, p < 0.01) compared with control (Me2SO). There was no significant difference between the control and FSK+H89 conditions (104 ± 9% of control). Data are expressed as mean ± S.E. One-way analysis of variance with post-hoc analysis was used for comparison, n = 3.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Candidate PKA consensus sites within the SK2 channel subunit. The amino acid sequence of the SK2 channel subunit is shown with the intracellular regions underlined. The full-length SK2 subunit consists of 580 amino acid residues with a putative tertiary structure of six transmembrane regions, two intracellular loops and cytoplasmic amino- and carboxyl-terminal domains. We inspected the primary amino acid sequence of the intracellular domains of the SK2 subunit for known PKA consensus sequences (RX1-2(S/T)X) as these regions may be accessible to intracellular PKA. Seven amino acid residues (Ser15, Ser136, Thr431, Ser465, Thr510, Ser561, and Ser568) were predicted as candidate PKA phosphorylation sites within SK2 by NetPhos (www.cbs.dtu.dk/services/NetPhos/). The predicted sites are indicated in bold and are numbered.

Western Blot Analysis—Gels were blotted electrophoretically to Immobilon filter paper using a transfer tank maintained at 4 °C as described previously (32). Blots were blocked for 1 h at room temperature in a blocking solution containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 5% milk, 0.01% thimerosal. The blots were incubated at room temperature with one of the following primary antibodies: total SK2 antibody, 1:500, Alomone Labs, Jerusalem, Israel; phospho-SK2 antibody, 1:500; phospho-PKA substrate antibody, 1:1000 (Cell Signaling Technology, Beverly, MA) for 1 h. After washing, blots were incubated in a horseradish peroxidase-conjugated secondary antibody (1:20,000, Cell Signaling Technology) for 60 min. Blots were washed extensively in TTBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) after incubation with the secondary antibody. Then enhanced chemiluminescence (ECL) or SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, IL) was used for detection of immunoreactivity.

Data Analysis—Immunoreactivity was quantified by densitometry (Scion Corp., version Beta4). The GraphPad Prism software package was used for statistical analysis of the data. Data are expressed as standard error of mean (S.E.). Representative examples are shown under "Results." One-way analysis of variance with post-hoc analysis or Student's t test was used for comparison. Statistical significance was taken as p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The PKA Pathway Modulates SK2 Channel Surface Expression in COS7 Cells—We first investigated PKA pathway regulation of SK2 channel trafficking and localization in the COS7 cell expression system. Immunostaining was performed on the SK2-transfected COS7 cells with the commercial SK2 antibody after treating the cells with PKA pathway modulators (Fig. 1A). 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). PKA activation using forskolin (FSK (50 µM) together with the phosphodiesterase inhibitor Ro-201724 (100 µM)) attenuated surface expression and increased perinuclear expression (presumptive ER and Golgi complex) of the SK2 subunits, whereas the PKA inhibitor, H89 (10 µM), facilitated SK2 channel trafficking to the cell surface. The decrease in surface expression of SK2 channels by forskolin is attenuated by preincubation with H89. These data suggest that PKA regulates the surface expression of SK2. As controls and in parallel with the studies presented above, we processed untransfected and sham-transfected COS7 cells for SK2 immunohistochemistry. These COS7 cells showed no SK2 staining (sham-transfected COS7 cells shown in supplemental Fig. S1).

