(Received for publication, November 22, 1996)
From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280
Voltage-dependent potentiation of
skeletal muscle L-type calcium channels requires phosphorylation by
cAMP-dependent protein kinase (PKA) that is localized by binding to a
cMP-dependent protein
inase-
nchoring
rotein (AKAP).
L-type calcium channels purified from rabbit skeletal muscle contain an
endogenous co-purifying protein kinase activity that phosphorylates the
1 and
subunits of the channel. The co-purifying kinase also
phosphorylates a known PKA peptide substrate, is stimulated by cAMP,
and is inhibited by PKA inhibitor peptide-(5-24), indicating that it
is PKA. PKA activity co-immunoprecipitates with the calcium channel,
suggesting that the channel and the kinase are physically associated.
Using biotinylated type II regulatory subunit of PKA (RII) as a probe, we have identified a 15-kDa RII-binding protein in purified calcium channel preparations, which we have designated AKAP-15. Anti-peptide antibodies directed against the
1 subunit of the calcium channel co-immunoprecipitate AKAP-15. Together, these findings demonstrate a
physical link between PKA and the calcium channel and suggest that
AKAP-15 may mediate their interaction.
The activation of PKA1 following transient increases in intracellular levels of cAMP represents a fundamental mechanism for regulating protein function via phosphorylation (1, 2). A wide variety of proteins including enzymes, membrane receptors, ion channels, and transcription factors have been shown to be PKA substrates with activities reversibly modulated by phosphorylation and dephosphorylation. It is clear that despite its broad substrate specificity, PKA activity is highly selective in a physiological setting and that specific hormones, each capable of raising intracellular cAMP, can result in the preferential phosphorylation of different target substrates (3). Understanding how the activation of a single signaling pathway can lead to multiple but varying cellular effects has become an important goal in cAMP signaling research. Recent work has demonstrated that both cAMP and its target kinase are specifically localized within the cell (4, 5). Together, these findings emphasize that PKA phosphorylation of various target substrates depends not only on whether cAMP levels are increased, but also on where within the cell this increase occurs and whether PKA and its substrates are localized at the site.
The PKA holoenzyme consists of two regulatory subunits and two catalytic subunits forming an inactive heterotetramer (6). Each regulatory subunit binds two cAMP molecules, causing the release of active catalytic subunits. Two classes of regulatory subunits exist, giving rise to type I and II holoenzymes. While type I PKA is generally soluble and cytoplasmic, the type II isoform is predominantly associated with the cell particulate fraction (7). Type II PKA activity has been shown to be associated with a variety of subcellular structures including the plasma membrane, cystoskeleton, Golgi apparatus, and nucleus (8). The type II regulatory subunit (RII) mediates kinase localization via an N-terminal region absent in the type I regulatory subunit (9). Therefore, the RII protein plays a pivotal role both in regulating type II PKA activity and in specifically localizing the type II holoenzyme within the cell.
cMP-dependent protein
inase-
nchoring
roteins (AKAPs)
comprise a diverse group of proteins defined by their high
affinity for RII and proposed role in mediating the attachment of type II PKA to subcellular structures (10, 11). AKAPs retain their ability
to bind RII following SDS-PAGE and electrotransfer to nitrocellulose
(12). Using purified RII protein to probe blots has allowed the
detection of AKAPs from a variety of tissues ranging in size from 34 to
300 kDa (13). The RII protein has also been radiolabeled and used to
screen cDNA expression libraries, allowing the primary structure of
several AKAPs to be determined (14, 15). Despite their diversity, AKAPs
share certain structural features. It has been shown that AKAPs bind to
a common site at the N terminus of the RII protein and that
dimerization of the RII protein is required for binding (16). Critical
RII-binding regions within several AKAPs have been identified and
consist in each case of a single amphipathic helix (17). A short
peptide containing the RII-binding amphipathic helix of the AKAP Ht-31 has been shown to bind RII with nanomolar affinity and has been termed
an "anchoring inhibitor peptide" based on its ability to competitively inhibit RII-AKAP interactions (18). In addition to an
RII-binding domain, each AKAP is proposed to have a unique targeting
domain responsible for determining the subcellular localization of type
II PKA. The targeting domains of the AKAPs MAP-2 (19), AKAP-75 (20),
and AKAP-220 (21) have been identified, and the locations and
structures of other AKAP targeting domains are currently under
investigation.