We further investigated this effect with surface biotinylation experiments to quantify the cell-surface expression of SK2 following PKA cascade manipulation in COS7 cells. Western blotting with the commercial SK2 antibody was performed to compare the biotinylated and total pools of SK2 channels in each experimental group (Fig. 1B). Western blotting showed no difference in the total SK2 channel expression in lysates of all treated groups. However, there was a significant reduction in SK2 channel expression on the cell surface following forskolin application relative to control (65 ± 4% of Me₂SO control, p < 0.05, n = 3) (Fig. 1C). Western blotting with a second SK2 antibody (generated in the laboratory of J.P.A.) against a different epitope (SK2 residues 538-547) revealed similar results (data not shown). In contrast, following H89 treatment, there was a significant elevation of SK2 channel expression on the cell surface compared with control (168 ± 13% of Me₂SO control, p < 0.01, n = 3). There was no significant difference in surface expression of cells treated with both FSK and H89 (104 ± 9% of Me₂SO control). Together these results demonstrate that, in SK2-transfected COS7 cells, activation of the PKA cascade attenuates surface expression of SK2 channel subunits, whereas a reduction of PKA activity increases SK2 subunit surface expression. Based on the observation that there are PKA consensus sites within the SK2 amino acid sequence, we hypothesized that PKA regulates SK2 surface expression by direct phosphorylation of the SK2 subunits.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. PKA phosphorylates the SK2 amino- and carboxyl-terminal cytoplasmic domains in vitro. GST fusion protein constructs of the SK2 amino-terminal (NT, amino acid residues 1-140) and carboxyl-terminal (CT, amino acid residues 395-580) regions were incubated with the PKA catalytic subunit and [-32P]ATP in vitro. Reaction products were separated using SDS-PAGE and visualized by Coomassie Blue staining (Coomassie) and autoradiography (Autorad). The initial reactions were performed using an incubation period of 30 min (A), and subsequently a time course of the phosphorylation events was performed (B). A, Coomassie Blue-stained gels demonstrate bands at 42 kDa (GST SK2-NT) and at 50 kDa (GST SK2-CT) representing the NT and CT constructs, respectively (left panel). The autoradiogram shows 32P incorporation into the GST SK2-NT and -CT constructs, respectively (right panel), suggesting that both the SK2-NT and -CT domains are PKA substrates. Note that GST alone (28 kDa) and PKA alone (without peptide substrates) were not phosphorylated by PKA in vitro. B, the time course of phosphorylation of the GST SK2-NT (top) or GST SK2-CT (bottom) fusion proteins by PKA was performed. The average normalized optical density of the autoradiographs is shown. By 60 min, the phosphorylation of both NT and CT domains reached saturation levels. Error bars represent S.E. Three independent experiments were performed.

View larger version (54K): [in this window] [in a new window]   FIGURE 4. PKA phosphorylates a single amino acid residue within the SK2 amino-terminal cytoplasmic domain. The SK2 amino-terminal (NT) cytoplasmic domain contained only two predicted PKA consensus sites (Ser15 and Ser136). Therefore, we used serine to alanine mutants (S15A, S136A, and S15A/S136A) within the GST SK2-NT fusion protein to evaluate PKA phosphorylation of these sites in vitro. The top panel shows a Coomassie Blue-stained gel (Coomassie) with bands at 42 kDa representing the SK2-NT WT and mutant constructs. The lower panel represents an autoradiogram (Autorad) of the Coomassie Blue-stained gel, which shows 32P incorporation into the WT and S15A mutant but not the S136A and S15A/S136A mutant constructs. These findings suggest that Ser136 is the only amino acid residue phosphorylated by PKA within the SK2-NT region.

Prior to sequencing the PKA phosphorylation sites within the SK2 domains we determined the time course of phosphorylation of the constructs to ensure that no phosphorylation sites were missed in the mapping studies. The time course of phosphorylation of the SK2 domains by PKA showed that, by 60 min, both the NT and CT fusion proteins had reached saturation levels (Fig. 3B). Therefore, incubations of 60 min are sufficient for complete phosphorylation of the fusion proteins by PKA in vitro.

Identification of the PKA Phosphorylation Sites within the SK2-NT Domain—Because Ser¹⁵ and Ser¹³⁶ were the only two predicted PKA phosphorylation sites within the SK2 NT region, we used site-directed mutagenesis to map the sites within the GST SK2-NT fusion protein. The Ser¹⁵ and Ser¹³⁶ residues were mutated to alanine to block phosphorylation at these sites. Three mutant constructs, single mutants S15A and S136A and the double mutant S15A/S136A were made and incubated with active PKA and [-³²P]ATP in vitro. There was ³²P incorporation within the S15A mutant, but not within the S136A or S15A/S136A mutants (Fig. 4). These data suggest PKA phosphorylates the SK2-NT domain at Ser¹³⁶.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. PKA phosphorylates the SK2 carboxyl-terminal cytoplasmic domain. A, to map the PKA phosphorylation sites within the SK2-CT domain we performed a large scale phosphorylation reaction with the PKA catalytic subunit and the GST SK2-CT construct. The reaction product was digested in-gel by Lys-C and analyzed with tandem electrospray ionization mass spectroscopy for peptide-associated phosphate groups. A-79 precursor ion scan and tandem mass spectroscopy revealed that a phosphate group was associated with a peptide corresponding to SK2 residues 455KFLQAIHQLRSVK467 with the m/z ratio of 823.0 (arrow). These results indicate that Ser465 within the GST SK2-CT construct is phosphorylated by PKA. Mass spectroscopy also identified peptides containing SK2 amino acid residues Thr431 and Thr510 (predicted PKA sites), but these peptides were not phosphorylated. B, we evaluated phosphorylation of GST SK2-CT wild-type compared with a S465A GST SK2-CT mutant construct. Coomassie Blue (Coomassie) staining shows 49-kDa bands, representing the GST SK2-CT wild-type (WT) and mutant (S465A) constructs. Autoradiography (Autorad) demonstrates robust 32P incorporation into both of the constructs. These findings suggest that Ser465 may be a minor site and that there are additional phosphorylation sites within the SK2 CT. C, serine to alanine mutants of the candidate PKA sites, Ser561 and Ser568 in combination with Ser465 within the GST SK2-CT construct were tested for PKA phosphorylation. Coomassie Blue staining and autoradiography show 32P incorporation into GST SK2-CT mutants: S561A, S568A, S465A/S561A, S465A/S568A, and S465A/S561A/S568A. These data suggest that there are additional PKA phosphorylation sites located within the SK2-CT region.

As an alternative approach, we used site-directed mutagenesis to determine if the remaining predicted sites were phosphorylated by PKA. Site-directed mutation of serine to alanine at position 465, to remove the PKA phosphorylation site did not decrease ³²P incorporation into the GST SK2-CT construct (Fig. 5B). These data suggest that additional PKA phosphorylation sites exist within the SK2-CT region, possibly in the serine- and arginine-rich CT tail. To identify the remaining PKA phosphorylation site(s), GST SK2-CT constructs with mutations of the predicted Ser⁵⁶¹ and Ser⁵⁶⁸ sites and the mapped S465 site were generated and incubated with activated PKA and [-³²P]ATP in vitro (Fig. 5C). Phosphorylation was still observed in these constructs. These findings suggest that additional PKA phosphorylation sites within the SK2-CT domain are present. In addition, these data also suggest that Ser⁴⁶⁵ is a minor phosphorylation site.

We hypothesized that the additional PKA sites reside in the serine- and arginine-rich distal SK2-CT tail. Thus, we assessed ³²P incorporation within the GST SK2-CT wild-type compared with three truncated GST SK2-CT fusion proteins, S559stop, S568stop, and T575stop following incubation with activated PKA (Fig. 6A, upper panels). The ³²P incorporation was found within the GST SK2-CT wild-type and T575stop construct but not within the S559stop and S568stop constructs (Fig. 6A, lower panels). These findings suggest that additional PKA phosphorylation site(s) are located distal to residue 567 within the SK2-CT tail. To further define the PKA phosphorylation sites within this region, we mutated residues ⁵⁶⁸SSST⁵⁷¹ to ⁵⁶⁸AAAA⁵⁷¹ within the GST SK2-CT construct (Fig. 6B, upper panel number 2) and performed PKA phosphorylation reactions with [-³²P]ATP. No ³²P incorporation was found within the GST SK2-CT ⁵⁶⁸AAAA⁵⁷¹ construct (Fig. 6B, lower panel, lane 2), suggesting that the major phosphorylation sites were restricted to the ⁵⁶⁸SSST⁵⁷¹ region. A series of serine to alanine mutations were made within the ⁵⁶⁸SSST⁵⁷¹ region to identify the specific residues phosphorylated by PKA (Fig. 6B, upper panel). There was ³²P incorporation within the SK2-CT ⁵⁶⁸SAAA⁵⁷¹, ⁵⁶⁸ASAA⁵⁷¹, and ⁵⁶⁸AASA⁵⁷¹ constructs but not in ⁵⁶⁸AAAT⁵⁷¹ construct (Fig. 6B, lower panels, lanes 3-6)_. These findings suggest that the residues Ser⁵⁶⁸, Ser⁵⁶⁹, and Ser⁵⁷⁰ within SK2 are PKA substrates.