Anchoring inhibitor peptides have been used in physiological
experiments to demonstrate the functional importance of PKA anchoring. -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate
glutamate receptor ion channels in hippocampal neurons require PKA
phosphorylation for the maintenance of inward currents (22), and the
presence of peptides designed to disrupt RII-AKAP binding results in a diminishment of the current probably by displacing anchored PKA from
the channel (23). The importance of PKA anchoring via AKAPs has also
been established in the regulation of L-type calcium channels. In
skeletal muscle transverse tubules, these channels initiate muscle
contraction by interacting directly with ryanodine receptors to cause
the release of calcium from the sarcoplasmic reticulum (24). The
calcium entering directly through L-type calcium channels, on the other
hand, is thought to be important in regulating the force of
contraction. High frequency stimulation of muscle fibers causes
potentiation of skeletal muscle calcium channel activity, an effect
that may mediate the increased force of muscle contraction during
tetanus (25). This frequency- and voltage-dependent
potentiation of calcium channel activity has been shown to require
phosphorylation by PKA since an inhibitor peptide of PKA blocks the
effect (25). Recent electrophysiological studies have shown that this
effect requires the anchoring of PKA near the channel. The introduction
of an anchoring inhibitor peptide into skeletal muscle myotubes via the
recording pipette eliminates potentiation in skeletal muscle myotubes,
presumably by displacing PKA from an endogenous AKAP (26). The
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor
and L-type calcium channel therefore provide two examples demonstrating
the physiological importance of PKA anchoring.
The electrophysiological evidence that PKA is anchored to the L-type calcium channel complements biochemical studies. It was demonstrated that an endogenous protein kinase activity co-purifies with the calcium channel through several chromatographic steps, suggesting that it might be physically associated with the channel (27). In the present study, we identify this co-purifying kinase as PKA and show that it is physically linked to the channel. Furthermore, we have identified a previously undescribed 15-kDa AKAP that is linked to the L-type calcium channel and may mediate its association with PKA.
10-20% Tricine gels and molecular weight
markers were obtained from Novex (San Diego, CA). cAMP, cAMP-agarose,
protein A-Sepharose, and MAP-2 were purchased from Sigma. EZTM
sulfosuccinimidyl-6-(biotinamido)hexanoate, horseradish
peroxidase-linked avidin, and chemiluminescent substrate (SupersignalTM) were from Pierce. [-32P]ATP (3000 Ci/mmol) was obtained from DuPont NEN. PKA inhibitor peptide-(5-24)
(PKI-(5-24)) was purchased from Peninsula Laboratories, Inc. (Belmont,
CA). HT-31, HT-31-P (18, 26), SP48 (LTYEKRFSSPHQSLLSIR), and SP44
(KYMKKLGSKKPQK) peptides were synthesized and purified in the
University of Washington Molecular Pharmacology Protein Core Facility.
DSDD peptide (RRRDDDSDDD) was a gift from Ed Krebs (University of
Washington).
Skeletal muscle calcium channels were purified as described by Curtis and Catterall (28) from skeletal muscle microsomes prepared according to Fernandez et al. (29). All buffers contained the following protease inhibitors: 4-(aminoethyl)benzenesulfonyl fluoride (1 mM), leupeptin (2 µM), pepstatin A (1 µM), antipain (1.6 µM), calpain inhibitor I (10 µg/ml), calpain inhibitor II (10 µg/ml), and o-phenanthroline (0.9 mM). The picomoles of purified calcium channel were estimated by assuming a molecular mass of 429 kDa for the channel complex.
Phosphorylation of the Calcium Channel and Synthetic Peptides by the Endogenous KinasePurified calcium channels were
phosphorylated by incubation at 37 °C in 50 mM Tris-HCl
(pH 7.5), 0.1% Triton X-100, 10 mM MgCl2, 1 mM EGTA, and 0.15 µM
[-32P]ATP (3000 Ci/mmol) for the times indicated.
Calcium channel phosphorylation reactions were terminated by heating at
65 °C for 3 min in 80 mM Tris-HCl (pH 6.8), 10%
glycerol, 10 mM dithiothreitol, and 2% SDS and
subsequently analyzed by SDS-PAGE. In some cases, individual
phosphoprotein bands were excised from the gels, and 32P
was quantified by liquid scintillation counting.