A summary of all of the PKA sites that we mapped within the SK2 cytoplasmic domains are presented in a model diagram of the SK2 channel subunit (Fig. 7). As indicated, only one phospho-site was identified within the SK2-NT domain, while four additional sites were identified within the SK2-CT domain. For the studies testing the functional significance of direct phosphorylation of the SK2 channel by PKA presented below, we focused on the three sites (Ser⁵⁶⁸, Ser⁵⁶⁹, and Ser⁵⁷⁰) located within the distal SK2-CT domain. 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).

View larger version (65K): [in this window] [in a new window]   FIGURE 6. Mapping the PKA phosphorylation sites within the distal region of the CT cytoplasmic domain of SK2. Wild-type and mutant constructs of the GST SK2-CT fusion protein were reacted with [-32P]ATP and activated PKA in vitro. The sequences shown (upper panels in A and B) highlight the region of the GST SK2-CT fusion protein with the mutations. All mutant constructs also contained the S465A mutation. Coomassie Blue staining (Coomassie) and autoradiography (Autorad) was performed on the reaction products. A, the sequences of the GST SK2-CT wild-type (WT) construct (1) and the stop truncation mutants (2-4) are shown in the upper panel. There was 32P incorporation in WT (lane 1) but no incorporation into S559stop (lane 2) and S568stop (lane 3) mutants (lower panel). The T575stop mutant also showed 32P incorporation (lane 4). These data suggest that the PKA phosphorylation sites are in 568SSST571 sequence or in the very distal tail of the SK2-CT. B, 32P incorporation was seen in GST SK2-CT WT construct (lane 1) but was blocked in the quadruple site-mutant (568AAAA571, upper panel, 2) (lane 2), which suggested that the PKA sites were located within the 568SSST571 sequence. A series of mutations within these residues revealed 32P incorporation in the 568SAAA571 (lane 3), 568ASAA571 (lane 4), and 568AASA571 (lane 5) constructs but not the 568AAAT571 construct (lane 6). These findings suggest that residues Ser568, Ser569, and Ser570 within the SK2-CT region are phosphorylated by PKA.

The antibody was used to assess PKA phosphorylation of the full-length SK2 channel subunit in the COS7 expression system. COS7 cells transiently expressing wild-type SK2 were incubated with forskolin (50 µM, FSK, together with 100 µM Ro-201724) or H89 (10 µM) for PKA activation or inhibition, respectively, of the endogenous PKA cascade. Membranes were prepared from SK2-transfected COS7 cells after PKA pathway manipulations. Western blots were probed with the phospho-SK2 antibody and a commercial SK2 antibody that is not sensitive to the phosphorylation state of SK2 (same antibody used in Fig. 1 studies). Actin immunoreactivity was used for normalization. Densitometry revealed a significant increase in immunoreactivity with the phospho-SK2 antibody following forskolin stimulation compared with vehicle (Me₂SO)-treated control COS7 cells (259 ± 21% of control, p < 0.001, n = 4). This effect was blocked by preincubation with H89 (H89 plus FSK, 149 ± 16% of control, not significant compared with control, compared with FSK: p < 0.001, n = 4) (Fig. 9A, left panels). There was no significant change in immunoreactivity using the commercial SK2 antibody following PKA pathway manipulation (Fig. 9A, right panels), indicating that this manipulation does not result in a change in SK2 protein expression. 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). Based on this series of experiments, we conclude that PKA activation couples to direct phosphorylation of residues Ser⁵⁶⁸, Ser⁵⁶⁹, and Ser⁵⁷⁰ within the SK2 channel subunit.

View larger version (45K): [in this window] [in a new window]   FIGURE 7. Summary of the PKA phosphorylation sites within the SK2 channel subunit. A model diagram of the SK2 channel is shown with putative six transmembrane domains and the intracellular amino- and carboxyl-terminal (NT and CT, respectively) domains. Five PKA phosphorylation sites were identified within intracellular domains: Serine (S) 136 within the NT and Ser465, Ser568, Ser569, and Ser570 within the CT. The Ser136 site lies within a region of SK2 that is at the junction of the first transmembrane domain. Ser465 is located in the proximal CT, whereas the Ser568, Ser569, and Ser570 sites are located in the distal tail of the CT cytoplasmic domain. The PKA phosphorylation sites are represented by open circles.

View larger version (23K): [in this window] [in a new window]   FIGURE 8. Development and screening of the phospho-selective SK2 antisera. To measure PKA phosphorylation of the SK2 subunits, phospho-selective antisera were generated and the affinity-purified antibody was screened by Western blotting. A, a phosphorylated SK2 CT peptide fragment representing amino acids 558-580 of the SK2 CT cytoplasmic domain was synthesized for antibody generation (phosphorylated residues are marked in bold and italics). This peptide and the unphosphorylated form of the synthetic peptide were used for antibody screening. B, the phosphorylated and unphosphorylated synthetic peptide fragments of the SK2-CT were coupled to ovalbumin for use in Western blotting. In each gel shown, lanes 1 and 3 contain the unphospho-ovalpeptide (full-strength and 1:5 dilution, respectively), whereas lanes 2 and 4 contain the phospho-ovalpeptide (full-strength and 1:5 dilution, respectively). The purified phospho-SK2 antibody showed recognition of the phosphorylated but not the unphosphorylated ovalbumin-coupled peptide (right panel labeled Ab only). The immunoreactivity with the purified phospho-SK2 antibody was blocked by preincubation of the antigenic peptide (Pep) in a concentration-dependent manner (panels labeled Pep (2.6 nM), Pep (0.26 nM), and Pep (0.1 nM)). C, phospho-selectivity of the affinity-purified antibody was screened by Western blotting using the unphospho- and phospho-SK2-CT GST constructs. The left panel (Ab) shows antibody recognition of the phospho-SK2-CT GST construct (lane 1) with no recognition of the unphospho-SK2-CT GST fusion protein (lane 2). In parallel studies immunoreactivity of the phospho-antibody to the phospho-SK2-CT GST construct was blocked by preincubation of the antibody with the phospho-peptide antigen (right panel, Ab+Pep (0.26 nM)).

View larger version (33K): [in this window] [in a new window]   FIGURE 9. PKA pathway modulation couples to altered levels of SK2 channel subunit phosphorylation in COS7 cells. PKA phosphorylation studies were performed on COS7 cells transfected with the full-length SK2 wild-type (SK2 WT) or full-length SK2 channel subunits with Ser568, Ser569, and Ser570 residues mutated to alanine (SK2-AAA). A, following PKA manipulations, PKA phosphorylation of SK2 WT in transfected COS7 cells was measured by Western blotting membrane fractions using the phospho-SK2 antibody (P-SK2, left panel). PKA activation with forskolin (FSK;50 µM together with 100 µM Ro-201724, a phosphodiesterase inhibitor) significantly increased SK2 phosphorylation relative to vehicle (0.2% Me2SO) control (259 ± 21% of control; ***, p < 0.001), which was significantly attenuated by PKA inhibition with H89 preincubation (10 µM, H89+FSK)(149 ± 16% of control, compared with FSK; ***, p < 0.001). Phospho-SK2 immunoreactivity following preincubation with H89 alone (H89) was unchanged compared with control. In parallel studies, blots probed with the commercial SK2 antibody (not sensitive to phosphorylation sites) showed no differences in SK2 expression (SK2, right panels). Untreated (Sham) and FSK-stimulated (Sham + FSK) untransfected COS7 cells showed no SK2 immunoreactivity. B, since there were basal levels of immunoreactivity in the control and H89 conditions, we evaluated whether the affinity-purified phospho-SK2 antibody recognized unphosphorylated channel proteins. Aliquots of SK2 WT-transfected COS7 cells treated with PKA pathway modulators were incubated with or without alkaline phosphatase (AP, 5 unit/µl). Representative Western blots of total lysates probed with the phospho-SK2 (P-SK2), a commercial phospho-PKA substrate (PKA substrate), or actin (Actin) antibody are shown. Treatment of the samples with AP following PKA manipulation (AP treated) eliminated immunoreactivity with the phospho-SK2 antibody. Probing the blots with the actin antibody showed equal protein loading across lanes. Blots probed with the PKA substrate antibody showed that there was an increase in phosphorylated proteins following PKA activation (FSK) compared with basal levels in the control (Me2SO (DMSO)). H89+FSK reduced the increase in PKA substrate phosphorylation compared with FSK only. In parallel experiments, application of AP to the samples eliminated the immunoreactivity seen with the PKA substrate antibody. C, as an additional confirmation of the phospho-selectivity of the P-SK2 antibody, we performed PKA activation experiments in COS7 cells expressing the full-length SK2-AAA mutant channel subunit. Western blotting with the P-SK2 antibody following PKA cascade activation (FSK) compared with control (Me2SO (DMSO)) demonstrated no immunoreactivity with whole cell lysates (top panel). Parallel lysates probed with the SK2 antibody (not phospho-selective) demonstrated that the SK2-AAA subunit was expressed (bottom panel). Data are expressed as mean ± S.E. One-way analysis of variance with post-hoc analysis was used for comparison, n = 4.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The studies presented here demonstrate that the PKA pathway regulates surface expression of SK2 homomeric channels in the COS7 expression system. PKA activation decreased SK2 surface localization, whereas PKA inhibition had the opposite effect. To determine the mechanisms of this effect, we further investigated whether SK2 was directly phosphorylated by PKA and whether this phosphorylation was responsible for the effect. Through a series of mapping and surface biotinylation experiments, residues Ser⁵⁶⁸, Ser⁵⁶⁹, and Ser⁵⁷⁰ within the distal CT of the SK2 cytoplasmic domain were identified as the PKA phosphorylation sites responsible for the trafficking effect of PKA activation on SK2 surface expression. We also showed that PKA activation increases phosphorylation of expressed SK2 channel subunits at these sites in COS7 cells using a phospho-selective antibody. Furthermore, biotinylation and immunostaining studies showed a decrease in surface expression of SK2 following activation of the endogenous PKA cascade. Modulation of SK2 channel surface expression by PKA was eliminated in the SK2 construct with serine to alanine mutation of the three PKA sites within the distal CT tail. These results support an important role for these sites in PKA-mediated alterations in localization and trafficking of the SK2 channel subunit. The functional significance of PKA phospho-regulation at Ser¹³⁶ and Ser⁴⁶⁵ sites in the full-length SK2 subunit in a cell system remains to be shown.