Purified synthetic peptides were phosphorylated by incubation at
37 °C in 50 mM Tris-HCl (pH 7.5), 10 mM
MgCl2, 1 mM dithiothreitol, and 0.5 mM [-32P]ATP (80 mCi/mmol) for 10 min in
the presence of 1-5 pmol of purified calcium channel. Phosphorylation
reactions were terminated by acidification to 16% acetic acid, spotted
on P-81 paper, washed with 0.1% phosphoric acid, and quantified by
liquid scintillation counting.
The
expression plasmid pET11d containing the mouse RII cDNA (30) was
transformed into Escherichia coli BL21(DE3) competent cells
(Novagen). The RII
vector was kindly provided by John Scott (Vollum
Institute, Oregon Health Sciences University). 25-ml overnight cultures
were grown in LB medium containing 100 µg/ml ampicillin (LBA) and
used to inoculate 1 liter of LBA. Cells were then grown by shaking at
37 °C until the A550 of the 1-liter culture
reached 0.300. Expression of RII was subsequently induced with 1 mM isopropyl-
-D-thiogalactopyranoside, and
incubation was continued for an additional 2.5 h. Cells were pelleted by centrifugation at 2200 × g for 10 min and
resuspended in 20 ml of 10 mM MOPS (pH 6.9), 100 mM NaCl, and 1 mM dithiothreitol containing
aprotinin (10 µg/ml), leupeptin (10 µg/ml), pepstatin A (1 µM), o-phenanthroline (0.2 mg/ml),
4-(aminoethyl)benzenesulfonyl fluoride (1 mM), and
benzamidine (15.7 µg/ml) (lysis buffer). Resuspended cells were lysed
by mild sonication with a probe sonicator for 30 s on ice and
centrifuged at 10,000 × g for 15 min. Pellets were
discarded, and 10 g of ammonium sulfate were added to the supernatant while stirring on ice to achieve 80% saturation. Stirring on ice was continued for 15 min to allow protein precipitation to reach
equilibrium, and precipitated proteins were separated from soluble
material by centrifugation at 10,000 × g for 30 min. Supernatants were discarded, and ammonium sulfate precipitates were
resuspended in 20 ml of lysis buffer containing 10 µM
isobutylmethylxanthine in addition to the protease inhibitors listed
above. Resuspended material was mixed with 5 ml of cAMP-agarose and
rotated for 16 h at 4 °C. Nonspecifically bound proteins were
removed by washing the cAMP-agarose with 50 ml of lysis buffer,
followed by washing with 50 ml of lysis buffer containing 1 M NaCl and finally washing again with 50 ml of lysis
buffer. Bound RII protein was eluted by incubating cAMP-agarose with 15 ml of lysis buffer containing 25 mM free cAMP for 30 min at
room temperature. The eluate was concentrated by centrifugation in a
Centriprep 30 concentrator (Amicon, Inc.) according to the
manufacturer's instructions.
Purified recombinant RII was biotinylated by dialyzing 0.5 ml of pure RII protein (0.5-1.5 mg/ml) against 2 liters of 50 mM sodium bicarbonate (pH 8.5) for 2-4 h. EZTM sulfosuccinimidyl-6-(biotinamido)hexanoate was then added at a 10-fold molar ratio over protein and incubated on ice for 2 h. Excess unreacted biotin was removed by dialysis overnight against 10 mM Tris-HCl (pH 7.4) and 0.15 M NaCl (TBS).
Polyacrylamide Gel Electrophoresis, Immunoblotting, and RII OverlaySDS-PAGE was carried out under reducing conditions on 10-20% polyacrylamide-Tris/Tricine gels. Electroblotting to nitrocellulose membranes was carried out in a Novex Xcell II apparatus for 2.5 h at 195 mA (constant current) with 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol (pH 8.3) as the transfer buffer. Unbound sites were blocked either for 30 min at room temperature or overnight at 4 °C with 5% (w/v) skim milk powder or 10% normal horse serum in TBS. Blots were blocked in 10% normal horse serum only prior to detection of RII, which involved the use of an anti-goat antibody that cross-reacted with proteins from milk, but not horse serum.