Ion channel trafficking involves the secretory and endocytic pathways to and from the surface membrane and the regulatory elements in this process are diverse. Recently it has been shown that the activity of protein kinase pathways is an important determinant of potassium channel trafficking (34). Our findings demonstrate that PKA activation leads to a decrease in SK2 channel subunit expression at the COS7 cell surface through direct phosphorylation of the SK2 subunits. Kinase regulation of SK2 surface expression has not previously been shown; however, SK2 association with calmodulin is obligatory for SK2 surface expression in COS7 cells (35). It is possible that phosphorylation of the sites that we mapped leads to conformation changes that disrupt calmodulin binding. It is unlikely that the PKA effect is related to phosphorylation of calmodulin, because there are no PKA consensus sequences within the calmodulin amino acid sequence.

View larger version (18K): [in this window] [in a new window]   FIGURE 10. Serine to alanine mutation of the SK2 distal CT phosphorylation sites blocks the PKA-mediated decrease in SK2 surface expression. To evaluate the effects of PKA phosphorylation of the three contiguous serines Ser568, Ser569, and Ser570 on SK2 trafficking, phosphorylation reactions were performed on COS7 cells transfected with the full-length SK2 triple-site mutant (SK2-AAA), which eliminated the PKA phosphorylation sites within the CT. As a control, parallel experiments were performed with the full-length SK2 wild-type channel. COS7 surface expression of the SK2-AAA subunit following vehicle or PKA activation with forskolin (50 µM) was quantified by Western blotting of biotin-captured surface proteins with the commercial SK2 antibody (SK2) that is not sensitive to the phosphorylation state of SK2. Representative blots of surface (Surface) proteins and total (Total) lysates from COS7 cells expressing the SK-AAA mutant channel subunit treated with vehicle (0.2% Me2SO (DMSO)) or forskolin (FSK) are shown. Normalized data of the changes in the surface expression of the SK2-AAA channel subunit following PKA stimulation are shown in the bar graph. PKA activation had no significant effect on surface localization of the SK2-AAA mutant subunit (Me2SO: 100 ± 18% and FSK: 92 ± 9% of Me2SO, n = 3) in COS7 cells. Forskolin stimulation significantly decreased surface expression of the wild-type SK2 channel subunit compared with vehicle control (not shown; same results as shown in Fig. 1). There was no difference in total SK2-AAA channel protein (blot marked as Total) or actin (Actin). All conditions were normalized to actin. These findings suggest that the contiguous phosphorylation sites (568SSS570) are critical for PKA-induced down-regulation of SK2 surface expression. Data are expressed as mean ± S.E. Student's paired t test was used for analysis.

The effects of PKA phosphorylation of SK2 in excitable tissues where the channels are natively expressed have not been elucidated; however, our findings suggest a molecular locus for previous reports of the regulation of the I_mAHP, which is thought to be formed at least in part, by SK2 channels (18). The enhancement of theta-burst LTP induction in hippocampal slices by brain-derived neurotrophic factor (BDNF) is associated with an increase in direct serine phosphorylation within SK2 and a reduction in the I_mAHP (43). The specific kinase that is activated and serines that are phosphorylated within SK2 in BDNF-enhanced theta-burst LTP remain to be elucidated. However, BDNF transiently activates PKA in the hippocampus (44). Thus, SK2 may be a direct target of the PKA cascade in BDNF-modulated neuronal plasticity. Moreover, functional studies in rat jaw-closing spinal motor neurons have coupled activation of the metabotrophic 5-HT_1A receptor to down-regulation of the apamin-sensitive I_mAHP (45). The exact pathway mediating this effect in spinal motor neurons is unknown, but activation of the 5-HT_1A receptor induces downstream cAMP accumulation and PKA activation (46). Therefore, activation of 5-HT_1A receptors in spinal motor neurons may result in direct phosphorylation of the SK2 subunit and modulation of the I_mAHP.

The phospho-selective antibody that we have generated may provide a tool to assess PKA phosphorylation of SK2 in cell types that natively express SK2 channels. In preliminary studies we have begun to evaluate PKA pathway coupling to SK2 phosphorylation in hippocampal tissue.³ In these studies, the phospho-SK2 antibody recognizes a band at roughly 49 kDa that increases with PKA pathway activation by forskolin. The commercial SK2 antibody recognizes a band at the same molecular weight that does not modulate with forskolin stimulation. These findings suggest that PKA phosphorylation of SK2 occurs in a physiologically relevant system and that the phospho-antibody is selective in a tissue type where SK2 channels are natively expressed.

In conclusion, we have demonstrated that direct PKA phosphorylation of SK2 regulates the localization of SK2 channel proteins in the COS7 expression system. The altered cellular distribution of SK2 channels may play a role in the modulation of the I_mAHP and regulate excitability in tissues where the channels are expressed, such as the nervous system. Given the recently defined role of SK2 in regulation of NMDA receptor-mediated synaptic potentials and Ca²⁺ influx (13, 14), modulation of SK2 surface expression by PKA phosphorylation may have dramatic consequences on cellular plasticity and learning and memory.

	FOOTNOTES

 
^* This work was supported by grants from the NINDS, National Institutes of Health (to A. E. A., L. F. B., P. J. P., J. P. A., and F. D. L.), the National Institute of Mental Health (to L. A. S.), the Child Neurology Foundation (to A. E. A. and L. A. S.), and the Epilepsy Foundation of America (to L. F. B. and F. D. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Baylor College of Medicine, Feigin Center 955, 1102 Bates St., MC 3-6365, Houston, TX 77030. Tel.: 832-824-3976; Fax: 832-825-4217; E-mail: annea{at}bcm.tmc.edu.

² The abbreviations used are: SK, small conductance Ca²⁺-activated K⁺ channels; PKA, cAMP-dependent protein kinase; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; I_AHP, afterhyperpolarization current; NT, N-terminal; CT, C-terminal; PBS, phosphate-buffered saline; FSK, forskolin; ER, endoplasmic reticulum; MS, mass spectrometry; MALDI, matrix-assisted laser desorption ionization; AP, alkaline phosphatase; BDNF, brain-derived neurotrophic factor.

³ Y. Ren, L. F. Barnwell, J. C. Alexander, F. D. Lubin, J. P. Adelman, P. J. Pfaffinger, L. A. Schrader, and A. E. Anderson, unpublished preliminary results.

	ACKNOWLEDGMENTS

 
We thank Drs. Wai Ling Lee and Xianghua Xu for helpful discussions.

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