For AKAP detection, blocked membranes were washed three times for 10 min each with TBS containing 0.05% Tween 20 (TBST) and then incubated
for 1 h with 5 nM RII-biotin with or without a 30-min
room temperature preincubation with 0.4 µM HT-31 or
HT-31-P peptide in TBST. Membranes were then washed three times for 10 min each with TBST and incubated for 30 min with 2 µg/ml horseradish peroxidase-linked avidin in TBST. Blots were washed three times for 10 min each with TBST, and reactive bands were visualized using the Pierce
SupersignalTM ECL detection system. For RII detection, nitrocellulose
membranes blocked in horse serum were incubated for 1 h with a
goat anti-RII antibody (kindly provided by John Scott, Vollum
Institute, Oregon Health Sciences University), followed by a 30-min
incubation with a horseradish peroxidase-linked anti-goat IgG (Jackson
ImmunoResearch Laboratories, Inc.). Detection was via ECL, and TBST
washes were included between steps as described above.
Electrotransferred calcium channel was detected following a 1-h
incubation with the rabbit anti-CP11 peptide antibody directed against
the C-terminal region of the 1 subunit, followed by a 30-min
incubation with horseradish peroxidase-linked protein A, ECL, and film
exposure.
Approximately 10 pmol of calcium channel, purified as described above, were incubated with 20 µg of anti-CP11 antibody or control antibody for 2 h at 4 °C in a final volume of 0.5 ml in 50 mM Tris-HCl (pH 7.4), 75 mM NaCl, 2.5 mM EDTA, and 0.1% digitonin (Buffer A). Each 0.5-ml reaction was added to 5 mg of protein A-Sepharose beads and incubated for an additional 1 h at 4 °C. Protein A-Sepharose beads containing precipitated complexes were washed three times with 1 ml Buffer A, and associated proteins were either used in endogenous phosphorylation experiments as described above or eluted following incubation in SDS sample buffer for 15 min at 65 °C. SDS-eluted proteins were separated by SDS-PAGE and probed for AKAPs as described above.
Incubation of purified calcium channels with
[-32P]ATP followed by SDS-PAGE and autoradiography as
described under "Experimental Procedures" yielded three prominent
phosphoprotein bands of ~170, 65, and 60 kDa, with a less prominent
band appearing at 90 kDa (Fig. 1A). Fig.
1A also shows that the phosphorylation of these proteins by
the endogenous kinase increased over time. To identify the three
phosphorylated proteins, we performed immunoblotting experiments on
identical purified calcium channel preparations. The 170-kDa
phosphoprotein seen in Fig. 1A displayed the same apparent
mobility as the
1 subunit of the calcium channel recognized by
anti-CP11 antibody (Fig. 1B, first lane). The
65-kDa phosphoprotein was similarly identified as the
subunit of
the calcium channel based on comparison with the migration of the
subunit as recognized by a
subunit-specific antibody (Fig.
1B, second lane). Finally, the ~60-kDa
phosphoprotein was identified as RII as its gel position was the same
as that of the RII protein recognized by RII-specific antibodies (Fig.
1B, third lane). The faint band at ~90 kDa was not identified and may represent a degradation product of the calcium
channel
1 subunit.
Endogenous Kinase Activity Resembles PKA
The presence of RII
in purified calcium channel preparations as well as the fact that the
calcium channel is a known substrate for PKA (31) led us to test
whether the endogenous kinase was in fact PKA. Fig.
2A demonstrates the effect of cAMP on the
endogenous kinase activity as measured by phosphorylation of the
calcium channel 1 subunit. The endogenous kinase activity was
stimulated following the addition of cAMP. This experiment was repeated
five times, and in each case, cAMP stimulated the phosphorylation of the
1 subunit. cAMP-dependent stimulation ranged from
~1.5 to 3-fold. We further established the identity of the kinase
present in calcium channel preparations using specific kinase substrate and inhibitor peptides. Fig. 2B shows that the endogenous
kinase phosphorylated the known PKA substrate SP48 (32) and that
phosphorylation of this peptide was stimulated by the addition of cAMP.
cAMP consistently (n = 5) increased the phosphorylation
of SP48, with the stimulation ranging from ~2.5 to 8-fold. In
addition, PKI-(5-24) completely blocked the cAMP-stimulated kinase
activity (Fig. 2B). PKI-(5-24), however, did not completely
block all endogenous protein kinase activity. The endogenous kinase
activity failed to phosphorylate peptides that are substrates for PKC
and casein kinase II (Fig. 2B). Together, these data
strongly suggest that PKA is co-purifying with the calcium channel.
PKA Activity Co-immunoprecipitates with the Calcium Channel
We performed co-immunoprecipitation experiments to test
whether the co-purifying kinase is physically associated with the calcium channel. The channel was first immunoprecipitated from calcium
channel preparations with the 1 subunit-specific anti-CP11 antibody
and then incubated under phosphorylating conditions with [
-32P]ATP as described under "Experimental
Procedures." Fig. 3A shows that following
immunoprecipitation and addition of [
-32P]ATP, both
the
1 and
subunits of the calcium channel were phosphorylated by
a co-precipitating kinase. The PKA substrate peptide SP48 was also
phosphorylated by the immunoprecipitated calcium channel complex (Fig.
3B). When the immunoprecipitation was performed with a
control antibody (IgG), phosphorylation of the peptide was not detected
(Fig. 3B). Therefore, based on co-immunoprecipitation experiments, the calcium channel is physically associated with a
co-purifying kinase.
Detection of AKAPs with RII-Biotin Overlay
Recombinant RII
protein expressed, purified, and biotinylated as described under
"Experimental Procedures" was employed in an RII overlay assay
adapted from the procedure of Lohmann et al. (12). Pure
MAP-2 protein resolved by SDS-PAGE and immobilized on nitrocellulose
was probed with RII-biotin and visualized with horseradish
peroxidase-linked avidin and ECL as described under "Experimental
Procedures." Fig. 4 demonstrates that RII-biotin can
be used in an overlay assay to detect the AKAP MAP-2. Preincubation of
RII-biotin with a 24-amino acid anchoring inhibitor peptide (HT-31)
derived from the AKAP Ht-31 (18) prevented recognition of MAP-2,
indicating a specific RII-AKAP interaction (Fig. 4). HT-31-P is a
control proline-substituted peptide derived from HT-31 that does not
bind RII (18). Preincubation of RII-biotin with HT-31-P peptide did not
prevent recognition of MAP-2 (Fig. 4). Thus, RII-biotin binds to AKAPs
in a specific manner in an overlay assay. The protein detected at ~70
kDa is likely a degradation product of full-length MAP-2 in the
commercial preparation.
Identification of a 15-kDa AKAP in Purified Calcium Channel
Proteins present in purified calcium channel
preparations were separated by SDS-PAGE, transferred to nitrocellulose,
and screened for AKAPs using the RII-biotin overlay as described under
"Experimental Procedures." In Fig. 5, we identify a
15-kDa AKAP that co-purified with the calcium channel. Preincubation of
RII-biotin with HT-31 peptide abolished recognition of AKAP-15, while
preincubation with the control HT-31-P peptide did not affect
recognition (Fig. 5). Together, these data demonstrate the presence of
a previously undescribed RII-binding protein present in purified
calcium channel preparations, which we designate AKAP-15.
AKAP-15 Co-immunoprecipitates with the Calcium Channel
To demonstrate a physical association between
AKAP-15 and the calcium channel, we immunoprecipitated the calcium
channel with anti-CP11 antibody and assayed for the co-precipitation of AKAP-15. The calcium channel and associated proteins precipitated from
calcium channel preparations were separated by SDS-PAGE, transferred to
nitrocellulose, and probed for the presence of AKAP-15 as described
under "Experimental Procedures." Fig. 6 shows the
presence of AKAP-15 among precipitated proteins as detected by the
RII-biotin overlay assay. Detection of AKAP-15 was prevented by
preincubation of RII-biotin with HT-31 peptide (Fig. 6). Moreover, AKAP-15 was not detected when a control antibody (IgG) was employed in
place of anti-CP11 antibody (Fig. 6). Co-immunoprecipitation of AKAP-15
with the calcium channel confirms its physical association with the
calcium channel complex.
Skeletal muscle L-type calcium channel activity is enhanced by PKA
phosphorylation (25, 26, 33); also, purified calcium channels
reconstituted into phospholipid vesicles or bilayers display an
increase in ion flux activity following phosphorylation by PKA (34,
35). The channel complex purified from skeletal muscle transverse
tubule membranes consists of five subunits: the principal pore-forming
1 subunit and auxiliary
,
,
2, and
subunits (24). Both
the
1 and
subunits are phosphorylated by PKA in vitro
(31, 34, 36, 37). The
1 subunit is phosphorylated on multiple serine
residues by PKA both in vitro and in intact cells in
response to cAMP stimulation (33, 38, 39). While the precise sites
responsible for physiological modulation of calcium channel activity by
PKA phosphorylation have not been identified, it is likely that
critical sites lie on the
1 and/or
subunit of the calcium
channel.
In this study, we have identified an endogenous kinase activity that co-purifies with the calcium channel. We have further provided evidence that this co-purifying kinase is PKA and that it is physically associated with the channel complex. Using a novel RII-biotin overlay assay, we have detected an AKAP of ~15 kDa that also co-purifies and co-immunoprecipitates with the calcium channel. Together, these results suggest a role for AKAP-15 in anchoring type II PKA to the calcium channel and thereby allowing discrete phosphorylation of the channel in response to cAMP.
Our results are consistent with previous physiological experiments demonstrating a close association between L-type calcium channels and PKA (26). Voltage-dependent phosphorylation by PKA increases the subsequent activity of the calcium channel (25). This voltage-dependent potentiation represents a measurable effect of PKA on calcium channels in intact cells and has been used to assess the requirement of kinase anchoring. Johnson et al. (26) showed that the AKAP-derived anchoring inhibitor peptide (HT-31) that disrupts RII-AKAP interactions completely blocks voltage-dependent potentiation of calcium channels in skeletal muscle myotubes. Blockade of voltage-dependent potentiation by disruption of anchoring was overcome by the introduction of excess catalytic subunit of PKA into cells at concentrations high enough to obviate the requirement for specific localization of PKA near the calcium channel (26). The ability of HT-31 peptide to block potentiation of the calcium channel by PKA strongly suggests that PKA is anchored near the channel by an AKAP. Our experiments are consistent with this hypothesis and suggest that AKAP-15 mediates the anchoring of PKA near the calcium channel.
The kinase activity we detect in purified calcium channel preparations
resembles PKA in several respects. Our initial observation shows that
this activity phosphorylates three PKA substrates, the 1 and
subunits of the calcium channel as well as RII. The endogenous kinase
activity is also stimulated by cAMP, and the stimulated activity is
inhibited by PKI(5-24). We measured the phosphorylation of various
substrate peptides by the endogenous kinase and found that the PKA
substrate SP48 was phosphorylated, while SP44, DSDD, PKC, and casein
kinase II substrates, respectively, were not. Furthermore,
two-dimensional tryptic phosphopeptide maps of the skeletal muscle
calcium channel
1 subunit phosphorylated by either exogenous PKA or
the endogenous kinase were similar (data not shown). While we believe
we have identified PKA, it is important to note that we were unable to
completely block the endogenous kinase activity with PKI(5-24),
indicating that there may also be another kinase in these
preparations.
The endogenous kinase activity not only co-purifies with the calcium channel, but co-immunoprecipitates with it as well. This result indicates that the kinase and calcium channel are physically linked to each other and provided the impetus to examine purified calcium channel preparations for AKAPs. Various approaches to detect AKAPs have been described, each involving the use of purified RII protein in an overlay assay (40). In this study, we have introduced a variation of the RII overlay using biotinylated RII. This new assay, which involves detection of RII-biotin with peroxidase-conjugated avidin, has proven faster and easier than detection with antibodies or with radiolabeled RII. We validated the RII-biotin overlay by using it to detect the known AKAP MAP-2. Preincubation of RII-biotin with an AKAP-derived anchoring inhibitor peptide (HT-31) prevented MAP-2 recognition, indicating that this is a specific RII-AKAP interaction. In addition to its usefulness in detecting AKAPs, we anticipate that RII-biotin will serve as a useful affinity reagent for purification of AKAPs.
Using the RII-biotin overlay, we have identified a novel 15-kDa AKAP in purified calcium channel preparations that we believe mediates the association of PKA and the calcium channel. Detection of AKAP-15 is blocked by preincubation of RII-biotin with HT-31 peptide, consistent with it being an AKAP. Co-purification of AKAP-15 with the calcium channel suggests that these two proteins are physically associated. We confirmed this association by co-immunoprecipitating AKAP-15 using a calcium channel-specific anti-peptide antibody. AKAP-15 was not co-precipitated by a control antibody, and its detection was blocked by preincubation of RII-biotin with HT-31 peptide. AKAP-15 is the only RII-binding protein we detected in purified calcium channel preparations, making it an excellent candidate for mediating the role of attaching PKA to the calcium channel complex. Unlike many AKAPs, which remain poorly characterized with respect to their specific targets and functional importance, AKAP-15 possesses both a likely target (L-type calcium channels) and functional importance (voltage-dependent potentiation). Purification and characterization of this AKAP may allow determination of its role in anchoring PKA to the calcium channel.